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Migration of T cells is essential for their ability to mount immune responses. Chemokine-induced T cell migration requires WNK1, a kinase that regulates ion influx into the cell. However, it is not known why ion entry is necessary for T cell movement. Here we show that signaling from the chemokine receptor CCR7 leads to activation of WNK1 and its downstream pathway at the leading edge of migrating CD4 + T cells, resulting in ion influx and water entry by osmosis. We propose that WNK1-induced water entry is required to swell the membrane at the leading edge, generating space into which actin filaments can polymerize, thereby facilitating forward movement of the cell. Given the broad expression of WNK1 pathway proteins, our study suggests that ion and water influx are likely to be essential for migration in many cell types, including leukocytes and metastatic tumor cells. The ability of T cells to migrate is a central component of their functionality and is known to require WNK1 kinase that is linked to the influx of ions into the cell. Here the authors show that T cell migration requires WNK1 mediated ion and water influx to swell the membrane of the leading edge and support actin polymerisation and forward motility.

Signaling from the T cell antigen receptor (TCR) on CD4 +  T cells plays a critical role in adaptive immune responses by inducing T cell activation, proliferation, and differentiation. Here we demonstrate that WNK1, a kinase implicated in osmoregulation in the kidney, is required in T cells to support T-dependent antibody responses. We show that the canonical WNK1-OXSR1-STK39 kinase signaling pathway is required for TCR signaling in CD4 +  T cells, their subsequent entry into the cell cycle, and suppression of the ATR-mediated G2/M cell cycle checkpoint. We show that the WNK1 pathway regulates ion influx leading to water influx, potentially through AQP3, and that water influx is required for TCR-induced signaling and cell cycle entry. Thus, TCR signaling via WNK1, OXSR1, STK39 and AQP3 leads to water entry that is essential for CD4 +  T cell proliferation and hence T cell-dependent antibody responses. T cell antigen receptor stimulation by the ligand antigen triggers multiple downstream pathways that affect CD4 + T cell function. Here authors show that activation of the downstream WNK1 kinase causes water entry into the cells, which is essential for CD4 + T cell proliferation.

WNK1 is a kinase that has been implicated in ion homeostasis in cells through the activation of the kinases OXSR1 and STK39, which in turn phosphorylate the NKCC- and KCC-families of ion cotransporters leading to their activation and inhibition respectively. Mutations in the human WNK1 gene that cause overexpression of WNK1 result in pseudohypoaldosteronism type II, a condition where individuals present with hypertension and high concentrations of potassium in their blood. WNK1 has been implicated in migration and cell division in cancer cells. Work in CD4+ T cells has shown that WNK1 is a negative regulator of adhesion and a positive regulator of migration, but the function of WNK1 in other immune cells remains unknown. The work presented in this thesis describes the function of WNK1 in B cells. I have used inducible deletion of WNK1 in both naïve and activated B cells, as well as an inhibitor of WNK1, to assess the role of WNK1 in mature B cell biology. I have shown that WNK1 is a crucial kinase for several aspects of B cell biology in mice, since loss of Wnk1 expression caused dysregulation of B cell survival, adhesion, migration and development. WNK1 is required in B cells during an immune response as it positively regulates proliferation after activation. Furthermore, WNK1- deficient B cells display defects in antigen presentation to CD4+ T cells as well as defective responses to stimulation with CD40L, highlighting a role for WNK1 in the regulation of crosstalk between B and CD4+ T cells. WNK1-deficient B cells are not able to mount a T-dependent antibody response nor differentiate into germinal centre B cells. Taken together this work indicates that WNK1 is absolutely required for multiple aspects of B cell function.

