A scalable, spin-free approach to generate enhanced induced pluripotent stem cell-derived natural killer cells for cancer immunotherapy.

Natural killer (NK) cells play a vital role in innate immunity and show great promise in cancer immunotherapy. Traditional sources of NK cells, such as the peripheral blood, are limited by availability and donor variability. In addition, in vitro expansion can lead to functional exhaustion and gene editing challenges. This study aimed to harness induced pluripotent stem cell (iPSC) technology to provide a consistent and scalable source of NK cells, overcoming the limitations of traditional sources and enhancing the potential for cancer immunotherapy applications. We developed human placental-derived iPSC lines using reprogramming techniques. Subsequently, an optimized two-step differentiation protocol was introduced to generate high-purity NK cells. Initially, iPSCs were differentiated into hematopoietic-like stem cells using spin-free embryoid bodies (EBs). Subsequently, the EBs were transferred to ultra-low attachment plates to induce NK cell differentiation. iPSC-derived NK (iNK) cells expressed common NK cell markers (NKp46, NKp30, NKp44, CD16 and eomesodermin) at both RNA and protein levels. iNK cells demonstrated significant resilience to cryopreservation and exhibited enhanced cytotoxicity. The incorporation of a chimeric antigen receptor (CAR) construct further augmented their cytotoxic potential. This study exemplifies the feasibility of generating iNK cells with high purity and enhanced functional capabilities, their improved resilience to cryopreservation and the potential to have augmented cytotoxicity through CAR expression. Our findings offer a promising pathway for the development of potential cellular immunotherapies, highlighting the critical role of iPSC technology in overcoming challenges associated with traditional NK cell sources.

Growth Hormone Stops Excessive Inflammation After Partial Hepatectomy, Allowing Liver Regeneration and Survival Through Induction of H2-Bl/HLA-G.

BACKGROUND AND AIMS: Growth hormone (GH) is important for liver regeneration after partial hepatectomy (PHx). We investigated this process in C57BL/6 mice that express different forms of the GH receptor (GHR) with deletions in key signaling domains. APPROACH AND RESULTS: PHx was performed on C57BL/6 mice lacking GHR (Ghr-/- ), disabled for all GH-dependent Janus kinase 2 signaling (Box1-/- ), or lacking only GH-dependent signal transducer and activator of transcription 5 (STAT5) signaling (Ghr391-/- ), and wild-type littermates. C57BL/6 Ghr-/- mice showed striking mortality within 48 hours after PHx, whereas Box1-/- or Ghr391-/- mice survived with normal liver regeneration. Ghr-/- mortality was associated with increased apoptosis and elevated natural killer/natural killer T cell and macrophage cell markers. We identified H2-Bl, a key immunotolerance protein, which is up-regulated by PHx through a GH-mediated, Janus kinase 2-independent, SRC family kinase-dependent pathway. GH treatment was confirmed to up-regulate expression of the human homolog of H2-Bl (human leukocyte antigen G [HLA-G]) in primary human hepatocytes and in the serum of GH-deficient patients. We find that injury-associated innate immune attack by natural killer/natural killer T cell and macrophage cells are instrumental in the failure of liver regeneration, and this can be overcome in Ghr-/- mice by adenoviral delivery of H2-Bl or by infusion of HLA-G protein. Further, H2-Bl knockdown in wild-type C57BL/6 mice showed elevated markers of inflammation after PHx, whereas Ghr-/- backcrossed on a strain with high endogenous H2-Bl expression showed a high rate of survival following PHx. CONCLUSIONS: GH induction of H2-Bl expression is crucial for reducing innate immune-mediated apoptosis and promoting survival after PHx in C57BL/6 mice. Treatment with HLA-G may lead to improved clinical outcomes following liver surgery or transplantation.

Novel drivers and modifiers of MPL-dependent oncogenic transformation identified by deep mutational scanning.

