Colibactin leads to a bacteria-specific mutation pattern and self-inflicted DNA damage.

Colibactin produced primarily by Escherichia coli strains of the B2 phylogroup cross-links DNA and can promote colon cancer in human hosts. Here, we investigate the toxin's impact on colibactin producers and on bacteria cocultured with producing cells. Using genome-wide genetic screens and mutation accumulation experiments, we uncover the cellular pathways that mitigate colibactin damage and reveal the specific mutations it induces. We discover that although colibactin targets A/T-rich motifs, as observed in human colon cells, it induces a bacteria-unique mutation pattern. Based on this pattern, we predict that long-term colibactin exposure will culminate in a genomic bias in trinucleotide composition. We test this prediction by analyzing thousands of E. coli genomes and find that colibactin-producing strains indeed show the predicted skewness in trinucleotide composition. Our work reveals a bacteria-specific mutation pattern and suggests that the resistance protein encoded on the colibactin pathogenicity island is insufficient in preventing self-inflicted DNA damage.

Colibactin-induced damage in bacteria is cell contact independent.

The bacterial toxin colibactin, produced primarily by the B2 phylogroup of Escherichia coli, underlies some cases of colorectal cancers. Colibactin crosslinks DNA and induces genotoxic damage in both mammalian and bacterial cells. While the mechanisms facilitating colibactin delivery remain unclear, results from multiple studies supported a delivery model that necessitates cell-cell contact. We directly tested this requirement in bacterial cultures by monitoring the spatiotemporal dynamics of the DNA damage response using a fluorescent transcriptional reporter. We found that in mixed-cell populations, DNA damage saturated within twelve hours and was detectable even in reporter cells separated from colibactin producers by hundreds of microns. Experiments with distinctly separated producer and reporter colonies revealed that the intensity of DNA damage decays similarly with distance regardless of colony contact. Our work reveals that cell contact is inconsequential for colibactin delivery in bacteria and suggests that contact-dependence needs to be reexamined in mammalian cells as well. Importance: Colibactin is a bacteria-produced toxin that binds and damages DNA. It has been widely studied in mammalian cells due to its potential role in tumorigenesis. However, fundamental questions about its impact in bacteria remain underexplored. We used E. coli as a model system to study colibactin toxicity in neighboring bacteria and directly tested if cell-cell contact is required for toxicity, as has previously been proposed. We found that colibactin can induce DNA damage in bacteria hundreds of microns away and that the intensity of DNA damage presents similarly regardless of cell-cell contact. Our work further suggests that the requirement for cell-cell contact for colibactin-induced toxicity also needs to be reevaluated in mammalian cells.

Embryonic motor neuron programming factors reactivate immature gene expression and suppress ALS pathologies in postnatal motor neurons.

Aging is a major risk factor in amyotrophic lateral sclerosis (ALS) and other adult-onset neurodegenerative disorders. Whereas young neurons are capable of buffering disease-causing stresses, mature neurons lose this ability and degenerate over time. We hypothesized that the resilience of young motor neurons could be restored by re-expression of the embryonic motor neuron selector transcription factors ISL1 and LHX3. We found that viral re-expression of ISL1 and LHX3 reactivates aspects of the youthful gene expression program in mature motor neurons and alleviates key disease-relevant phenotypes in the SOD1G93A mouse model of ALS. Our results suggest that redeployment of lineage-specific neuronal selector transcription factors can be an effective strategy to attenuate age-dependent phenotypes in neurodegenerative disease.

CXCL17 is a proinflammatory chemokine and promotes neutrophil trafficking.

CXCL17, a novel member of the CXC chemokine class, has been implicated in several human pathologies, but its role in mediating immune response is not well understood. Characteristic features of immune response include resident macrophages orchestrating successive and structured recruitment of neutrophils and monocytes to the insult site. Here, we show that Cxcl17 knockout (KO) mice, compared with the littermate wild-type control mice, were significantly impaired in peritoneal neutrophil recruitment post-lipopolysaccharide (LPS) challenge. Further, the KO mice show dysregulated Cxcl1, Cxcr2, and interleukin-6 levels, all of which directly impact neutrophil recruitment. Importantly, the KO mice showed no difference in monocyte recruitment post-LPS challenge or in peritoneal macrophage levels in both unchallenged and LPS-challenged mice. We conclude that Cxcl17 is a proinflammatory chemokine and that it plays an important role in the early proinflammatory response by promoting neutrophil recruitment to the insult site.

