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Abstract Background Increased neutrophil extracellular trap (NET) formation and abundant NET-associated proteins are frequently found in the inflamed colon of patients with inflammatory bowel disease. Peptidyl arginine deiminase 4 (PAD4) activation is essential for the generation of NET and NET-mediated pathogenesis. However, the role of PAD4-dependent NET formation in murine inflammatory bowel disease models and the molecular mechanisms responsible for the altered gut barrier function are unknown. Methods Wild-type and Pad4 knockout (Pad4-/-) mice were administrated 3% dextran sulfate sodium (DSS) in their drinking water. Caco-2 monolayers were used to test the effect of NETs on intestinal barrier function and cytotoxicity. Histones were intrarectally administrated to wild-type mice to determine their effects on intestinal barrier function and cytotoxicity in vivo. Results PAD4 deficiency reduced the severity of DSS-induced colitis with decreased intestinal NET formation and enhanced gut barrier function and integrity in mice. NETs disrupted the barrier function in intestinal epithelial Caco-2 monolayers through their protein, rather than DNA, components. Pretreatment of NETs with histone inhibitors abrogated the effects on epithelial permeability. Consistent with these observations, adding purified histone proteins to Caco-2 monolayers significantly damaged epithelial barrier function, which was associated with the abnormal distribution and integrity of tight junctions as well as with increased cell death. Furthermore, intrarectal administration of histones damaged the intestinal barrier integrity and induced cytotoxicity in the mouse colon epithelium. Conclusions PAD4-mediated NET formation has a detrimental role in acute colitis. NET-associated histones directly inhibit intestinal barrier function, resulting in cytotoxicity in vitro and in vivo. Lay Summary Peptidyl arginine deiminase 4–dependent neutrophil extracellular trap formation is detrimental to intestinal barrier function in acute colitis. Neutrophil extracellular trap–associated histones altered the integrity of tight junction and adherens junction proteins as well as induced intestinal epithelial cell death that resulted in increased gut epithelium permeability.

Aberrant neutrophil extracellular trap (NET) formation and the loss of barrier integrity in inflamed intestinal tissues have long been associated with inflammatory bowel disease (IBD). However, whether NETs alter intestinal epithelium permeability during colitis remains elusive. Here, we demonstrated that NETs promote the breakdown in intestinal barrier function for the pathogenesis of intestinal inflammation in mouse models of colitis. NETs were abundant in the colon of mice with colitis experimentally induced by dextran sulfate sodium (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS). Analysis of the intestinal barrier integrity revealed that NETs impaired gut permeability, enabling the initiation of luminal bacterial translocation and inflammation. Furthermore, NETs induced the apoptosis of epithelial cells and disrupted the integrity of tight junctions and adherens junctions. Intravenous administration of DNase I, an enzyme that dissolves the web-like DNA filaments of NETs, during colitis restored the mucosal barrier integrity which reduced the dissemination of luminal bacteria and attenuated intestinal inflammation in both DSS and TNBS models. We conclude that NETs serve a detrimental factor in the gut epithelial barrier function leading to the pathogenesis of mucosal inflammation during acute colitis.

Production of a functional peripheral T cell compartment typically involves massive expansion of the bone marrow progenitors that seed the thymus. There are two main phases of expansion during T cell development, following T lineage commitment of double-negative (DN) 2 cells and after successful rearrangement and selection for functional TCRβ chains in DN3 thymocytes, which promotes the transition of DN4 cells to the DP stage. The signals driving the expansion of DN2 thymocytes are well studied. However, factors regulating the proliferation and survival of DN4 cells remain poorly understood. Here, we uncover an unexpected link between the transcription factor Zfp335 and control of cGAS/STING-dependent cell death in post-β-selection DN4 thymocytes. Zfp335 controls survival by sustaining expression of Ankle2, which suppresses cGAS/STING-dependent cell death. Together, this study identifies Zfp335 as a key transcription factor regulating the survival of proliferating post-β-selection thymocytes and demonstrates a key role for the cGAS/STING pathway in driving apoptosis of developing T cells. T cell development involves extensive proliferation of developing thymocytes. Here, the authors demonstrate that the transcription factor Zfp335 regulates the survival post-β-selection thymocytes via the cGAS/STING pathway.

