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The antimycobacterial drug clofazimine (CFZ) is used as a single agent at high doses, to suppress the exaggerated inflammation associated with leprosy. Paradoxically, increasing doses of CFZ leads to bioaccumulation of CFZ in the spleen and other organs under physiologically relevant dosing regimens, without accompanying dose-dependent elevation in the concentrations of the circulating drug in the blood. In long-term oral dosing regimens, CFZ induces immunological and metabolic changes resulting in splenomegaly, while the mass of other organs decreases or remains unchanged. As an organ that extensively sequesters CFZ as insoluble drug precipitates, the spleen likely influences drug-induced inflammatory signaling. To probe the role of systemic drug concentrations vs. drug bioaccumulation in the spleen, healthy mice were treated with six different dosing regimens. A subgroup of these mice underwent surgical splenectomies prior to drug treatment to assess the bioaccumulation-dependent changes in immune system signaling and immune-system-mediated drug distribution. Under increasing drug loading, the spleen was observed to grow up to six times in size, sequestering over 10% of the total drug load. Interestingly, when the spleen was removed prior to CFZ administration, drug distribution in the rest of the organism was unaffected. However, there were profound cytokine elevations in the serum of asplenic CFZ-treated mice, indicating that the spleen is primarily involved in suppressing the inflammatory signaling mechanisms that are upregulated during CFZ bioaccumulation. Thus, beyond its role in drug sequestration, the spleen actively modulates the systemic effect of CFZ on the immune system, without impacting its blood concentrations or distribution to the rest of the organism.

Synaptotagmin, complexin, and neuronal SNARE (soluble N -ethylmaleimide sensitive factor attachment protein receptor) proteins mediate evoked synchronous neurotransmitter release, but the molecular mechanisms mediating the cooperation between these molecules remain unclear. Here we determine crystal structures of the primed pre-fusion SNARE–complexin–synaptotagmin-1 complex. These structures reveal an unexpected tripartite interface between synaptotagmin-1 and both the SNARE complex and complexin. Simultaneously, a second synaptotagmin-1 molecule interacts with the other side of the SNARE complex via the previously identified primary interface. Mutations that disrupt either interface in solution also severely impair evoked synchronous release in neurons, suggesting that both interfaces are essential for the primed pre-fusion state. Ca 2+ binding to the synaptotagmin-1 molecules unlocks the complex, allows full zippering of the SNARE complex, and triggers membrane fusion. The tripartite SNARE–complexin–synaptotagmin-1 complex at a synaptic vesicle docking site has to be unlocked for triggered fusion to start, explaining the cooperation between complexin and synaptotagmin-1 in synchronizing evoked release on the sub-millisecond timescale. An atomic model of the primed pre-fusion SNARE–complexin–synaptotagmin-1 complex in neuronal exocytosis accounting for vesicle priming and cooperation in synchronizing and activating evoked release on the sub-millisecond timescale. Three-way complex primes synapses for membrane fusion For rapid neurotransmitter release upon the arrival of an action potential, synaptic vesicles are 'primed' to undergo synchronous fusion with the pre-synaptic membrane, but the molecular basis of such priming is unknown. Now, with two large co-crystal structures, Axel Brunger and colleagues reveal a new and unexpected three-way interface between the proteins synaptotagmin (Syt1), complexin (Cpx) and the SNARE complex, beside a previously identified primary interface involving another molecule of Syt1 with the same SNARE complex. Through a combination of mutagenesis, biochemistry and electrophysiology, the authors show how this tripartite interface locks the primed complex into a state of low fusion probability and how action-potential-driven Ca 2+ binds to the Syt1 molecules to unlock the complex, allowing full zippering of the SNARE complex and triggering membrane fusion in a highly synchronized fashion on the sub-millisecond timescale.

There is abundant, physiologically relevant knowledge about protein cores; they are hydrophobic, exquisitely well packed, and nearly all hydrogen bonds are satisfied. An equivalent understanding of protein surfaces has remained elusive because proteins are almost exclusively studied in vitro in simple aqueous solutions. Here, we establish the essential physiological roles played by protein surfaces by measuring the equilibrium thermodynamics and kinetics of protein folding in the complex environment of living Escherichia coli cells, and under physiologically relevant in vitro conditions. Fluorine NMR data on the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) show that charge–charge interactions are fundamental to protein stability and folding kinetics in cells. Our results contradict predictions from accepted theories of macromolecular crowding and show that cosolutes commonly used to mimic the cellular interior do not yield physiologically relevant information. As such, we provide the foundation for a complete picture of protein chemistry in cells.