Signaling from the T cell antigen receptor (TCR) on CD4+ T cells plays a critical role in adaptive immune responses by inducing T cell activation, proliferation, and differentiation. We demonstrate that WNK1, a kinase implicated in osmoregulation in the kidney, is required in T cells to support T-dependent antibody responses. WNK1-deficient CD4+ T cells are severely impaired in their ability to proliferate and to generate antigen-specific T follicular helper cells in response to immunization with a T-dependent antigen. We show that WNK1 and its downstream OXSR1 and STK39 kinases are required for TCR signaling in CD4+ T cells and for entry into cell cycle. Additionally, by preventing ATR activation, this pathway is required for T cells to progress from G2 into M phase of the cell cycle. Unexpectedly, we show that this WNK1 pathway regulates water influx, most likely through AQP3, which is required for TCR-induced signaling and cell cycle entry. Thus, TCR signaling via WNK1, OXSR1, STK39 and AQP3 leads to water entry that is essential for CD4+ T cell proliferation and hence T cell-dependent antibody responses. Given the broad expression of WNK1, WNK1-dependent water influx may be a common feature of mitogenic pathways in many cell types, both within the immune system and beyond. T cell antigen receptor signaling via the WNK1 kinase causes water entry which is essential for CD4+ T cell proliferation.

Down syndrome (DS) is one of the most common birth defects and the most prevalent genetic form of intellectual disability. DS arises from trisomy of chromosome 21, but its molecular and pathological consequences are not fully understood. In this study, we compared Dp1Tyb mice, a DS model, against their wild-type (WT) littermates of both sexes to investigate the impact of DS-related genetic abnormalities on the brain phenotype. We performed in vivo whole brain magnetic resonance imaging (MRI) and hippocampal 1H magnetic resonance spectroscopy (MRS) on the animals at 3 months of age. Subsequently, ex vivo MRI scans and histological analyses were conducted post-mortem. Our findings unveiled distinct neuroanatomical and biochemical alterations in the Dp1Tyb brains. Dp1Tyb brains exhibited a smaller surface area and a rounder shape compared to WT brains. Regional volumetric analysis revealed significant changes in 26 out of 72 examined brain regions, including the medial prefrontal cortex and dorsal hippocampus. These alterations were consistently observed in both in vivo and ex vivo imaging data. Additionally, high-resolution ex vivo imaging enabled us to investigate cerebellar layers and hippocampal subregions, revealing selective areas of decrease and remodelling in these structures. An analysis of hippocampal metabolites revealed an elevation in glutamine and the glutamine/glutamate ratio in the Dp1Tyb mice compared to controls, suggesting a possible imbalance in the excitation/inhibition ratio. This was accompanied by the decreased levels of taurine. Histological analysis revealed fewer neurons in the hippocampal CA3 and DG layers, along with an increase in astrocytes and microglia. These findings recapitulate multiple neuroanatomical and biochemical features associated with DS, enriching our understanding of the potential connection between chromosome 21 trisomy and the resultant phenotype.

Migration of T cells is essential for their ability to mount immune responses. Chemokine-induced T cell migration requires WNK1, a kinase that regulates ion influx into the cell. However, it is not known why ion entry is necessary for T cell movement. Here we show that signaling from the chemokine receptor CCR7 leads to activation of WNK1 and its downstream pathway at the leading edge of migrating CD4+ T cells, resulting, we propose, in ion influx and consequent water entry by osmosis through AQP3. This WNK1-induced water entry is required to swell the membrane at the leading edge, generating space into which actin filaments can polymerize, thereby facilitating forward movement of the cell. Given the broad expression of WNK1 pathway proteins, our study shows that ion and water influx are likely to be essential for migration in many cell types, including leukocytes and metastatic tumor cells. Competing Interest Statement The authors have declared no competing interest. Footnotes * Figure 4C and 4E are new additions in this version. In addition, the Discussion has been expanded somewhat.

Down syndrome (DS), trisomy 21, is a gene dosage disorder which results in multiple phenotypes including congenital heart defects (CHD). This clinically important pathology is caused by a third copy of one or more of the ∼230 genes on human chromosome 21 (Hsa21), but the identity of the causative dosage-sensitive genes is unknown and hence pathological mechanisms remain obscure. We show that embryonic hearts from human fetuses with DS and mouse models of DS have reduced expression of mitochondrial respiration and cell proliferation genes correlating with CHD. Using systematic genetic mapping, we determine that three copies of the Dyrk1a gene, encoding a serine/threonine protein kinase, are required to cause CHD. Reducing Dyrk1a copy number from three to two reverses defects in proliferation and mitochondrial respiration in embryonic cardiomyocytes and rescues septation defects in DS hearts. Furthermore, treatment of pregnant mice with a DYRK1A inhibitor developed for clinical use partially reduces the incidence of CHD among Dp1Tyb embryos. Thus, increased dosage of DYRK1A is required to impair mitochondrial function and cause CHD in DS, revealing a therapeutic target for this common human condition. Increased dosage of DYRK1A causes mitochondrial dysfunction and congenital heart defects in Down syndrome and is ameliorated in utero by a drug.

