Orthotopic Models Using New, Murine Lung Adenocarcinoma Cell Lines Simulate Human Non-Small Cell Lung Cancer Treated with Immunotherapy.

Understanding tumor-host immune interactions and the mechanisms of lung cancer response to immunotherapy is crucial. Current preclinical models used to study this often fall short of capturing the complexities of human lung cancer and lead to inconclusive results. To bridge the gap, we introduce two new murine monoclonal lung cancer cell lines for use in immunocompetent orthotopic models. We demonstrate how our cell lines exhibit immunohistochemical protein expression (TTF-1, NapA, PD-L1) and common driver mutations (KRAS, p53, and p110α) seen in human lung adenocarcinoma patients, and how our orthotopic models respond to combination immunotherapy in vivo in a way that closely mirrors current clinical outcomes. These new lung adenocarcinoma cell lines provide an invaluable, clinically relevant platform for investigating the intricate dynamics between tumor and the immune system, and thus potentially contributes to a deeper understanding of immunotherapeutic approaches to lung cancer treatment.

The lowdown on breakdown: Open questions in plant proteolysis.

Proteolysis, including post-translational proteolytic processing as well as protein degradation and amino acid recycling, is an essential component of the growth and development of living organisms. In this article, experts in plant proteolysis pose and discuss compelling open questions in their areas of research. Topics covered include the role of proteolysis in the cell cycle, DNA damage response, mitochondrial function, the generation of N-terminal signals (degrons) that mark many proteins for degradation (N-terminal acetylation, the Arg/N-degron pathway, and the chloroplast N-degron pathway), developmental and metabolic signaling (photomorphogenesis, abscisic acid and strigolactone signaling, sugar metabolism, and post-harvest regulation), plant responses to environmental signals (endoplasmic-reticulum associated degradation, chloroplast-associated degradation, drought tolerance, the growth-defense tradeoff)), and the functional diversification of peptidases. We hope these thought-provoking discussions help to stimulate further research.

CD39 delineates chimeric antigen receptor regulatory T cell subsets with distinct cytotoxic & regulatory functions against human islets.

Human regulatory T cells (Treg) suppress other immune cells. Their dysfunction contributes to the pathophysiology of autoimmune diseases, including type 1 diabetes (T1D). Infusion of Tregs is being clinically evaluated as a novel way to prevent or treat T1D. Genetic modification of Tregs, most notably through the introduction of a chimeric antigen receptor (CAR) targeting Tregs to pancreatic islets, may improve their efficacy. We evaluated CAR targeting of human Tregs to monocytes, a human ß cell line and human islet ß cells in vitro. Targeting of HLA-A2-CAR (A2-CAR) bulk Tregs to HLA-A2+ cells resulted in dichotomous cytotoxic killing of human monocytes and islet ß cells. In exploring subsets and mechanisms that may explain this pattern, we found that CD39 expression segregated CAR Treg cytotoxicity. CAR Tregs from individuals with more CD39low/- Tregs and from individuals with genetic polymorphism associated with lower CD39 expression (rs10748643) had more cytotoxicity. Isolated CD39- CAR Tregs had elevated granzyme B expression and cytotoxicity compared to the CD39+ CAR Treg subset. Genetic overexpression of CD39 in CD39low CAR Tregs reduced their cytotoxicity. Importantly, ß cells upregulated protein surface expression of PD-L1 and PD-L2 in response to A2-CAR Tregs. Blockade of PD-L1/PD-L2 increased ß cell death in A2-CAR Treg co-cultures suggesting that the PD-1/PD-L1 pathway is important in protecting islet ß cells in the setting of CAR immunotherapy. In summary, introduction of CAR can enhance biological differences in subsets of Tregs. CD39+ Tregs represent a safer choice for CAR Treg therapies targeting tissues for tolerance induction.

Older Adults with Cancer and Common Comorbidities-Challenges and Opportunities in Improving Their Cancer Treatment Outcomes.