Whereas ferromagnets have been known and used for millennia, antiferromagnets were only discovered in the 1930s 1 . At large scale, because of the absence of global magnetization, antiferromagnets may seem to behave like any non-magnetic material. At the microscopic level, however, the opposite alignment of spins forms a rich internal structure. In topological antiferromagnets, this internal structure leads to the possibility that the property known as the Berry phase can acquire distinct spatial textures 2 , 3 . Here we study this possibility in an antiferromagnetic axion insulator—even-layered, two-dimensional MnBi 2 Te 4 —in which spatial degrees of freedom correspond to different layers. We observe a type of Hall effect—the layer Hall effect—in which electrons from the top and bottom layers spontaneously deflect in opposite directions. Specifically, under zero electric field, even-layered MnBi 2 Te 4 shows no anomalous Hall effect. However, applying an electric field leads to the emergence of a large, layer-polarized anomalous Hall effect of about 0.5 e 2 / h (where e is the electron charge and h is Planck’s constant). This layer Hall effect uncovers an unusual layer-locked Berry curvature, which serves to characterize the axion insulator state. Moreover, we find that the layer-locked Berry curvature can be manipulated by the axion field formed from the dot product of the electric and magnetic field vectors. Our results offer new pathways to detect and manipulate the internal spatial structure of fully compensated topological antiferromagnets 4 – 9 . The layer-locked Berry curvature represents a first step towards spatial engineering of the Berry phase through effects such as layer-specific moiré potential. A new type of Hall effect—the layer Hall effect—is produced in a 2D antiferromagnet that does not exhibit any net magnetization.

The genomes of cancers deficient in mismatch repair contain exceptionally high numbers of somatic mutations. In a proof-of-concept study, we previously showed that colorectal cancers with mismatch repair deficiency were sensitive to immune checkpoint blockade with antibodies to programmed death receptor–1 (PD-1). We have now expanded this study to evaluate the efficacy of PD-1 blockade in patients with advanced mismatch repair–deficient cancers across 12 different tumor types. Objective radiographic responses were observed in 53% of patients, and complete responses were achieved in 21% of patients. Responses were durable, with median progression-free survival and overall survival still not reached. Functional analysis in a responding patient demonstrated rapid in vivo expansion of neoantigen-specific T cell clones that were reactive to mutant neopeptides found in the tumor. These data support the hypothesis that the large proportion of mutant neoantigens in mismatch repair–deficient cancers make them sensitive to immune checkpoint blockade, regardless of the cancers’ tissue of origin.

In this trial, high-frequency oscillatory ventilation was compared with conventional ventilation with a lung-protective protocol. When the study was stopped early, hospital mortality was 47% with HFOV versus 35% with the control ventilation strategy. The acute respiratory distress syndrome (ARDS) is a common complication of critical illness. 1 , 2 Mortality is high, and survivors often have long-term complications. 3 , 4 Although mechanical ventilation is life-sustaining for patients with ARDS, it can perpetuate lung injury. Basic research suggests that repetitive overstretching or collapse of lung units with each respiratory cycle can generate local and systemic inflammation, contributing to multiorgan failure and death. 5 Consistent with these findings are data from clinical trials that support the use of smaller tidal volumes (6 vs. 12 ml per kilogram of predicted body weight) 6 and higher levels of positive end-expiratory pressure (PEEP). . . .