The single transmembrane domain (TMD) of the human thrombopoietin receptor (TpoR/myeloproliferative leukemia [MPL] protein), encoded by exon 10 of the MPL gene, is a hotspot for somatic mutations associated with myeloproliferative neoplasms (MPNs). Approximately 6% and 14% of JAK2 V617F- essential thrombocythemia and primary myelofibrosis patients, respectively, have "canonical" MPL exon 10 driver mutations W515L/K/R/A or S505N, which generate constitutively active receptors and consequent loss of Tpo dependence. Other "noncanonical" MPL exon 10 mutations have also been identified in patients, both alone and in combination with canonical mutations, but, in almost all cases, their functional consequences and relevance to disease are unknown. Here, we used a deep mutational scanning approach to evaluate all possible single amino acid substitutions in the human TpoR TMD for their ability to confer cytokine-independent growth in Ba/F3 cells. We identified all currently recognized driver mutations and 7 novel mutations that cause constitutive TpoR activation, and a much larger number of second-site mutations that enhance S505N-driven activation. We found examples of both of these categories in published and previously unpublished MPL exon 10 sequencing data from MPN patients, demonstrating that some, if not all, of the new mutations reported here represent likely drivers or modifiers of myeloproliferative disease.

Bright spots among the world's coral reefs.

Ongoing declines in the structure and function of the world's coral reefs require novel approaches to sustain these ecosystems and the millions of people who depend on them3. A presently unexplored approach that draws on theory and practice in human health and rural development is to systematically identify and learn from the 'outliers'­places where ecosystems are substantially better ('bright spots') or worse ('dark spots') than expected, given the environmental conditions and socioeconomic drivers they are exposed to. Here we compile data from more than 2,500 reefs worldwide and develop a Bayesian hierarchical model to generate expectations of how standing stocks of reef fish biomass are related to 18 socioeconomic drivers and environmental conditions. We identify 15 bright spots and 35 dark spots among our global survey of coral reefs, defined as sites that have biomass levels more than two standard deviations from expectations. Importantly, bright spots are not simply comprised of remote areas with low fishing pressure; they include localities where human populations and use of ecosystem resources is high, potentially providing insights into how communities have successfully confronted strong drivers of change. Conversely, dark spots are not necessarily the sites with the lowest absolute biomass and even include some remote, uninhabited locations often considered near pristine6. We surveyed local experts about social, institutional, and environmental conditions at these sites to reveal that bright spots are characterized by strong sociocultural institutions such as customary taboos and marine tenure, high levels of local engagement in management, high dependence on marine resources, and beneficial environmental conditions such as deep-water refuges. Alternatively, dark spots are characterized by intensive capture and storage technology and a recent history of environmental shocks. Our results suggest that investments in strengthening fisheries governance, particularly aspects such as participation and property rights, could facilitate innovative conservation actions that help communities defy expectations of global reef degradation.

Landscape-scale patterns of nutrient enrichment in a coral reef ecosystem: implications for coral to algae phase shifts.

Nutrient pollution is altering coastal ecosystems worldwide. On coral reefs, excess nutrients can favor the production of algae at the expense of reef-building corals, yet the role of nutrients in driving community changes such as shifts from coral to macroalgae is not well understood. Here we investigate the potential role of anthropogenic nutrient loading in driving recent coral-to-macroalgae phase shifts on reefs in the lagoons surrounding the Pacific island of Moorea, French Polynesia. We use nitrogen (N) tissue content and stable isotopes (δ15 N) in an abundant macroalga (Turbinaria ornata) together with empirical models of nutrient discharge to describe spatial and temporal patterns of nutrient enrichment in the lagoons. We then employ time series data to test whether recent increases in macroalgae are associated with nutrients. Our results revealed that patterns of N enrichment were linked to several factors, including rainfall, wave-driven circulation, and distance from anthropogenic nutrient sources, especially human sewage. Reefs near large watersheds, where inputs of N from sewage and agriculture are high, have been consistently enriched in N for at least the last decade. In many of these areas, corals have decreased and macroalgae have increased, while reefs with lower levels of N input have maintained high cover of coral and low cover of macroalgae. Importantly, these patchy phase shifts to macroalgae have occurred despite substantial island-wide increases in the density and biomass of herbivorous fishes over the time period. Together, these results indicate that nutrient loading may be an important driver of coral-to-macroalgae phase shifts in the lagoons of Moorea even though the reefs harbor an abundant and diverse herbivore assemblage. These results emphasize the important role that bottom-up factors can play in driving coral-to-macroalgae phase shifts and underscore the critical importance of watershed management for reducing inputs of nutrients and other land-based pollutants to coral reef ecosystems.