The older American population is rapidly increasing, and millions of older adults will be cancer survivors with comorbidities. This population faces specific challenges regarding treatment and has unique clinical needs. Recognizing this need, the National Cancer Institute (NCI), in collaboration with the National Institute on Aging (NIA), hosted a webinar series, entitled "Cancer, Aging, and Comorbidities." This commentary provides a reflection of five thematic areas covered by the webinar series, which was focused on improving cancer treatment for older adults with cancer and comorbidities: i) the impact of comorbidities on treatment tolerability and patient outcomes; ii) the impact of comorbidities on cancer clinical trial design; iii) the development of wearable devices in measuring comorbidities in cancer treatment; iv) the effects of nutrition and the microbiome on cancer therapy and; v) the role of senescence and senotherapy in age-related diseases. While advances have been made in these areas, many gaps and challenges exist and are discussed in this commentary. To improve cancer survivorship in older populations with comorbidities, aging and comorbidities must be jointly considered and incorporated across the spectrum of cancer research. This includes more basic research of the mechanisms linking comorbidities and cancer development and treatment response, building critical resources and infrastructure (eg, preclinical models and patient samples), conducting clinical trials focused on the older population, integrating geriatric assessment into cancer treatment, and incorporating novel technologies, such as wearable devices into clinical trials and cancer care.

Hydroxyurea to prevent brain injury in children with sickle cell disease (HU Prevent)-A randomized, placebo-controlled phase II feasibility/pilot study.

Central nervous system (CNS) injury is common in sickle cell disease (SCD) and occurs early in life. Hydroxyurea is safe and efficacious for treatment of SCD, but high-quality evidence from randomized trials to estimate its neuroprotective effect is scant. HU Prevent was a randomized (1:1), double-blind, phase II feasibility/pilot trial of dose-escalated hydroxyurea vs. placebo for the primary prevention of CNS injury in children with HbSS or HbS-ß0-thalassemia subtypes of SCD age 12-48 months with normal neurological examination, MRI of the brain, and cerebral blood flow velocity. We hypothesized that hydroxyurea would reduce by 50% the incidence of CNS injury. Two outcomes were compared: primary-a composite of silent cerebral infarction, elevated cerebral blood flow velocity, transient ischemic attack, or stroke; secondary-a weighted score estimating the risk of suffering the consequences of stroke (the Stroke Consequences Risk Score-SCRS), based on the same outcome events. Six participants were randomized to each group. One participant in the hydroxyurea group had a primary outcome vs. four in the placebo group (incidence rate ratio [90% CI] 0.216 [0.009, 1.66], p = .2914) (~80% reduction in the hydroxyurea group). The mean SCRS score was 0.078 (SD 0.174) in the hydroxyurea group, 0.312 (SD 0.174) in the placebo group, p = .072, below the p-value of .10 often used to justify subsequent phase III investigations. Serious adverse events related to study procedures occurred in 3/41 MRIs performed, all related to sedation. These results suggest that hydroxyurea may have profound neuroprotective effect in children with SCD and support a definitive phase III study to encourage the early use of hydroxyurea in all infants with SCD.

Dietary Quality and Diet-Related Factors Among Emerging Adults (18-23 y) in the United States Are a Cause for Concern: National Health and Nutrition Examination Survey 2015-2018.

BACKGROUND: Poor dietary quality is a risk factor for diet-related chronic disease and suboptimal nutritional patterns often begin early in the life course. Although the dietary intakes of young children, adolescents, and middle-aged and older adults are well established, much less is known about emerging adults, who represent a unique time point in life, as they are undergoing significant changes in food environments, autonomy, finances, and caregiver and parental involvement. OBJECTIVES: This study aimed to examine dietary quality, as assessed via the Healthy Eating Index (HEI), by demographic, socioeconomic, and health-related characteristics among emerging adults (18-23 y) in the United States who participated in the 2015-2018 National Health and Nutrition Examination Survey (NHANES). METHODS: NHANES data were collected via a household interview and 2 24-h dietary recalls (24HR). Usual dietary intakes from the 24HRs were approximated using the multivariate National Cancer Institute Method to compute mean HEI-2015 overall and component scores (range: 0-100; higher scores indicating higher dietary quality). RESULTS: Overall dietary quality among emerging adults (HEI-2015: 50.3 ± 1.3) was significantly lower than other adults (≥24 y) (HEI-2015: 56.3 ± 0.5; P < 0.0001) in the United States, with differences primarily driven by lower intakes of whole fruit, vegetables, and whole grains and higher intakes of sodium, refined grains, and saturated fat. Few differences in HEI-2015 scores were noted across population subgroups by sex, food security, family income, and food assistance program participation, except for added sugar; intakes of added sugar were significantly higher among women, food insecure, and food assistance program participants than those in their counterparts, respectively. CONCLUSIONS: Dietary quality is poor among emerging adults in the United States and persists across all population subgroups, suggesting a significant need for tailored public health interventions to improve dietary quality among this population. Future research investigating to what extent emerging adults prioritize healthful behaviors and exploring other indicators for identifying nutritionally vulnerable subgroups may be impactful for identifying disparities among this life stage.