Extracellular matrix (ECM) plays an important developmental role by regulating cell behaviour through structural and biochemical stimulation. Tissue-specific ECM, attained through decellularization, has been proposed in several strategies for tissue and organ replacement. Decellularization of animal pancreata has been reported, but the same methods applied to human pancreas are less effective due to higher lipid content. Moreover, ECM-derived hydrogels can be obtained from many decellularized tissues, but methods have not been reported to obtain human pancreas-derived hydrogel. Using novel decellularization methods with human pancreas we produced an acellular, 3D biological scaffold (hP-ECM) and hydrogel (hP-HG) amenable to tissue culture, transplantation and proteomic applications. The inclusion of a homogenization step in the decellularization protocol significantly improved lipid removal and gelation capability of the resulting ECM, which was capable of gelation at 37 °C in vitro and in vivo , and is cytocompatible with a variety of cell types and islet-like tissues in vitro . Overall, this study demonstrates the characterisation of a novel protocol for the decellularization and delipidization of human pancreatic tissue for the production of acellular ECM and ECM hydrogel suitable for cell culture and transplantation applications. We also report a list of 120 proteins present within the human pancreatic matrisome.

Neutrophil infiltration and subsequent extracellular trap formation (NETosis) is a contributing factor in sterile inflammation. Furthermore, neutrophil extracellular traps (NETs) are prothrombotic, as they provide a scaffold for platelets and red blood cells to attach to. In circulation, neutrophils are constantly exposed to hemodynamic forces such as shear stress, which in turn regulates many of their biological functions such as crawling and NETosis. However, the mechanisms that mediate mechanotransduction in neutrophils are not fully understood. In this study, we demonstrate that shear stress induces NETosis, dependent on the shear stress level, and increases the sensitivity of neutrophils to NETosis-inducing agents such as adenosine triphosphate and lipopolysaccharides. Furthermore, shear stress increases intracellular calcium levels in neutrophils and this process is mediated by the mechanosensitive ion channel Piezo1. Activation of Piezo1 in response to shear stress mediates calpain activity and cytoskeleton remodeling, which consequently induces NETosis. Thus, activation of Piezo1 in response to shear stress leads to a stepwise sequence of cellular events that mediates NETosis and thereby places neutrophils at the centre of localized inflammation and prothrombotic effects. Neutrophils undergo shear stress in the circulation and mechano-sensing is known to impact neutrophil biology. Here the authors show that activation of Piezo1 by shear stress triggers a sequence of cellular events leading to NETosis.

Accurate and timely information about land cover pattern and change in urbanareas is crucial for urban land management decision-making, ecosystem monitoring andurban planning. This paper presents the methods and results of an object-basedclassification and post-classification change detection of multitemporal high-spatialresolution Emerge aerial imagery in the Gwynns Falls watershed from 1999 to 2004. TheGwynns Falls watershed includes portions of Baltimore City and Baltimore County,Maryland, USA. An object-based approach was first applied to implement the land coverclassification separately for each of the two years. The overall accuracies of theclassification maps of 1999 and 2004 were 92.3% and 93.7%, respectively. Following theclassification, we conducted a comparison of two different land cover change detectionmethods: traditional (i.e., pixel-based) post-classification comparison and object-basedpost-classification comparison. The results from our analyses indicated that an objectbasedapproach provides a better means for change detection than a pixel based methodbecause it provides an effective way to incorporate spatial information and expertknowledge into the change detection process. The overall accuracy of the change mapproduced by the object-based method was 90.0%, with Kappa statistic of 0.854, whereasthe overall accuracy and Kappa statistic of that by the pixel-based method were 81.3% and0.712, respectively.

Animals associate cues with outcomes and update these associations as new information is presented. This requires the hippocampus, yet how hippocampal neurons track changes in cue–outcome associations remains unclear. Using two-photon calcium imaging, we tracked the same dCA1 and vCA1 neurons across days to determine how responses evolve across phases of odor–outcome learning. Initially, odors elicited robust responses in dCA1, whereas, in vCA1, odor responses primarily emerged after learning and embedded information about the paired outcome. Population activity in both regions rapidly reorganized with learning and then stabilized, storing learned odor representations for days, even after extinction or pairing with a different outcome. Additionally, we found stable, robust signals across CA1 when mice anticipated outcomes under behavioral control but not when mice anticipated an inescapable aversive outcome. These results show how the hippocampus encodes, stores and updates learned associations and illuminates the unique contributions of dorsal and ventral hippocampus. By tracking the activity of CA1 neurons during associative learning, Biane, Ladow et al. reveal the distinct contribution of neurons along the dorsoventral axis of CA1 in the encoding and updating of task-related representations throughout learning.