Chemokine Cxcl1-Cxcl2 heterodimer is a potent neutrophil chemoattractant.

Microbial infection is characterized by release of multiple proinflammatory chemokines that direct neutrophils to the insult site. How collective function of these chemokines orchestrates neutrophil recruitment is not known. Here, we characterized the role for heterodimer and show that the Cxcl1-Cxcl2 heterodimer is a potent neutrophil chemoattractant in mice and can recruit more neutrophils than the individual chemokines. Chemokine-mediated neutrophil recruitment is determined by Cxcr2 receptor signaling, Cxcr2 endocytosis, and binding to glycosaminoglycans. We have now determined heterodimer's Cxcr2 activity using cellular assays and Cxcr2 density in blood and recruited neutrophils in heterodimer-treated mice. We have shown that the heterodimer binds glycosaminoglycans with higher affinity and more efficiently than Cxcl1 or Cxcl2. These data collectively indicate that optimal glycosaminoglycan interactions and dampened receptor activity acting in concert in a dynamic fashion promote heterodimer-mediated robust neutrophil recruitment. We propose that this could play a critical role in combating infection.

Habituation for professional learning: a qualitative study of physiotherapy students' experiences working with anatomy cadavers.

INTRODUCTION: Cadaveric dissection shapes the ways in which healthcare students understand the human body and the attitudes, identities and behaviors they exhibit as health professionals. There is however a paucity of related research with physiotherapy (PT) students. PURPOSE: The purpose of this interpretivist study was to investigate PT students' conceptions of the human body in relation to experiences with human cadavers in anatomy education. METHODS: Ten semi-structured interviews were conducted with PT students along with four optional written reflections completed. Data was thematically analyzed. RESULTS: Students engaged in a continuous process of habituation involving oscillation between "humanization" and "dehumanization" of cadavers in the anatomy lab. We describe the contextual mediators that shaped the process, the multi-sensory and emotional experience of the students, and the "interruptions" that contributed to the variability in their conceptions over time and contexts. Students ultimately habituated toward dehumanization which had multiple effects on learning and professionalization. CONCLUSION: Study findings highlight the complexity of PT students' experiences and learning within the cadaver lab outside of the formal goals of anatomy education. We discuss the implications for anatomy curricula, including the potential advantages of incorporating a biopsychosocial approach.

Discovery of a Potent and Selective CDKL5/GSK3 Chemical Probe That Is Neuroprotective.

Despite mediating several essential processes in the brain, including during development, cyclin-dependent kinase-like 5 (CDKL5) remains a poorly characterized human protein kinase. Accordingly, its substrates, functions, and regulatory mechanisms have not been fully described. We realized that availability of a potent and selective small molecule probe targeting CDKL5 could enable illumination of its roles in normal development as well as in diseases where it has become aberrant due to mutation. We prepared analogs of AT-7519, a compound that has advanced to phase II clinical trials and is a known inhibitor of several cyclin-dependent kinases (CDKs) and cyclin-dependent kinase-like kinases (CDKLs). We identified analog 2 as a highly potent and cell-active chemical probe for CDKL5/GSK3 (glycogen synthase kinase 3). Evaluation of its kinome-wide selectivity confirmed that analog 2 demonstrates excellent selectivity and only retains GSK3α/ß affinity. We next demonstrated the inhibition of downstream CDKL5 and GSK3α/ß signaling and solved a co-crystal structure of analog 2 bound to human CDKL5. A structurally similar analog (4) proved to lack CDKL5 affinity and maintain potent and selective inhibition of GSK3α/ß, making it a suitable negative control. Finally, we used our chemical probe pair (2 and 4) to demonstrate that inhibition of CDKL5 and/or GSK3α/ß promotes the survival of human motor neurons exposed to endoplasmic reticulum stress. We have demonstrated a neuroprotective phenotype elicited by our chemical probe pair and exemplified the utility of our compounds to characterize the role of CDKL5/GSK3 in neurons and beyond.