Based on rodent models, researchers have theorized that the hippocampus supports episodic memory and navigation via the theta oscillation, a ~4–10 Hz rhythm that coordinates brain-wide neural activity. However, recordings from humans have indicated that hippocampal theta oscillations are lower in frequency and less prevalent than in rodents, suggesting interspecies differences in theta’s function. To characterize human hippocampal theta, we examine the properties of theta oscillations throughout the anterior–posterior length of the hippocampus as neurosurgical subjects performed a virtual spatial navigation task. During virtual movement, we observe hippocampal oscillations at multiple frequencies from 2 to 14 Hz. The posterior hippocampus prominently displays oscillations at ~8-Hz and the precise frequency of these oscillations correlates with the speed of movement, implicating these signals in spatial navigation. We also observe slower ~3 Hz oscillations, but these signals are more prevalent in the anterior hippocampus and their frequency does not vary with movement speed. Our results converge with recent findings to suggest an updated view of human hippocampal electrophysiology. Rather than one hippocampal theta oscillation with a single general role, high- and low-frequency theta oscillations, respectively, may reflect spatial and non-spatial cognitive processes. We show that the human hippocampus exhibits two distinct theta oscillations during spatial navigation with the faster oscillation in posterior regions showing movement modulation. This result suggests a distinct feature of the human hippocampus compared to rodents, which generally show a single 8 Hz rhythm.

Homozygous deletion of methylthioadenosine phosphorylase ( MTAP ) in cancers such as glioblastoma represents a potentially targetable vulnerability. Homozygous MTAP -deleted cell lines in culture show elevation of MTAP’s substrate metabolite, methylthioadenosine (MTA). High levels of MTA inhibit protein arginine methyltransferase 5 (PRMT5), which sensitizes MTAP -deleted cells to PRMT5 and methionine adenosyltransferase 2A (MAT2A) inhibition. While this concept has been extensively corroborated in vitro, the clinical relevance relies on exhibiting significant MTA accumulation in human glioblastoma. In this work, using comprehensive metabolomic profiling, we show that MTA secreted by MTAP- deleted cells in vitro results in high levels of extracellular MTA. We further demonstrate that homozygous MTAP -deleted primary glioblastoma tumors do not significantly accumulate MTA in vivo due to metabolism of MTA by MTAP-expressing stroma. These findings highlight metabolic discrepancies between in vitro models and primary human tumors that must be considered when developing strategies for precision therapies targeting glioblastoma with homozygous MTAP deletion. The metabolite methylthioadenosine (MTA) inhibits PRMT5. Therefore, MTA accumulation due to MTA phosphorylase (MTAP) deletion has been proposed as a vulnerability for PRMT5-targeted therapy in cancer. Here, the authors show that MTA does not accumulate in MTAP-deficient cancer cells but is secreted and metabolized by MTAP-intact cells in the tumour microenvironment.

Sensorimotor control in vertebrates relies on internal models. When extending an arm to reach for an object, the brain uses predictive models of both limb dynamics and target properties. Whether invertebrates use such models remains unclear. Here we examine to what extent prey interception by dragonflies ( Plathemis lydia ), a behaviour analogous to targeted reaching, requires internal models. By simultaneously tracking the position and orientation of a dragonfly’s head and body during flight, we provide evidence that interception steering is driven by forward and inverse models of dragonfly body dynamics and by models of prey motion. Predictive rotations of the dragonfly’s head continuously track the prey’s angular position. The head–body angles established by prey tracking appear to guide systematic rotations of the dragonfly’s body to align it with the prey’s flight path. Model-driven control thus underlies the bulk of interception steering manoeuvres, while vision is used for reactions to unexpected prey movements. These findings illuminate the computational sophistication with which insects construct behaviour. This study tracks dragonfly head and body movements during high-velocity and high-precision prey-capture flights, and shows that the dragonfly uses predictive internal models and reactive control to build an interception trajectory that complies with biomechanical constraints. Dragonflies use complex internal models Primates and other vertebrates use internal models to control and predict the consequences of their movements, but invertebrates were thought to rely mostly on reflexes. In a study using motion capture to track the head and body movements of dragonflies catching flying prey, Anthony Leonardo and colleagues demonstrate a complexity of behaviour not previously seen in an insect. Approaching from below, the dragonfly's head locks onto its target while its body manoeuvres to align to the prey's flight path and reduce the distance to the prey. Rather than generating these steering movements reactively, by responding only to changes in prey motion, dragonflies using both reactive and predictive control. These predictions account for the motion of the prey and the dragonfly's own body, and are consistent with generation by internal models. The experimental accessibility of the insect nervous system, combined with the load-carrying capacity of the dragonfly, opens these general principles of motor control to detailed mechanistic dissection.