Unexpected roles for AMPK in the suppression of autophagy and the reactivation of MTORC1 signaling during prolonged amino acid deprivation.

AMPK promotes catabolic and suppresses anabolic cell metabolism to promote cell survival during energetic stress, in part by inhibiting MTORC1, an anabolic kinase requiring sufficient levels of amino acids. We found that cells lacking AMPK displayed increased apoptotic cell death during nutrient stress caused by prolonged amino acid deprivation. We presumed that impaired macroautophagy/autophagy explained this phenotype, as a prevailing view posits that AMPK initiates autophagy (often a pro-survival response) through phosphorylation of ULK1. Unexpectedly, however, autophagy remained unimpaired in cells lacking AMPK, as monitored by several autophagic readouts in several cell lines. More surprisingly, the absence of AMPK increased ULK1 signaling and MAP1LC3B/LC3B lipidation during amino acid deprivation while AMPK-mediated phosphorylation of ULK1 S555 (a site proposed to initiate autophagy) decreased upon amino acid withdrawal or pharmacological MTORC1 inhibition. In addition, activation of AMPK with compound 991, glucose deprivation, or AICAR blunted autophagy induced by amino acid withdrawal. These results demonstrate that AMPK activation and glucose deprivation suppress autophagy. As AMPK controlled autophagy in an unexpected direction, we examined how AMPK controls MTORC1 signaling. Paradoxically, we observed impaired reactivation of MTORC1 in cells lacking AMPK upon prolonged amino acid deprivation. Together these results oppose established views that AMPK promotes autophagy and inhibits MTORC1 universally. Moreover, they reveal unexpected roles for AMPK in the suppression of autophagy and the support of MTORC1 signaling in the context of prolonged amino acid deprivation. These findings prompt a reevaluation of how AMPK and its control of autophagy and MTORC1 affect health and disease.

Exploring Cultural Competence, Inclusivity, and Diversity in Ketamine Assisted Psychotherapy: A Phenomenological Study.

Black, Indigenous, and People of Color (BIPOC), and other minoritized populations are insufficiently represented in research on therapeutic psychedelics. This research was a phenomenological qualitative exploration of a culturally diverse (Hispanic, African American, Asian, Native American, biracial, or LGBTQIA+) and low-income sample of 15 individuals receiving ketamine-assisted psychotherapy (KAP) at a sliding-scale fee community clinic. Participants were interviewed after a ketamine session, after a ketamine integration session, and one month later. The interviews inquired about mental and emotional state prior to treatment and the treatment context (traditionally called set and setting), preparation for treatment, experiences during the ketamine and integration sessions, barriers to treatment, perceived stigma if any, reflections on KAPs' impact, and relevance of culture to the treatment. The current analysis, which focuses on participant comments related to diversity, equity, and inclusion that are uniquely relevant to this sample and the research goals, yielded four major themes: Insufficient Financial Resources, Race, Ethnicity, and LGBTQIA+, Stigma, and Culture and Ritual. Themes and subthemes are presented accompanied by representative quotes. Results demonstrate the high salience of culture in the KAP experience and the need to incorporate issues of race, culture, stigma, ritual, and socioeconomic status into treatment planning and outcome research.

AnnoSpat annotates cell types and quantifies cellular arrangements from spatial proteomics.

Cellular composition and anatomical organization influence normal and aberrant organ functions. Emerging spatial single-cell proteomic assays such as Image Mass Cytometry (IMC) and Co-Detection by Indexing (CODEX) have facilitated the study of cellular composition and organization by enabling high-throughput measurement of cells and their localization directly in intact tissues. However, annotation of cell types and quantification of their relative localization in tissues remain challenging. To address these unmet needs for atlas-scale datasets like Human Pancreas Analysis Program (HPAP), we develop AnnoSpat (Annotator and Spatial Pattern Finder) that uses neural network and point process algorithms to automatically identify cell types and quantify cell-cell proximity relationships. Our study of data from IMC and CODEX shows the higher performance of AnnoSpat in rapid and accurate annotation of cell types compared to alternative approaches. Moreover, the application of AnnoSpat to type 1 diabetic, non-diabetic autoantibody-positive, and non-diabetic organ donor cohorts recapitulates known islet pathobiology and shows differential dynamics of pancreatic polypeptide (PP) cell abundance and CD8+ T cells infiltration in islets during type 1 diabetes progression.