A Potent and Selective CDKL5/GSK3 Chemical Probe is Neuroprotective.

Despite mediating several essential processes in the brain, including during development, cyclin-dependent kinase-like 5 (CDKL5) remains a poorly characterized human protein kinase. Accordingly, its substrates, functions, and regulatory mechanisms have not been fully described. We realized that availability of a potent and selective small molecule probe targeting CDKL5 could enable illumination of its roles in normal development as well as in diseases where it has become aberrant due to mutation. We prepared analogs of AT-7519, a known inhibitor of several cyclin dependent and cyclin-dependent kinase-like kinases that has been advanced into Phase II clinical trials. We identified analog 2 as a highly potent and cell-active chemical probe for CDKL5/GSK3 (glycogen synthase kinase 3). Evaluation of its kinome-wide selectivity confirmed that analog 2 demonstrates excellent selectivity and only retains GSK3α/ß affinity. As confirmation that our chemical probe is a high-quality tool to use in directed biological studies, we demonstrated inhibition of downstream CDKL5 and GSK3α/ß signaling and solved a co-crystal structure of analog 2 bound to CDKL5. A structurally similar analog ( 4 ) proved to lack CDKL5 affinity and maintain potent and selective inhibition of GSK3α/ß. Finally, we used our chemical probe pair ( 2 and 4 ) to demonstrate that inhibition of CDKL5 and/or GSK3α/ß promotes the survival of human motor neurons exposed to endoplasmic reticulum (ER) stress. We have demonstrated a neuroprotective phenotype elicited by our chemical probe pair and exemplified the utility of our compounds to characterize the role of CDKL5/GSK3 in neurons and beyond.

Rapid signaling reactivation after targeted BRAF inhibition predicts the proliferation of individual melanoma cells from an isogenic population.

Cancer cells within tumors display a high degree of phenotypic variability. This variability is thought to allow some of the cells to survive and persist after seemingly effective drug treatments. Studies on vemurafenib, a signaling inhibitor that targets an oncogenic BRAF mutation common in melanoma, suggested that cell-to-cell variation in drug resistance, measured by long-term proliferation, originates from epigenetic differences in gene expression that pre-exist treatment. However, it is still unknown whether reactivation of signaling downstream to the inhibited BRAF, thought to be a key step for resistance, is heterogeneous across cells. While previous studies established that signaling reactivation takes place many hours to days after treatment, they monitored reactivation with bulk-population assays unsuitable for detecting cell-to-cell heterogeneity. We hypothesized that signaling reactivation is heterogeneous and is almost instantaneous for a small subpopulation of resistant cells. We tested this hypothesis by monitoring signaling dynamics at a single-cell resolution and observed that despite highly uniform initial inhibition, roughly 15% of cells reactivated signaling within an hour of treatment. Moreover, by tracking cell lineages over multiple days, we established that these cells indeed proliferated more than neighboring cells, thus establishing that rapid signaling reactivation predicts long-term vemurafenib resistance.

Genome-scale screens identify factors regulating tumor cell responses to natural killer cells.

To systematically define molecular features in human tumor cells that determine their degree of sensitivity to human allogeneic natural killer (NK) cells, we quantified the NK cell responsiveness of hundreds of molecularly annotated 'DNA-barcoded' solid tumor cell lines in multiplexed format and applied genome-scale CRISPR-based gene-editing screens in several solid tumor cell lines, to functionally interrogate which genes in tumor cells regulate the response to NK cells. In these orthogonal studies, NK cell-sensitive tumor cells tend to exhibit 'mesenchymal-like' transcriptional programs; high transcriptional signature for chromatin remodeling complexes; high levels of B7-H6 (NCR3LG1); and low levels of HLA-E/antigen presentation genes. Importantly, transcriptional signatures of NK cell-sensitive tumor cells correlate with immune checkpoint inhibitor (ICI) resistance in clinical samples. This study provides a comprehensive map of mechanisms regulating tumor cell responses to NK cells, with implications for future biomarker-driven applications of NK cell immunotherapies.

