Neutrophil extracellular traps protect the kidney from ascending infection and are required for a positive leukocyte dipstick test.

Lower urinary tract infection (UTI) is common but only rarely complicated by pyelonephritis. However, the mechanisms preventing extension to the kidney are unclear. Here, we identified neutrophil extracellular traps (NETs) in healthy human urine that provide an antibacterial defense strategy within the urinary tract. In both in vivo murine models of UTI where uropathogenic E. coli are inoculated into the bladder and ex vivo human urine models, NETs interacted with uromodulin to form large webs that entrapped the bacteria. Peptidyl arginine deiminase 4 (PADI4) inhibition in mice blocked NETosis and resulted in progression of cystitis into pyelonephritis, suggesting that NETosis of urinary neutrophils acts to prevent bacterial ascent into the kidney. Analysis of UK Biobank data revealed that genetic variants in PADI4 that associated with increased risk of rheumatoid arthritis in multiple genome-wide association studies were consistently associated with reduced susceptibility to UTI. Last, we showed that urine dipstick testing for leukocyte esterase was negative in the presence of intact blood neutrophils but became positive when neutrophils were stimulated to NET, and this could be prevented by selective PADI4 inhibition, demonstrating that this test does not detect absolute neutrophil count, as has long been assumed, but specifically detects neutrophils that have undergone NETosis. These findings highlight the role of NETosis in preventing ascending infections in the urinary tract and improve our understanding of one of the most common clinical tests in medicine.

A Phase 1 single ascending dose study of pure oral harmine in healthy volunteers.

BACKGROUND: Harmine is a component of the hallucinogenic brew, Ayahuasca, which also contains the psychoactive compound, N, N-dimethyltryptamine. Whether pharmaceutical-grade harmine hydrochloride (HCl) has psychoactive effects, the doses at which these might occur, and the dose-response relationship to side effects and safety in humans are unknown. METHODS: We conducted a Phase 1, open-label single ascending dose trial in healthy adults with normal body mass index and no prior psychiatric illness. The primary goal was to determine the maximum tolerated dose (MTD) of oral pharmaceutical-grade harmine HCl and to characterize safety and tolerability. A secondary goal was to ascertain whether any oral dose has psychoactive effects. RESULTS: Thirty-four adult participants, aged 18-55 years, were screened for study eligibility. Twenty-five participants met eligibility criteria and were randomized to a single dose of 100, 200, 300, or 500 mg of harmine HCl, respectively, using a continuous reassessment method. The most common adverse events (AEs) observed were gastrointestinal and/or neurological, dose-related, and of mild to moderate severity. The MTD was determined to be between 100 and 200 mg and is weight-based, with 90% of those participants receiving >2.7 mg/kg experiencing a dose-limiting toxicity. No serious AEs of harmine HCl were identified. CONCLUSIONS: Harmine HCl can be orally administered to healthy participants in doses <2.7 mg/kg with minimal or no AEs. Doses >2.7 mg/kg are associated with vomiting, drowsiness, and limited psychoactivity. This study is the first to systematically characterize the psychoactive effects of pharmaceutical quality harmine in healthy participants.

Hippocampal γCaMKII dopaminylation promotes synaptic-to-nuclear signaling and memory formation.

Protein monoaminylation is a class of posttranslational modification (PTM) that contributes to transcription, physiology and behavior. While recent analyses have focused on histones as critical substrates of monoaminylation, the broader repertoire of monoaminylated proteins in brain remains unclear. Here, we report the development/implementation of a chemical probe for the bioorthogonal labeling, enrichment and proteomics-based detection of dopaminylated proteins in brain. We identified 1,557 dopaminylated proteins - many synaptic - including γCaMKII, which mediates Ca 2+ -dependent cellular signaling and hippocampal-dependent memory. We found that γCaMKII dopaminylation is largely synaptic and mediates synaptic-to-nuclear signaling, neuronal gene expression and intrinsic excitability, and contextual memory. These results indicate a critical role for synaptic dopaminylation in adaptive brain plasticity, and may suggest roles for these phenomena in pathologies associated with altered monoaminergic signaling.

Combined high rates of alternative breeding strategies unexpectedly found among populations of a solitary nesting raptor.