Gravity of human impacts mediates coral reef conservation gains.

Coral reefs provide ecosystem goods and services for millions of people in the tropics, but reef conditions are declining worldwide. Effective solutions to the crisis facing coral reefs depend in part on understanding the context under which different types of conservation benefits can be maximized. Our global analysis of nearly 1,800 tropical reefs reveals how the intensity of human impacts in the surrounding seascape, measured as a function of human population size and accessibility to reefs ("gravity"), diminishes the effectiveness of marine reserves at sustaining reef fish biomass and the presence of top predators, even where compliance with reserve rules is high. Critically, fish biomass in high-compliance marine reserves located where human impacts were intensive tended to be less than a quarter that of reserves where human impacts were low. Similarly, the probability of encountering top predators on reefs with high human impacts was close to zero, even in high-compliance marine reserves. However, we find that the relative difference between openly fished sites and reserves (what we refer to as conservation gains) are highest for fish biomass (excluding predators) where human impacts are moderate and for top predators where human impacts are low. Our results illustrate critical ecological trade-offs in meeting key conservation objectives: reserves placed where there are moderate-to-high human impacts can provide substantial conservation gains for fish biomass, yet they are unlikely to support key ecosystem functions like higher-order predation, which is more prevalent in reserve locations with low human impacts.

Loss of growth hormone-mediated signal transducer and activator of transcription 5 (STAT5) signaling in mice results in insulin sensitivity with obesity.

Growth hormone (GH) has an important function as an insulin antagonist with elevated insulin sensitivity evident in humans and mice lacking a functional GH receptor (GHR). We sought the molecular basis for this sensitivity by utilizing a panel of mice possessing specific deletions of GHR signaling pathways. Metabolic clamps and glucose homeostasis tests were undertaken in these obese adult C57BL/6 male mice, which indicated impaired hepatic gluconeogenesis. Insulin sensitivity and glucose disappearance rate were enhanced in muscle and adipose of mice lacking the ability to activate the signal transducer and activator of transcription (STAT)5 via the GHR (Ghr-391-/-) as for GHR-null (GHR-/-) mice. These changes were associated with a striking inhibition of hepatic glucose output associated with altered glycogen metabolism and elevated hepatic glycogen content during unfed state. The enhanced hepatic insulin sensitivity was associated with increased insulin receptor ß and insulin receptor substrate 1 activation along with activated downstream protein kinase B signaling cascades. Although phosphoenolpyruvate carboxykinase (Pck)-1 expression was unchanged, its inhibitory acetylation was elevated because of decreased sirtuin-2 expression, thereby promoting loss of PCK1. Loss of STAT5 signaling to defined chromatin immunoprecipitation targets would further increase lipogenesis, supporting hepatosteatosis while lowering glucose output. Finally, up-regulation of IL-15 expression in muscle, with increased secretion of adiponectin and fibroblast growth factor 1 from adipose tissue, is expected to promote insulin sensitivity.-Chhabra, Y., Nelson, C. N., Plescher, M., Barclay, J. L., Smith, A. G., Andrikopoulos, S., Mangiafico, S., Waxman, D. J., Brooks, A. J., Waters, M. J. Loss of growth hormone-mediated signal transducer and activator of transcription 5 (STAT5) signaling in mice results in insulin sensitivity with obesity.

Just add water: rapid assembly of new communities in previously dry riverbeds, and limited long-distance effects on existing communities.

Barriers preventing species from dispersing to a location can have a major influence on how communities assemble. Dispersal success may also depend on whether dispersers have to colonise an established community or a largely depauperate location. In freshwater systems, dams and weirs have fragmented rivers, potentially limiting dispersal of biota along rivers. Decommissioning aqueducts on two weirs, each within a tributary of different regulated rivers, delivered flow to previously dry riverbeds and additional flows to the main stem, regulated rivers further downstream. This provided an opportunity to test how removal of dispersal constraints affected community assembly in new habitats and whether changed dispersal can alter existing communities. The results were very similar for the two systems. Even with dispersal constrained via reduced drift rates, the new communities in the newly formed habitat in tributaries rapidly resembled unimpacted reference communities that were the source of colonists. For established communities (regulated rivers), greater flow increased the densities of filter feeders but this was due to greater areas of fast-flowing habitat (a change in environmental constraints) rather than higher dispersal rates. Our study illustrates that communities can quickly re-assemble when natural channels that have been dry for decades are re-wetted by flows that deliver dispersers from intact locations upstream. Nevertheless, boosting flows and concomitant densities of dispersers had no strong effects on existing communities. Instead, increased discharges effected a reduction in environmental constraints, which altered trophic structure. Thus, increases in discharge and dispersal produced different outcomes in new versus established communities.