Nutritional Beliefs and Practices of Arabic-Speaking Middle Eastern Mothers Living in the United States.

INTRODUCTION: Culture and acculturation influence nutritional beliefs. Little is known about the Arabic population in the United States. In this study, Arabic-speaking Middle Eastern mothers' perceptions of motherhood and childhood nutritional beliefs and practices are explored. METHOD: Semi-structured interviews with 12 mothers from Arabic-speaking Middle Eastern countries. RESULTS: Food and family are central to everyday life. Mothers worked hard to maintain traditional nutritional practices with their school-age children. DISCUSSION: Findings can enhance school nurses' ability to collaborate with mothers in nutritional education and address any issues in the classroom.

Enhancing implementation of the I-PASS Handoff Tool Using a Provider Handoff Task Force at a Comprehensive Cancer Center.

BACKGROUND: Communication failures are among the most common causes of harmful medical errors. At one Comprehensive Cancer Center, patient handoffs varied among services. The authors describe the implementation and results of an Organization-wide project to improve handoffs and implement an evidence-based handoff tool across all inpatient services. METHODS: The research team created a task force composed of members from 22 hospital services-advanced practice providers (APPs), trainees, some faculty members, electronic health record (EHR) staff, education and training specialists, and nocturnal providers. Over two years, the task force expanded to include consulting services and Anesthesiology. Factors contributing to ineffective handoffs were identified and organized into categories. The EHR I-PASS tool was used to standardize handoff documentation. Training was provided to staff on its use, and compliance was monitored using a customized dashboard. I-PASS champions in each service were responsible for the rollout of I-PASS in their respective services. The data were reported quarterly to the Quality Assessment and Performance Improvement (QAPI) governing committee. Provider handoff perception was assessed through the biennial Institution-wide safety culture survey. RESULTS: All fellows, residents, APPs, and physician assistants were trained in the use of I-PASS, either online or in person. Adherence to the I-PASS written tool improved from 41.6% in 2019 to 70.5% in 2022 (p < 0.05), with improvements seen in most services. The frequency of updating I-PASS elements and the action list in the handoff tool also increased over time. The handoff favorability score on the safety culture survey improved from 38% in 2018 to 59% in 2022. CONCLUSION: The implementation approach developed by the Provider Handoff Task Force led to increased use of the I-PASS EHR tool and improved safety culture survey handoff favorability.

Roadmap for the next decade of plant programmed cell death research.

Programmed cell death (PCD) is fundamentally important for plant development, abiotic stress responses and immunity, but our understanding of its regulation remains fragmented. Building a stronger research community is required to accelerate progress in this area through knowledge exchange and constructive debate. In this Viewpoint, we aim to initiate a collective effort to integrate data across a diverse set of experimental models to facilitate characterisation of the fundamental mechanisms underlying plant PCD and ultimately aid the development of a new plant cell death classification system in the future. We also put forward our vision for the next decade of plant PCD research stemming from discussions held during the 31st New Phytologist workshop, 'The Life and Death Decisions of Plant Cells' that took place at University College Dublin in Ireland (14-15 June 2023). We convey the key areas of significant progress and possible future research directions identified, including resolving the spatiotemporal control of cell death, isolation of its molecular and genetic regulators, and harnessing technical advances for studying PCD events in plants. Further, we review the breadth of potential impacts of plant PCD research and highlight the promising new applications of findings from this dynamically evolving field.

Unexpected roles for AMPK in the suppression of autophagy and the reactivation of mTORC1 signaling during prolonged amino acid deprivation.