Migration and adhesion play critical roles in B cells, regulating recirculation between lymphoid organs, migration within lymphoid tissue and interaction with CD4+ T cells. However, there is limited knowledge of how B cells integrate chemokine receptor and integrin signaling with B cell activation to generate efficient humoral responses. Here we show that the WNK1 kinase, a regulator of migration and adhesion, is essential in B cells for T-dependent antibody responses. We demonstrate that WNK1 transduces signals from the BCR, CXCR5 and CD40, and using intravital imaging we show that WNK1 regulates migration of naive and activated B cells, and their interactions with T cells. Unexpectedly, we show that WNK1 is required for BCR- and CD40-induced proliferation, acting through the OXSR1 and STK39 kinases, and for efficient B cell-T cell collaboration in vivo. Thus, WNK1 is critical for humoral immune responses, by regulating B cell migration, adhesion and T cell-dependent activation. Competing Interest Statement The authors have declared no competing interest.

Research that yields conflicting results rightly causes controversy. Where methodological weaknesses are apparent, there is ready opportunity for discord within the scientific community, which may undermine the entire study. We use the debate about the role of dingoes Canis dingo in conservation in Australia as a case study for a phenomenon that is relevant to all applied ecologists, where conflicting results have been published in high‐quality journals and yet the problems with the methods used in these studies have led to significant controversy. To alleviate such controversies, scientists need to use robust methods to ensure that their results are repeatable and defendable. To date, this has not occurred in Australia's dingo debate due to the use of unvalidated indices that rely on unsupported assumptions. We highlight the problems that poor methods have caused in this debate. We also reiterate our recommendations for practitioners, statisticians and researchers to work together to develop long‐term, multi‐site experimental research programmes using robust methods to understand the impacts of dingoes on mesopredators. Synthesis and applications. Incorporating robust methods and appropriate experimental designs is needed to ensure that conservation actions are appropriately focused and are supported with robust results. Such actions will go a long way towards resolving the debate about the role of dingoes in conservation in Australia, and other, ecological debates.

Elderly people (aged 65 years or more) are at increased risk of polypharmacy (five or more medications), inappropriate medication use, and associated increased health care costs. The use of clinical decision support (CDS) within an electronic medical record (EMR) could improve medication safety. Participatory action research methods were applied to preproduction design and development and postproduction optimization of an EMR-embedded CDS implementation of the Beers' Criteria for medication management and the Cockcroft-Gault formula for estimating glomerular filtration rates (GFR). The \"Seniors Medication Alert and Review Technologies\" (SMART) intervention was used in primary care and geriatrics specialty clinics. Passive (chart messages) and active (order-entry alerts) prompts exposed potentially inappropriate medications, decreased GFR, and the possible need for medication adjustments. Physician reactions were assessed using surveys, EMR simulations, focus groups, and semi-structured interviews. EMR audit data were used to identify eligible patient encounters, the frequency of CDS events, how alerts were managed, and when evidence links were followed. Analysis of subjective data revealed that most clinicians agreed that CDS appeared at appropriate times during patient care. Although managing alerts incurred a modest time burden, most also agreed that workflow was not disrupted. Prevalent concerns related to clinician accountability and potential liability. Approximately 36% of eligible encounters triggered at least one SMART alert, with GFR alert, and most frequent medication warnings were with hypnotics and anticholinergics. Approximately 25% of alerts were overridden and ~15% elicited an evidence check. While most SMART alerts validated clinician choices, they were received as valuable reminders for evidence-informed care and education. Data from this study may aid other attempts to implement Beers' Criteria in ambulatory care EMRs.