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The iterative bleaching extends multiplexity (IBEX) Knowledge-Base is a central portal for researchers adopting IBEX and related 2D and 3D immunofluorescence imaging methods. The design of the Knowledge-Base is modeled after efforts in the open-source software community and includes three facets: a development platform (GitHub), static website, and service for data archiving. The Knowledge-Base facilitates the practice of open science throughout the research life cycle by providing validation data for recommended and non-recommended reagents, such as primary and secondary antibodies. In addition to reporting negative data, the Knowledge-Base empowers method adoption and evolution by providing a venue for sharing protocols, videos, datasets, software, and publications. A dedicated discussion forum fosters a sense of community among researchers while addressing questions not covered in published manuscripts. Together, scientists from around the world are advancing scientific discovery at a faster pace, reducing wasted time and effort, and instilling greater confidence in the resulting data.

Whether antiretroviral therapy (ART) is always completely suppressive, or HIV might continue to replicate at low levels despite ART in some people with HIV (PWH), is still debated. Here, we intensified the ART regimen by doubling dolutegravir (DTG) dosage and investigated the impact of this strategy on HIV blood and tissue reservoirs, immune activation, and inflammation. Twenty HIV-infected adults, who had received a triple ART consisting of 50 mg DTG/600 mg abacavir/300 mg lamivudine pre-intensification and had been suppressed on ART for at least 2 years, were enrolled in a phase 2 randomized clinical trial (https://clinicaltrials.gov/ identifier: NCT05351684). Half of them received an additional 50 mg of DTG for a period of 84 days. As expected, plasma and tissue DTG concentrations significantly increased during the study period in the intensified group but not in the control group. Accordingly, significant decreases in total HIV DNA, intact HIV DNA, and cell-associated unspliced (US) HIV RNA in peripheral blood mononuclear cells, as well as in the US RNA/total DNA ratio, were observed in the intensified group but not in the control group. Intensification also modestly reduced markers of immune activation and exhaustion but had no measurable impact on systemic or tissue inflammation. Together with this, intensification resulted in a temporary decrease in the CD4/CD8 ratio that returned to baseline by day 84. Our results strongly suggest that the pre-intensification ART regimen was not completely suppressive. If confirmed in larger clinical trials, these results could have an impact on the clinical management of PWH and HIV curative strategies.

Striatal dopamine (DA) release regulates reward-related learning and motivation and is believed to consist of a short-lived phasic and continuous tonic component. Here, we build a large-scale three-dimensional model of extracellular DA dynamics in dorsal (DS) and ventral striatum (VS). The model predicts rapid dynamics in DS with little to no basal DA and slower dynamics in the VS enabling build-up of tonic DA levels. These regional differences do not reflect release-related phenomena but rather differential dopamine transporter (DAT) activity. Interestingly, our simulations posit DAT nanoclustering as a possible regulator of this activity. Receptor binding simulations show that D1 receptor occupancy follows extracellular DA concentration with milliseconds delay, while D2 receptors do not respond to brief pauses in firing but rather integrate DA signal over seconds. Summarised, our model distills recent experimental observations into a computational framework that challenges prevailing paradigms of striatal DA signalling.

Asymmetric partition of fate determinants during cell division is a hallmark of cell differentiation. Recent work suggested that such a mechanism is hijacked by cancer cells to increase both their phenotypic heterogeneity and plasticity and, in turn, their fitness. To quantify fluctuations in the partitioning of cellular elements, imaging-based approaches are used, whose accuracy is limited by the difficulty of detecting cell divisions. Our work addresses this gap, proposing a general method based on high-throughput flow cytometry measurements coupled with a theoretical framework. We applied our method to a panel of both normal and cancerous human colon cells, showing that different kinds of colon adenocarcinoma cells display very distinct extents of fluctuations in their cytoplasm partition, explained by an asymmetric division of their size. To test the accuracy of our population-level protocol, we directly measure the inherited fractions of cellular elements from extensive time lapses of live-cell laser scanning microscopy, finding excellent agreement across the cell types. Ultimately, our flow cytometry-based method promises to be accurate and easily applicable to a wide range of biological systems where the quantification of partition fluctuations would help account for the observed phenotypic heterogeneity and plasticity.

Antiretroviral therapy (ART) initiated in the acute phase of HIV infection (AHI) results in a smaller viral reservoir. However, the impact of early HIV-specific T-cell responses on long-term reservoir dynamics is less well characterized. Therefore, we measured the size of the viral reservoir and functionality of HIV-specific CD8+ T-cell responses after the acute phase at 24 and 156 weeks after ART initiation in people with HIV who started treatment during AHI. A significant reduction in total and defective HIV DNA and a trend toward a reduction in intact HIV DNA were observed between 24 and 156 weeks. Functional CD8+ T-cell responses against HIV peptides Env, Gag, Nef, and Pol were maintained over 3 years after treatment initiation. The proliferative capacity of HIV-specific CD8+ T-cells at 24 weeks of ART was predictive of the degree of reduction in total and defective HIV DNA between 24 and 156 weeks, suggesting HIV-specific CD8+ T-cells may at least partially drive the decline of the viral reservoir. Therefore, enforcing HIV-specific immune responses as early as possible after diagnosis of AHI should be a central focus of HIV cure strategies.