AMPK promotes catabolic and suppresses anabolic cell metabolism to promote cell survival during energetic stress, in part by inhibiting mTORC1, an anabolic kinase requiring sufficient levels of amino acids. We found that cells lacking AMPK displayed increased apoptotic cell death during nutrient stress caused by prolonged amino acid deprivation. We presumed that impaired autophagy explained this phenotype, as a prevailing view posits that AMPK initiates autophagy (often a pro-survival response) through phosphorylation of ULK1. Unexpectedly, however, autophagy remained unimpaired in cells lacking AMPK, as monitored by several autophagic readouts in several cell lines. More surprisingly, the absence of AMPK increased ULK1 signaling and LC3b lipidation during amino acid deprivation while AMPK-mediated phosphorylation of ULK1 S555 (a site proposed to initiate autophagy) decreased upon amino acid withdrawal or pharmacological mTORC1 inhibition. In addition, activation of AMPK with compound 991, glucose deprivation, or AICAR blunted autophagy induced by amino acid withdrawal. These results demonstrate that AMPK activation and glucose deprivation suppress autophagy. As AMPK controlled autophagy in an unexpected direction, we examined how AMPK controls mTORC1 signaling. Paradoxically, we observed impaired reactivation of mTORC1 in cells lacking AMPK upon prolonged amino acid deprivation. Together these results oppose established views that AMPK promotes autophagy and inhibits mTORC1 universally. Moreover, they reveal unexpected roles for AMPK in the suppression of autophagy and the support of mTORC1 signaling in the context of prolonged amino acid deprivation. These findings prompt a reevaluation of how AMPK and its control of autophagy and mTORC1 impact health and disease.

Distinct ancient structural polymorphisms control heterodichogamy in walnuts and hickories.

The maintenance of stable mating type polymorphisms is a classic example of balancing selection, underlying the nearly ubiquitous 50/50 sex ratio in species with separate sexes. One lesser known but intriguing example of a balanced mating polymorphism in angiosperms is heterodichogamy - polymorphism for opposing directions of dichogamy (temporal separation of male and female function in hermaphrodites) within a flowering season. This mating system is common throughout Juglandaceae, the family that includes globally important and iconic nut and timber crops - walnuts (Juglans), as well as pecan and other hickories (Carya). In both genera, heterodichogamy is controlled by a single dominant allele. We fine-map the locus in each genus, and find two ancient (>50 Mya) structural variants involving different genes that both segregate as genus-wide trans-species polymorphisms. The Juglans locus maps to a ca. 20 kb structural variant adjacent to a probable trehalose phosphate phosphatase (TPPD-1), homologs of which regulate floral development in model systems. TPPD-1 is differentially expressed between morphs in developing male flowers, with increased allele-specific expression of the dominant haplotype copy. Across species, the dominant haplotype contains a tandem array of duplicated sequence motifs, part of which is an inverted copy of the TPPD-1 3' UTR. These repeats generate various distinct small RNAs matching sequences within the 3' UTR and further downstream. In contrast to the single-gene Juglans locus, the Carya heterodichogamy locus maps to a ca. 200-450 kb cluster of tightly linked polymorphisms across 20 genes, some of which have known roles in flowering and are differentially expressed between morphs in developing flowers. The dominant haplotype in pecan, which is nearly always heterozygous and appears to rarely recombine, shows markedly reduced genetic diversity and is over twice as long as its recessive counterpart due to accumulation of various types of transposable elements. We did not detect either genetic system in other heterodichogamous genera within Juglandaceae, suggesting that additional genetic systems for heterodichogamy may yet remain undiscovered.

PROTAC for agriculture: learning from human medicine to generate new biotechnological weed control solutions.

Weed control has relied on the use of organic and inorganic molecules that interfere with druggable targets, especially enzymes, for almost a century. This approach, although effective, has resulted in multiple cases of herbicide resistance. Furthermore, the rate of discovery of new druggable targets that are selective and with favorable environmental profiles has slowed down, highlighting the need for innovative control tools. The arrival of the biotechnology and genomics era gave hope to many that all sorts of new control tools would be developed. However, the reality is that most efforts have been limited to the development of transgenic crops with resistance to a few existing herbicides, which in fact is just another form of selectivity. Proteolysis-targeting chimera (PROTAC) is a new technology developed to treat human diseases but that has potential for multiple applications in agriculture. This technology uses a small bait molecule linked to an E3 ligand. The 3-dimensional structure of the bait favors physical interaction with a binding site in the target protein in a manner that allows E3 recruitment, ubiquitination and then proteasome-mediated degradation. This system makes it possible to circumvent the need to find druggable targets because it can degrade structural proteins, transporters, transcription factors, and enzymes without the need to interact with the active site. PROTAC can help control herbicide-resistant weeds as well as expand the number of biochemical targets that can be used for weed control. In the present article, we provide an overview of how PROTAC works and describe the possible applications for weed control as well as the challenges that this technology might face during development and implementation for field uses. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Optimizing Care for Military Women: The Value of the Dual-Certified Family and Women's Health Nurse Practitioner.