Neutrophil recruitment by chemokines Cxcl1/KC and Cxcl2/MIP2: Role of Cxcr2 activation and glycosaminoglycan interactions.

Chemokines play a crucial role in combating microbial infection by recruiting blood neutrophils to infected tissue. In mice, the chemokines Cxcl1/KC and Cxcl2/MIP2 fulfill this role. Cxcl1 and Cxcl2 exist as monomers and dimers, and exert their function by activating the Cxcr2 receptor and binding glycosaminoglycans (GAGs). Here, we characterized Cxcr2 G protein and ß-arrestin activities, and GAG heparan sulfate (HS) interactions of Cxcl1 and Cxcl2 and of the trapped dimeric variants. To understand how Cxcr2 and GAG interactions impact in vivo function, we characterized their neutrophil recruitment activity to the peritoneum, Cxcr2 and CD11b levels on peritoneal and blood neutrophils, and transport profiles out of the peritoneum. Cxcl2 variants compared with Cxcl1 variants were more potent for Cxcr2 activity. Native Cxcl1 compared with native Cxcl2 and dimers compared with native proteins bound HS with higher affinity. Interestingly, recruitment activity between native Cxcl1 and Cxcl2, between dimers, and between the native protein and the dimer could be similar or very different depending on the dose or the time point. These data indicate that peritoneal neutrophil recruitment cannot be solely attributed to Cxcr2 or GAG interactions, and that the relationship between recruited neutrophils, Cxcr2 activation, GAG interactions, and chemokine levels is complex and highly context dependent. We propose that the ability of Cxcl1 and Cxcl2 to reversibly exist as monomers and dimers and differences in their Cxcr2 activity and GAG interactions coordinate neutrophil recruitment and activation, which play a critical role for successful resolution of inflammation.

Standardized Reporter Systems for Purification and Imaging of Human Pluripotent Stem Cell-derived Motor Neurons and Other Cholinergic Cells.

Reliable and consistent pluripotent stem cell reporter systems for efficient purification and visualization of motor neurons are essential reagents for the study of normal motor neuron biology and for effective disease modeling. To overcome the inherent noisiness of transgene-based reporters, we developed a new series of human induced pluripotent stem cell lines by knocking in tdTomato, Cre, or CreERT2 recombinase into the HB9 (MNX1) or VACHT (SLC18A3) genomic loci. The new lines were validated by directed differentiation into spinal motor neurons and immunostaining for motor neuron markers HB9 and ISL1. To facilitate efficient purification of spinal motor neurons, we further engineered the VACHT-Cre cell line with a validated, conditional CD14-GFP construct that allows for both fluorescence-based identification of motor neurons, as well as magnetic-activated cell sorting (MACS) to isolate differentiated motor neurons at scale. The targeting strategies developed here offer a standardized platform for reproducible comparison of motor neurons across independently derived pluripotent cell lines.

Micromolded Carbon Paste Microelectrodes for Electrogenerated Chemiluminescent Detection on Microfluidic Devices.

Micromolded carbon paste electrodes are easily fabricated, disposable, and can be integrated into microfluidic devices to fabricate inexpensive sensors and biosensors. In this work, carbon paste microelectrodes were fabricated in poly(dimethylsiloxane) using micromolding techniques and were coupled to a microfluidic channel to fabricate electrogenerated chemiluminescent (ECL) sensors. ECL was generated using both the tris(2,2'-bipyridyl)ruthenium(II)-tripropylamine system and the hydrogen peroxide and luminol system. For each of these ECL systems, the sensor fabrication method was optimized, along with key experimental parameters (applied voltage, solution flow rate, buffer species and luminol concentration). The limit of detection (S/N = 3) for TPrA was ~2.4 µM with a linear range of 10-100µM. For hydrogen peroxide the LOD was ~11 µM and the electrodes gave a linear response between 30 µM and 200 µM hydrogen peroxide. Electrodes containing glucose oxidase were fabricated using this new method, demonstrating that glucose could be indirectly detected via generation of hydrogen peroxide by the enzymatic reaction at the micromolded biosensor.

Development of MAP4 Kinase Inhibitors as Motor Neuron-Protecting Agents.