Social monogamy is the prevalent mating system in birds, but alternative strategies of extra-pair paternity (EPP) and conspecific brood parasitism (CBP) occur in many species. Raptors are virtually absent in discussions of broad taxonomic reviews regarding EPP and CBP likely because these strategies are mostly absent or at low frequency; CBP is unreported in solitary nesting raptors. In contrast, we found high frequencies of EPP (16%-31%) and CBP (15%-26%) nests among three populations of Cooper's Hawks (Accipiter cooperii) across the northern breeding range of this solitary nesting, socially monogamous species. EPP and CBP combined occurred in 42%-46% of all nests among populations and hence unexpectedly were nearly equivalent to proportions of genetically monogamous nests. Select covariates failed to predict presence of EPP and CBP in part because virtually all extra-pair adults were uncaught and likely were floaters. We found no support for the hypothesis that territorial females traded copulations for food to maximize energy intake for increased production. Our unique discoveries enhance knowledge of the extent and diversity of alternative breeding strategies among groups of avian and other animal species.

UK Reproducibility Network open and transparent research practices survey dataset.

Openness and transparency in the research process are a prerequisite to the production of high quality research outputs. Efforts to maximise these features have substantially accelerated in recent years, placing open and transparent research practices at the forefront of funding and related priorities, and encouraging investment in resources and infrastructure to enable such practices. Despite these efforts, there has been no systematic documentation of current practices, infrastructure, or training and resources that support open and transparent research in the UK. To address this gap, we developed and conducted the Open and Transparent Research Practices survey, a large-scale audit study completed by research-active staff in UK research institutions to better understand existing practices, needs, support, and barriers faced when implementing open and transparent research. The data presented here capture responses from over 2,500 research-active staff based at 15 institutions affiliated with the UK Reproducibility Network. The data provide a snapshot of open research practices that can be used to identify barriers, training needs, and areas that require greater investments.

Harmine and exendin-4 combination therapy safely expands human ß cell mass in vivo in a mouse xenograft system.

Five hundred thirty-seven million people globally suffer from diabetes. Insulin-producing ß cells are reduced in number in most people with diabetes, but most individuals still have some residual ß cells. However, none of the many diabetes drugs in common use increases human ß cell numbers. Recently, small molecules that inhibit dual tyrosine-regulated kinase 1A (DYRK1A) have been shown to induce immunohistochemical markers of human ß cell replication, and this is enhanced by drugs that stimulate the glucagon-like peptide 1 (GLP1) receptor (GLP1R) on ß cells. However, it remains to be demonstrated whether these immunohistochemical findings translate into an actual increase in human ß cell numbers in vivo. It is also unknown whether DYRK1A inhibitors together with GLP1R agonists (GLP1RAs) affect human ß cell survival. Here, using an optimized immunolabeling-enabled three-dimensional imaging of solvent-cleared organs (iDISCO+) protocol in mouse kidneys bearing human islet grafts, we demonstrate that combination of a DYRK1A inhibitor with exendin-4 increases actual human ß cell mass in vivo by a mean of four- to sevenfold in diabetic and nondiabetic mice over 3 months and reverses diabetes, without alteration in human α cell mass. The augmentation in human ß cell mass occurred through mechanisms that included enhanced human ß cell proliferation, function, and survival. The increase in human ß cell survival was mediated, in part, by the islet prohormone VGF. Together, these findings demonstrate the therapeutic potential and favorable preclinical safety profile of the DYRK1A inhibitor-GLP1RA combination for diabetes treatment.

Sexually discordant selection is associated with trait-specific morphological changes and a complex genomic response.

Sexes often have differing fitness optima, potentially generating intra-locus sexual conflict, as each sex bears a genetic "load" of alleles beneficial to the other sex. One strategy to evaluate conflict in the genome is to artificially select populations discordantly against established sexual dimorphism (SD), reintroducing attenuated conflict. We investigate a long-term artificial selection experiment reversing sexual size dimorphism in Drosophila melanogaster during ~350 generations of sexually discordant selection. We explore morphological and genomic changes to identify loci under selection between the sexes in discordantly and concordantly size-selected treatments. Despite substantial changes to overall size, concordant selection maintained ancestral SD. However, discordant selection altered size dimorphism in a trait-specific manner. We observe multiple possible soft selective sweeps in the genome, with size-related genes showing signs of selection. Patterns of genomic differentiation between the sexes within lineages identified potential sites maintained by sexual conflict. One discordant selected lineage shows a pattern of elevated genomic differentiation between males and females on chromosome 3L, consistent with the maintenance of sexual conflict. Our results suggest visible signs of conflict and differentially segregating alleles between the sexes due to discordant selection.

Select DYRK1A Inhibitors Enhance Both Proliferation and Differentiation in Human Pancreatic Beta Cells.