Predicting coral community recovery using multi-species population dynamics models.

Predicting whether, how, and to what degree communities recover from disturbance remain major challenges in ecology. To predict recovery of coral communities we applied field survey data of early recovery dynamics to a multi-species integral projection model that captured key demographic processes driving coral population trajectories, notably density-dependent larval recruitment. After testing model predictions against field observations, we updated the model to generate projections of future coral communities. Our results indicated that communities distributed across an island landscape followed different recovery trajectories but would reassemble to pre-disturbed levels of coral abundance, composition, and size, thus demonstrating persistence in the provision of reef habitat and other ecosystem services. Our study indicates that coral community dynamics are predictable when accounting for the interplay between species life-history, environmental conditions, and density-dependence. We provide a quantitative framework for evaluating the ecological processes underlying community trajectory and characteristics important to ecosystem functioning.

Dietary partitioning promotes the coexistence of planktivorous species on coral reefs.

Theories involving niche diversification to explain high levels of tropical diversity propose that species are more likely to co-occur if they partition at least one dimension of their ecological niche space. Yet, numerous species appear to have widely overlapping niches based upon broad categorizations of resource use or functional traits. In particular, the extent to which food partitioning contributes to species coexistence in hyperdiverse tropical ecosystems remains unresolved. Here, we use a molecular approach to investigate inter- and intraspecific dietary partitioning between two species of damselfish (Dascyllus flavicaudus, Chromis viridis) that commonly co-occur in branching corals. Species-level identification of their diverse zooplankton prey revealed significant differences in diet composition between species despite their seemingly similar feeding strategies. Dascyllus exhibited a more diverse diet than Chromis, whereas Chromis tended to select larger prey items. A large calanoid copepod, Labidocera sp., found in low density and higher in the water column during the day, explained more than 19% of the variation in dietary composition between Dascyllus and Chromis. Dascyllus did not significantly shift its diet in the presence of Chromis, which suggests intrinsic differences in feeding behaviour. Finally, prey composition significantly shifted during the ontogeny of both fish species. Our findings show that levels of dietary specialization among coral reef associated species have likely been underestimated, and they underscore the importance of characterizing trophic webs in tropical ecosystems at higher levels of taxonomic resolution. They also suggest that niche redundancy may not be as common as previously thought.

Predicting coral community recovery using multi-species population dynamics models.

Predicting whether, how, and to what degree communities recover from disturbance remain major challenges in ecology. To predict recovery of coral communities we applied field survey data of early recovery dynamics to a multi-species integral projection model that captured key demographic processes driving coral population trajectories, notably density-dependent larval recruitment. After testing model predictions against field observations, we updated the model to generate projections of future coral communities. Our results indicated that communities distributed across an island landscape followed different recovery trajectories but would reassemble to pre-disturbed levels of coral abundance, composition, and size, thus demonstrating persistence in the provision of reef habitat and other ecosystem services. Our study indicates that coral community dynamics are predictable when accounting for the interplay between species life-history, environmental conditions, and density-dependence. We provide a quantitative framework for evaluating the ecological processes underlying community trajectory and characteristics important to ecosystem functioning.

BioTIME: A database of biodiversity time series for the Anthropocene.