The number of women in the military has more than tripled over the past 50 years, increasing from 5% in the 1970s to 17% in 2023, making them essential for global health engagement and military operations. Provider competence and confidence are barriers to the consistent availability of preventive, gynecologic, and reproductive services for women across service locations and duty platforms. The Defense Health Board recommends standardizing services and improving the availability and scope of services for women at every point of care. In direct conflict with these recommendations, however, is a congressional call for a drawdown of medical forces, which creates a need for operationally trained clinicians with a broad skill set including comprehensive care for women. Advanced practice registered nurses, such as family and women's health nurse practitioners, are key assets to fill this gap on military medical health-care teams. At the request of the U.S. Air Force, the Graduate School of Nursing at the Uniformed Services University began offering a Women's Health Nurse Practitioner (WHNP) program in 2014. The WHNP curriculum was layered onto the existing Family Nurse Practitioner program so that Family Nurse Practitioner students receive enhanced education in women's health and WHNP students are prepared to meet the holistic, primary care needs of patients across the lifespan in addition to caring for women with obstetric and urogenital health concerns. This article highlights the value of dual-certified Family Nurse Practitioners and WHNPs in the military health-care system. These Uniformed Services University alumni are uniquely prepared to provide comprehensive primary and specialty care for female warfighters across the lifecycle from stable, well-resourced duty stations to austere, operational settings or deployment platforms.

An interdisciplinary synthesis of floodplain ecosystem dynamics in a rapidly deglaciating watershed.

Glacier retreat is rapidly transforming some watersheds, with ramifications for water supply, ecological succession, important species such as Pacific salmon (Oncorhynchus spp.), and cultural uses of landscapes. To advance a more holistic understanding of the evolution of proglacial landscapes, we integrate multiple lines of knowledge starting in the early 1900s with contemporary data from the Taaltsux̱éi (Tulsequah) Watershed in British Columbia, Canada. Our objectives were to: 1) synthesize recent historical geography and Indigenous Knowledge, including glacier dynamics, and hydrology; 2) describe the limnology of a proglacial lake; 3) quantify decadal-scale downstream physical floodplain change; and 4) characterize riverine physical, chemical, and biological differences relative to distance from the proglacial lake. Since 1982, the Tulsequah Glacier has receded 0.07 km/yr, exposing a cold, deep, and growing proglacial lake. The downstream floodplain is rapidly changing; satellite imagery analysis revealed a 14 % increase in vegetation from 2003 to 2017 and Indigenous Knowledge described increases in vegetation and wildlife habitat over the last century. Contemporary measurements of physical-chemical water properties differed across sites representing the upper and lower watershed, and mainstem and off-channel habitats. Catches of juvenile salmonids in the upper watershed (closer to the glacier) were mostly limited to warmer, clearer groundwater-fed channels, whereas in the lower watershed there were salmonids in both groundwater-fed and mainstem habitats. There was limited zooplankton taxa diversity from the proglacial lake and benthic macroinvertebrates in the river. Collectively, our synthesis suggests that the transformation of proglacial landscapes experiencing rapid ice loss can be influenced by interlinked abiotic processes of glacier retreat, lake formation, and altered hydrology, as well as corresponding biological processes such as beaver repopulation, wetland formation, and riparian vegetation growth. These factors, along with expected increases to proglacial lake productivity and salmon habitat suitability, are an important consideration for forward-looking watershed management of glacier-fed rivers.

Genetic risk converges on regulatory networks mediating early type 2 diabetes.