The small molecule DYRK1A inhibitor, harmine, induces human beta cell proliferation, expands beta cell mass, enhances expression of beta cell phenotypic genes, and improves human beta cell function i n vitro and in vivo . It is unknown whether the "pro-differentiation effect" is a DYRK1A inhibitor class-wide effect. Here we compare multiple commonly studied DYRK1A inhibitors. Harmine, 2-2c and 5-IT increase expression of PDX1, MAFA, NKX6.1, SLC2A2, PCSK1, MAFB, SIX2, SLC2A2, SLC30A8, ENTPD3 in normal and T2D human islets. Unexpectedly, GNF4877, CC-401, INDY, CC-401 and Leucettine fail to induce expression of these essential beta cell molecules. Remarkably, the pro-differentiation effect is independent of DYRK1A inhibition: although silencing DYRK1A induces human beta cell proliferation, it has no effect on differentiation; conversely, harmine treatment enhances beta cell differentiation in DYRK1A-silenced islets. A careful screen of multiple DYRK1A inhibitor kinase candidate targets was unable to identify pro-differentiation pathways. Overall, harmine, 2-2c and 5-IT are unique among DYRK1A inhibitors in their ability to enhance both beta cell proliferation and differentiation. While beta cell proliferation is mediated by DYRK1A inhibition, the pro-differentiation effects of harmine, 2-2c and 5-IT are distinct, and unexplained in mechanistic terms. These considerations have important implications for DYRK1A inhibitor pharmaceutical development.

Non-resolving neuroinflammation regulates axon regeneration in chronic spinal cord injury.

Chronic spinal cord injury (SCI) lesions retain increased densities of microglia and macrophages. In acute SCI, macrophages induce growth cone collapse, facilitate axon retraction away from lesion boundaries, as well as play a key role in orchestrating the growth-inhibitory glial scar. Little is known about the role of sustained inflammation in chronic SCI, or whether chronic inflammation affects repair and regeneration. We performed transcriptional analysis using the Nanostring Neuropathology panel to characterize the resolution of inflammation into chronic SCI, to characterize the chronic SCI microenvironment, as well as to identify spinal cord responses to macrophage depletion and repopulation using the CSF1R inhibitor, PLX-5622. We determined the ability for macrophage depletion and repopulation to augment axon growth into chronic lesions both with and without regenerative stimulation using neuronal-specific PTEN knockout (PTEN-KO). PTEN-KO was delivered with spinal injections of retrogradely transported adeno associated viruses (AAVrg's). Both transcriptional analyses and immunohistochemistry revealed the ability for PLX-5622 to significantly deplete inflammation around and within chronic SCI lesions, with a return to pre-depleted inflammatory densities after treatment removal. Neuronal-specific transcripts were significantly elevated in mice after inflammatory repopulation, but no significant effects were observed with macrophage depletion alone. Axon densities significantly increased within the lesion after PLX-5622 treatment with a more consistent effect observed in mice with inflammatory repopulation. PTEN-KO did not further increase axon densities within the lesion beyond effects induced by PLX-5622. We identified that PLX-5622 increased axon densities within the lesion that are histologically identified as 5-HT+and CGRP+, both of which are not robustly transduced by AAVrg's. Our work identified that increased macrophage/microglia densities in the chronic SCI environment may be actively retained by homeostatic mechanisms likely affiliated with a sustained elevated expression of CSF1 and other chemokines. Finally, we identify a novel role of sustained inflammation as a prospective barrier to axon regeneration in chronic SCI.

Life history changes associated with over 400 generations of artificial selection on body size in Drosophila.

Body size is a trait that shapes many aspects of a species' development and evolution. Larger body size is often beneficial in animals, but it can also be associated with life history costs in natural systems. Similarly, miniaturization, the evolution of extremely small adult body size, is found in every major animal group, yet carries its own life history trade-offs. Given that these effects can depend on an animal's environment and life stage and have mainly been studied in species that are already specialized for their size, the life history changes associated with evolutionary shifts in body size warrant additional investigation. Here, we used Drosophila melanogaster populations that had undergone over 400 generations of artificial selection on body size to investigate the changes in life history traits associated with the evolution of extremely large and extremely small body sizes. Populations selected for small body size experienced strong trade-offs in multiple life history traits, including reduced female fecundity and lower juvenile viability. Although we found positively correlated changes in egg size associated with selection for both large and small body size, after adjusting for female body size, females from populations selected for large size had the lowest relative investment per egg and females from populations selected for small size had the highest relative investment per egg. Taken together, our results suggest that egg size may be a key constraint on the evolution of body size in D. melanogaster, providing insight into the broader phenomenon of body size evolution in insects.