MOTIVATION: The BioTIME database contains raw data on species identities and abundances in ecological assemblages through time. These data enable users to calculate temporal trends in biodiversity within and amongst assemblages using a broad range of metrics. BioTIME is being developed as a community-led open-source database of biodiversity time series. Our goal is to accelerate and facilitate quantitative analysis of temporal patterns of biodiversity in the Anthropocene. MAIN TYPES OF VARIABLES INCLUDED: The database contains 8,777,413 species abundance records, from assemblages consistently sampled for a minimum of 2 years, which need not necessarily be consecutive. In addition, the database contains metadata relating to sampling methodology and contextual information about each record. SPATIAL LOCATION AND GRAIN: BioTIME is a global database of 547,161 unique sampling locations spanning the marine, freshwater and terrestrial realms. Grain size varies across datasets from 0.0000000158 km2 (158 cm2) to 100 km2 (1,000,000,000,000 cm2). TIME PERIOD AND GRAIN: BioTIME records span from 1874 to 2016. The minimal temporal grain across all datasets in BioTIME is a year. MAJOR TAXA AND LEVEL OF MEASUREMENT: BioTIME includes data from 44,440 species across the plant and animal kingdoms, ranging from plants, plankton and terrestrial invertebrates to small and large vertebrates. SOFTWARE FORMAT: .csv and .SQL.

Uropathogenic Escherichia coli Engages CD14-Dependent Signaling to Enable Bladder-Macrophage-Dependent Control of Acute Urinary Tract Infection.

BACKGROUND: CD14, a coreceptor for several pattern recognition receptors and a widely used monocyte/macrophage marker, plays a key role in host responses to gram-negative bacteria. Despite the central role of CD14 in the inflammatory response to lipopolysaccharide and other microbial products and in the dissemination of bacteria in some infections, the signaling networks controlled by CD14 during urinary tract infection (UTI) are unknown. METHODS: We used uropathogenic Escherichia coli (UPEC) infection of wild-type (WT) C57BL/6 and Cd14(-/-) mice and RNA sequencing to define the CD14-dependent transcriptional signature and the role of CD14 in host defense against UTI in the bladder. RESULTS: UPEC induced the upregulation of Cd14 and the monocyte/macrophage-related genes Emr1/F4/80 and Csf1r/c-fms, which was associated with lower UPEC burdens in WT mice, compared with Cd14(-/-) mice. Exacerbation of infection in Cd14(-/-) mice was associated with the absence of a 491-gene transcriptional signature in the bladder that encompassed multiple host networks not previously associated with this receptor. CD14-dependent pathways included immune cell trafficking, differential cytokine production in macrophages, and interleukin 17 signaling. Depletion of monocytes/macrophages in the bladder by administration of liposomal clodronate led to higher UPEC burdens. CONCLUSIONS: This study identifies new host protective and signaling roles for CD14 in the bladder during UPEC UTI.

Knowledge, attitudes and beliefs towards management of men with locally advanced prostate cancer following radical prostatectomy: an Australian survey of urologists.

OBJECTIVE: To investigate Australian urologists' knowledge, attitudes and beliefs, and the association of these with treatment preferences relating to guideline-recommended adjuvant radiotherapy for men with adverse pathologic features following radical prostatectomy. SUBJECTS AND METHODS: A nationwide mailed and web-based survey of Australian urologist members of the Urological Society of Australia and New Zealand (USANZ). RESULTS: 157 surveys were included in the analysis (45% response rate). Just over half of respondents (54%) were aware of national clinical practice guidelines for the management of prostate cancer. Urologists' attitudes and beliefs towards the specific recommendation for post-operative adjuvant radiotherapy for men with locally advanced prostate cancer were mixed. Just over half agreed the recommendation is based on a valid interpretation of the underpinning evidence (54.1%, 95% CI [46%, 62.2%]) but less than one third agreed adjuvant radiotherapy will lead to improved patient outcomes (30.2%, 95% CI [22.8%, 37.6%]). Treatment preferences were varied, demonstrating clinical equipoise. A positive attitude towards the clinical practice recommendation was significantly associated with treatment preference for adjuvant radiotherapy (rho = 0.520, P < 0.0001). There was stronger preference for adjuvant radiotherapy in more recently trained urologists (registrars) while preference for watchful waiting was greater in more experienced urologists (consultants) (b = 0.156, P = 0.034; 95% CI [0.048, 1.24]). Urologists' attitudes towards clinical practice guidelines in general were positive. CONCLUSION: There remains clinical equipoise among Australian urologists in relation to adjuvant radiotherapy for men with adverse pathologic features following radical prostatectomy.

JAK2 activation by growth hormone and other cytokines.