Disease-causing mutations in many neurodegenerative disorders lead to proteinopathies that trigger endoplasmic reticulum (ER) stress. However, few therapeutic options exist for patients with these diseases. Using an in vitro screening platform to identify compounds that protect human motor neurons from ER stress-mediated degeneration, we discovered that compounds targeting the mitogen-activated protein kinase kinase kinase kinase (MAP4K) family are neuroprotective. The kinase inhibitor URMC-099 (compound 1) stood out as a promising lead compound for further optimization. We coupled structure-based compound design with functional activity testing in neurons subjected to ER stress to develop a series of analogs with improved MAP4K inhibition and concomitant increases in potency and efficacy. Further structural modifications were performed to enhance the pharmacokinetic profiles of the compound 1 derivatives. Prostetin/12k emerged as an exceptionally potent, metabolically stable, and blood-brain barrier-penetrant compound that is well suited for future testing in animal models of neurodegeneration.

A Stem Cell-Based Screening Platform Identifies Compounds that Desensitize Motor Neurons to Endoplasmic Reticulum Stress.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease selectively targeting motor neurons in the brain and spinal cord. The reasons for differential motor neuron susceptibility remain elusive. We developed a stem cell-based motor neuron assay to study cell-autonomous mechanisms causing motor neuron degeneration, with implications for ALS. A small-molecule screen identified cyclopiazonic acid (CPA) as a stressor to which stem cell-derived motor neurons were more sensitive than interneurons. CPA induced endoplasmic reticulum stress and the unfolded protein response. Furthermore, CPA resulted in an accelerated degeneration of motor neurons expressing human superoxide dismutase 1 (hSOD1) carrying the ALS-causing G93A mutation, compared to motor neurons expressing wild-type hSOD1. A secondary screen identified compounds that alleviated CPA-mediated motor neuron degeneration: three kinase inhibitors and tauroursodeoxycholic acid (TUDCA), a bile acid derivative. The neuroprotective effects of these compounds were validated in human stem cell-derived motor neurons carrying a mutated SOD1 allele (hSOD1A4V). Moreover, we found that the administration of TUDCA in an hSOD1G93A mouse model of ALS reduced muscle denervation. Jointly, these results provide insights into the mechanisms contributing to the preferential susceptibility of ALS motor neurons, and they demonstrate the utility of stem cell-derived motor neurons for the discovery of new neuroprotective compounds.

Protein Prenylation Constitutes an Endogenous Brake on Axonal Growth.

Suboptimal axonal regeneration contributes to the consequences of nervous system trauma and neurodegenerative disease, but the intrinsic mechanisms that regulate axon growth remain unclear. We screened 50,400 small molecules for their ability to promote axon outgrowth on inhibitory substrata. The most potent hits were the statins, which stimulated growth of all mouse- and human-patient-derived neurons tested, both in vitro and in vivo, as did combined inhibition of the protein prenylation enzymes farnesyltransferase (PFT) and geranylgeranyl transferase I (PGGT-1). Compensatory sprouting of motor axons may delay clinical onset of amyotrophic lateral sclerosis (ALS). Accordingly, elevated levels of PGGT1B, which would be predicted to reduce sprouting, were found in motor neurons of early- versus late-onset ALS patients postmortem. The mevalonate-prenylation pathway therefore constitutes an endogenous brake on axonal growth, and its inhibition provides a potential therapeutic approach to accelerate neuronal regeneration in humans.

Modeling ALS with motor neurons derived from human induced pluripotent stem cells.

Directing the differentiation of induced pluripotent stem cells into motor neurons has allowed investigators to develop new models of amyotrophic lateral sclerosis (ALS). However, techniques vary between laboratories and the cells do not appear to mature into fully functional adult motor neurons. Here we discuss common developmental principles of both lower and upper motor neuron development that have led to specific derivation techniques. We then suggest how these motor neurons may be matured further either through direct expression or administration of specific factors or coculture approaches with other tissues. Ultimately, through a greater understanding of motor neuron biology, it will be possible to establish more reliable models of ALS. These in turn will have a greater chance of validating new drugs that may be effective for the disease.