Toxoplasma gondii is an obligate intracellular parasite that invades host cells and replicates within a unique parasitophorous vacuole. To maintain this intracellular niche, the parasite secretes an array of dense granule proteins (GRAs) into the nascent parasitophorous vacuole. These GRAs are believed to play key roles in vacuolar remodeling, nutrient uptake, and immune evasion while the parasite is replicating within the host cell. Despite the central role of GRAs in the Toxoplasma life cycle, only a subset of these proteins have been identified, and many of their roles have not been fully elucidated. In this report, we utilize the promiscuous biotin ligase BirA* to biotinylate GRA proteins secreted into the vacuole and then identify those proteins by affinity purification and mass spectrometry. Using GRA-BirA* fusion proteins as bait, we have identified a large number of known and candidate GRAs and verified localization of 13 novel GRA proteins by endogenous gene tagging. We proceeded to functionally characterize three related GRAs from this group (GRA38, GRA39, and GRA40) by gene knockout. While Δ gra38 and Δ gra40 parasites showed no altered phenotype, disruption of GRA39 results in slow-growing parasites that contain striking lipid deposits in the parasitophorous vacuole, suggesting a role in lipid regulation that is important for parasite growth. In addition, parasites lacking GRA39 showed dramatically reduced virulence and a lower tissue cyst burden in vivo . Together, the findings from this work reveal a partial vacuolar proteome of T. gondii and identify a novel GRA that plays a key role in parasite replication and pathogenesis. IMPORTANCE Most intracellular pathogens reside inside a membrane-bound vacuole within their host cell that is extensively modified by the pathogen to optimize intracellular growth and avoid host defenses. In Toxoplasma , this vacuole is modified by a host of secretory GRA proteins, many of which remain unidentified. Here we demonstrate that in vivo biotinylation of proximal and interacting proteins using the promiscuous biotin ligase BirA* is a powerful approach to rapidly identify vacuolar GRA proteins. We further demonstrate that one factor identified by this approach, GRA39, plays an important role in the ability of the parasite to replicate within its host cell and cause disease. Most intracellular pathogens reside inside a membrane-bound vacuole within their host cell that is extensively modified by the pathogen to optimize intracellular growth and avoid host defenses. In Toxoplasma , this vacuole is modified by a host of secretory GRA proteins, many of which remain unidentified. Here we demonstrate that in vivo biotinylation of proximal and interacting proteins using the promiscuous biotin ligase BirA* is a powerful approach to rapidly identify vacuolar GRA proteins. We further demonstrate that one factor identified by this approach, GRA39, plays an important role in the ability of the parasite to replicate within its host cell and cause disease.

Characterizing how cells in three-dimensional (3D) environments or natural tissues respond to biophysical stimuli is a longstanding challenge in biology and tissue engineering. We demonstrate a strategy to monitor morphological and mechanical responses of contractile fibroblasts in a 3D environment. Cells responded to stretch through specific, cell-wide mechanisms involving staged retraction and reinforcement. Retraction responses occurred for all orientations of stress fibers and cellular protrusions relative to the stretch direction, while reinforcement responses, including extension of cellular processes and stress fiber formation, occurred predominantly in the stretch direction. A previously unreported role of F-actin clumps was observed, with clumps possibly acting as F-actin reservoirs for retraction and reinforcement responses during stretch. Responses were consistent with a model of cellular sensitivity to local physical cues. These findings suggest mechanisms for global actin cytoskeleton remodeling in non-muscle cells and provide insight into cellular responses important in pathologies such as fibrosis and hypertension.

The coronavirus disease 2019 (COVID-19) pandemic has disproportionately affected patients with kidney disease, causing significant challenges in disease management, kidney research and trainee education. For patients, increased infection risk and disease severity, often complicated by acute kidney injury, have contributed to high mortality. Clinicians were faced with high clinical demands, resource shortages and novel ethical dilemmas in providing patient care. In this review, we address the impact of COVID-19 on the entire spectrum of kidney care, including acute kidney injury, chronic kidney disease, dialysis and transplantation, trainee education, disparities in health care, changes in health care policies, moral distress and the patient perspective. Based on current evidence, we provide a framework for the management and support of patients with kidney disease, infection mitigation strategies, resource allocation and support systems for the nephrology workforce.In this Review, the authors summarize the challenges associated with the care of patients with kidney disease during the COVID-19 pandemic. They describe the major challenges and missed opportunities, global inequalities in health care, and offer a framework for future pandemic preparedness.

Bacterial translocation from the gut microbiota is a source of sepsis in susceptible patients. Previous work suggests that overgrowth of gut pathobionts, including Klebsiella pneumoniae, increases the risk of disseminated infection. Our data from a human dietary intervention study found that, in the absence of fiber, K. pneumoniae bloomed during microbiota recovery from antibiotic treatment. We thus hypothesized that dietary nutrients directly support or suppress colonization of this gut pathobiont in the microbiota. Consistent with our study in humans, complex carbohydrates in dietary fiber suppressed the colonization of K. pneumoniae and allowed for recovery of competing commensals in mouse models. In contrast, through ex vivo and in vivo modeling, we identified simple carbohydrates as a limiting resource for K. pneumoniae in the gut. As proof of principle, supplementation with lactulose, a nonabsorbed simple carbohydrate and an FDA-approved therapy, increased colonization of K. pneumoniae. Disruption of the intestinal epithelium led to dissemination of K. pneumoniae into the bloodstream and liver, which was prevented by dietary fiber. Our results show that dietary simple and complex carbohydrates were critical not only in the regulation of pathobiont colonization but also disseminated infection, suggesting that targeted dietary interventions may offer a preventative strategy in high-risk patients.