Growth hormone (GH) and structurally related cytokines regulate a great number of physiological and pathological processes. They do this by coupling their single transmembrane domain (TMD) receptors to cytoplasmic tyrosine kinases, either as homodimers or heterodimers. Recent studies have revealed that many of these receptors exist as constitutive dimers rather than being dimerized as a consequence of ligand binding, which has necessitated a new paradigm for describing their activation process. In the present study, we describe a model for activation of the tyrosine kinase Janus kinase 2 (JAK2) by the GH receptor homodimer based on biochemical data and molecular dynamics simulations. Binding of the bivalent ligand reorientates and rotates the receptor subunits, resulting in a transition from a form with parallel TMDs to one where the TMDs separate at the point of entry into the cytoplasm. This movement slides the pseudokinase inhibitory domain of one JAK kinase away from the kinase domain of the other JAK within the receptor dimer-JAK complex, allowing the two kinase domains to interact and trans-activate. This results in phosphorylation and activation of STATs and other signalling pathways linked to this receptor which then regulate postnatal growth, metabolism and stem cell activation. We believe that this model will apply to most if not all members of the class I cytokine receptor family, and will be useful in the design of small antagonists and agonists of therapeutic value.

Intrinsically disordered cytoplasmic domains of two cytokine receptors mediate conserved interactions with membranes.

Class 1 cytokine receptors regulate essential biological processes through complex intracellular signalling networks. However, the structural platform for understanding their functions is currently incomplete as structure-function studies of the intracellular domains (ICDs) are critically lacking. The present study provides the first comprehensive structural characterization of any cytokine receptor ICD and demonstrates that the human prolactin (PRL) receptor (PRLR) and growth hormone receptor (GHR) ICDs are intrinsically disordered throughout their entire lengths. We show that they interact specifically with hallmark lipids of the inner plasma membrane leaflet through conserved motifs resembling immuno receptor tyrosine-based activation motifs (ITAMs). However, contrary to the observations made for ITAMs, lipid association of the PRLR and GHR ICDs was shown to be unaccompanied by changes in transient secondary structure and independent of tyrosine phosphorylation. The results of the present study provide a new structural platform for studying class 1 cytokine receptors and may implicate the membrane as an active component regulating intracellular signalling.

Exploiting hydrophobicity for efficient production of transmembrane helices for structure determination by NMR spectroscopy.

Despite the biological and pharmaceutical significance of membrane proteins, their tertiary structures constitute less than 3% of known structures. One of the major obstacles for initiating structural studies of membrane proteins by NMR spectroscopy is the generation of high amounts of isotope-labeled protein. In this work, we have exploited the hydrophobic nature of membrane proteins to develop a simple and efficient production scheme for isotope-labeled single-pass transmembrane domains (TMDs) with or without intrinsically disordered regions. We have evaluated the applicability and limitations of the strategy using seven membrane protein variants that differ in their overall hydrophobicity and length and show a recovery for suitable variants of >70%. The developed production scheme is cost-efficient and easy to implement and has the potential to facilitate an increase in the number of structures of single-pass TMDs, which are difficult to solve by other means.

Predation and landscape characteristics independently affect reef fish community organization.

Trophic island biogeography theory predicts that the effects of predators on prey diversity are context dependent in heterogeneous landscapes. Specifically, models predict that the positive effect of habitat area on prey diversity should decline in the presence of predators, and that predators should modify the partitioning of alpha and beta diversity across patchy landscapes. However, experimental tests of the predicted context dependency in top-down control remain limited. Using a factorial field experiment we quantify the effects of a focal predatory fish species (grouper) and habitat characteristics (patch size, fragmentation) on the partitioning of diversity and assembly of coral reef fish communities. We found independent effects of groupers and patch characteristics on prey communities. Groupers reduced prey abundance by 50% and gamma diversity by 45%, with a disproportionate removal of rare species relative to common species (64% and 36% reduction, respectively; an oddity effect). Further, there was a 77% reduction in beta diversity. Null model analysis demonstrated that groupers increased the importance of stochastic community assembly relative to patches without groupers. With regard to patch size, larger patches contained more fishes, but a doubling of patch size led to a modest (36%) increase in prey abundance. Patch size had no effect on prey diversity; however, fragmented patches had 50% higher species richness and modified species composition relative to unfragmented patches. Our findings suggest two different pathways (i.e., habitat or predator shifts) by which natural and/or anthropogenic processes can drive variation in fish biodiversity and community assembly.