Human vision is characterized by frequent eye movements and constant shifts in visual input, yet our perception of the world remains remarkably stable. Here, we directly demonstrate image-specific foveal feedback to primary visual cortex in the context of saccadic eye movements. To this end, we used a gaze-contingent fMRI paradigm, in which peripheral saccade targets disappeared before they could be fixated. Despite no direct foveal stimulation, we were able to decode peripheral saccade targets from foveal retinotopic areas, demonstrating that image-specific feedback during saccade preparation may underlie this effect. Decoding was sensitive to shape but not semantic category of natural images, indicating feedback of only low-to-mid-level information. Cross-decoding to a control condition with foveal stimulus presentation indicates a shared representational format between foveal feedback and direct stimulation. Moreover, eccentricity-dependent analyses showed a U-shaped decoding curve, confirming that these results are not explained by spillover of peripheral activity or large receptive fields. Finally, fluctuations in foveal decodability covaried with activity in the intraparietal sulcus, thus providing a candidate region for driving foveal feedback. These findings suggest that foveal cortex predicts the features of incoming stimuli through feedback from higher cortical areas, which offers a candidate mechanism underlying stable perception.

Rare and common genetic variants contribute to the risk of atrial fibrillation (AF). Although ion channels were among the first AF candidate genes identified, rare loss-of-function variants in structural genes, such as TTN, have also been implicated in AF pathogenesis, partly through the development of atrial myopathy; however, the underlying mechanisms are poorly understood. While TTN truncating variants (TTNtvs) have been causally linked to arrhythmia and cardiomyopathy syndromes, the role of missense variants (mvs) remains unclear. We show that rare TTNmvs are associated with worse clinical outcomes in a single-center ethnic minority clinical cohort and uncover a pathogenic mechanism by which the T32756I variant drives AF. Modeling the TTN-T32756I variant using human induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) revealed that the mutant cells display aberrant contractility, increased activity of a cardiac potassium channel (KCNQ1, Kv7.1), and dysregulated calcium homeostasis without compromising the sarcomeric integrity of the atrial cardiomyocytes. We also show that a titin-binding protein, the Four-and-a-Half Lim domains 2 (FHL2), has increased binding with KCNQ1 and its modulatory subunit KCNE1 in the TTN-T32756I-iPSC-aCMs, enhancing the slow delayed rectifier potassium current (I_ks). Suppression of FHL2 in mutant iPSC-aCMs normalized the I_ks, supporting FHL2 as an I_ks modulator. Our findings demonstrate that a single amino acid substitution in titin not only impairs its function but also remodels ion channels, contributing to AF. These findings underscore the importance of high-throughput screening to assess the pathogenicity of TTNmvs and establish a mechanistic connection between titin, potassium ion channels, and sarcomeric proteins, which may represent a novel therapeutic target.

Hierarchical communication between cells with distinct positional memories orchestrates the regeneration of missing limb structures in axolotls.

Cells process dynamic signaling inputs to regulate fate decisions during development. While oscillations or waves in key developmental pathways, such as Wnt, have been widely observed, the principles governing how cells decode these signals remain unclear. By leveraging optogenetic control of the Wnt signaling pathway in both HEK293T cells and H9 human embryonic stem cells, we systematically map the relationship between signal frequency and downstream pathway activation. We find that cells exhibit a minimal response to Wnt at certain frequencies, a behavior we term anti-resonance. We developed both detailed biochemical and simplified hidden variable models that explain how anti-resonance emerges from the interplay between fast and slow pathway dynamics. Remarkably, we find that frequency directly influences cell fate decisions involved in human gastrulation; signals delivered at anti-resonant frequencies result in dramatically reduced mesoderm differentiation. Our work reveals a previously unknown mechanism of how cells decode dynamic signals and how anti-resonance may filter against spurious activation. These findings establish new insights into how cells decode dynamic signals with implications for tissue engineering, regenerative medicine, and cancer biology.

The expression of antibiotic-inactivating enzymes, such as Pseudomonas-derived cephalosporinase-3 (PDC-3), is a major mechanism of intrinsic resistance in bacteria. Using reinforcement learning-driven molecular dynamics simulations and constant pH MD, we investigate how clinically observed mutations in the Ω-loop (at residues V211, G214, E219, and Y221) alter the structure and function of PDC-3. Our findings reveal that these substitutions modulate the dynamic flexibility of the Ω-loop and the R2-loop, reshaping the cavity of the active site. In particular, E219K and Y221A disrupt the tridentate hydrogen bond network around K67, thus lowering its pKa and promoting proton transfer to the catalytic residue S64. Markov state models reveal that E219K achieves enhanced catalysis by adopting stable, long-lived 'active' conformations, whereas Y221A facilitates activity by rapidly toggling between bond-formed and bond-broken states. In addition, substitutions influence key hydrogen bonds that control the opening and closure of the active-site pocket, consequently influencing the overall size. The pocket expands in all nine clinically identified variants, creating additional space to accommodate bulkier R1 and R2 cephalosporin side chains. Taken together, these results provide a mechanistic basis for how single residue substitutions in the Ω-loop affect catalytic activity. Insights into the structural dynamics of the catalytic site advance our understanding of emerging β-lactamase variants and can inform the rational design of novel inhibitors to combat drug-resistant P. aeruginosa.