Type 2 diabetes mellitus (T2D), a major cause of worldwide morbidity and mortality, is characterized by dysfunction of insulin-producing pancreatic islet ß cells1,2. T2D genome-wide association studies (GWAS) have identified hundreds of signals in non-coding and ß cell regulatory genomic regions, but deciphering their biological mechanisms remains challenging3-5. Here, to identify early disease-driving events, we performed traditional and multiplexed pancreatic tissue imaging, sorted-islet cell transcriptomics and islet functional analysis of early-stage T2D and control donors. By integrating diverse modalities, we show that early-stage T2D is characterized by ß cell-intrinsic defects that can be proportioned into gene regulatory modules with enrichment in signals of genetic risk. After identifying the ß cell hub gene and transcription factor RFX6 within one such module, we demonstrated multiple layers of genetic risk that converge on an RFX6-mediated network to reduce insulin secretion by ß cells. RFX6 perturbation in primary human islet cells alters ß cell chromatin architecture at regions enriched for T2D GWAS signals, and population-scale genetic analyses causally link genetically predicted reduced RFX6 expression with increased T2D risk. Understanding the molecular mechanisms of complex, systemic diseases necessitates integration of signals from multiple molecules, cells, organs and individuals, and thus we anticipate that this approach will be a useful template to identify and validate key regulatory networks and master hub genes for other diseases or traits using GWAS data.

Prenatal alcohol exposure promotes NLRP3 inflammasome-dependent immune actions following morphine treatment and paradoxically prolongs nerve injury-induced pathological pain in female mice.

BACKGROUND: Neuroimmune dysregulation from prenatal alcohol exposure (PAE) may contribute to neurological deficits associated with fetal alcohol spectrum disorders (FASD). PAE is a risk factor for developing peripheral immune and spinal glial sensitization and release of the proinflammatory cytokine IL-1ß, which lead to neuropathic pain (allodynia) from minor nerve injury. Although morphine acts on µ-opioid receptors, it also activates immune receptors, TLR4, and the NLRP3 inflammasome that induces IL-1ß. We hypothesized that PAE induces NLRP3 sensitization by morphine following nerve injury in adult mice. METHODS: We used an established moderate PAE paradigm, in which adult PAE and non-PAE control female mice were exposed to a minor sciatic nerve injury, and subsequent allodynia was measured using the von Frey fiber test. In control mice with standard sciatic damage or PAE mice with minor sciatic damage, the effects of the NLRP3 inhibitor, MCC950, were examined during chronic allodynia. Additionally, minor nerve-injured mice were treated with morphine, with or without MCC950. In vitro studies examined the TLR4-NLRP3-dependent proinflammatory response of peripheral macrophages to morphine and/or lipopolysaccharide, with or without MCC950. RESULTS: Mice with standard sciatic damage or PAE mice with minor sciatic damage developed robust allodynia. Blocking NLRP3 activation fully reversed allodynia in both control and PAE mice. Morphine paradoxically prolonged allodynia in PAE mice, while control mice with minor nerve injury remained stably non-allodynic. Allodynia resolved sooner in nerve-injured PAE mice without morphine treatment than in morphine-treated mice. MCC950 treatment significantly shortened allodynia in morphine-treated PAE mice. Morphine potentiated IL-1ß release from TLR4-activated PAE immune cells, while MCC950 treatment greatly reduced it. CONCLUSIONS: In female mice, PAE prolongs allodynia following morphine treatment through NLRP3 activation. TLR4-activated PAE immune cells showed enhanced IL-1ß release with morphine via NLRP3 actions. Similar studies are needed to examine the adverse impact of morphine in males with PAE. These results are predictive of adverse responses to opioid pain therapeutics in individuals with FASD.

Evidence for optimal semantic search throughout adulthood.

As people age, they learn and store new knowledge in their semantic memory. Despite learning a tremendous amount of information, people can still recall information relevant to the current situation with ease. To accomplish this, the mind must efficiently organize and search a vast store of information. It also must continue to retrieve information effectively despite changes in cognitive mechanisms due to healthy aging, including a general slowing in information processing and a decline in executive functioning. How effectively does the mind of an individual adjust its search to account for changes due to aging? We tested 746 people ages 25 through 69 on a semantic fluency task (free listing animals) and found that, on average, retrieval follows an optimal path through semantic memory. Participants tended to list a sequence of semantically related animals (e.g., lion, tiger, puma) before switching to a semantically unrelated animal (e.g., whale). We found that the timing of these transitions to semantically unrelated animals was remarkably consistent with an optimal strategy for maximizing the overall rate of retrieval (i.e., the number of animals listed per unit time). Age did not affect an individual's deviation from the optimal strategy given their general performance, suggesting that people adapt and continue to search memory optimally throughout their lives. We argue that this result is more likely due to compensating for a general slowing than a decline in executive functioning.