REDD1-dependent GSK3ß dephosphorylation promotes NF-κB activation and macrophage infiltration in the retina of diabetic mice.

Increasing evidence supports a role for inflammation in the early development and progression of retinal complications caused by diabetes. We recently demonstrated that the stress response protein regulated in development and DNA damage response 1 (REDD1) promotes diabetes-induced retinal inflammation by sustaining canonical activation of nuclear transcription factor, NF-κB. The studies here were designed to identify signaling events whereby REDD1 promotes NF-κB activation in the retina of diabetic mice. We observed increased REDD1 expression in the retina of mice after 16 weeks of streptozotocin (STZ)-induced diabetes and found that REDD1 was essential for diabetes to suppress inhibitory phosphorylation of glycogen synthase kinase 3ß (GSK3ß) at S9. In human retinal MIO-M1 Müller cell cultures, REDD1 deletion prevented dephosphorylation of GSK3ß and increased NF-κB activation in response to hyperglycemic conditions. Expression of a constitutively active GSK3ß variant restored NF-κB activation in cells deficient for REDD1. In cells exposed to hyperglycemic conditions, GSK3ß knockdown inhibited NF-κB activation and proinflammatory cytokine expression by preventing inhibitor of κB kinase complex autophosphorylation and inhibitor of κB degradation. In both the retina of STZ-diabetic mice and in Müller cells exposed to hyperglycemic conditions, GSK3 inhibition reduced NF-κB activity and prevented an increase in proinflammatory cytokine expression. In contrast with STZ-diabetic mice receiving a vehicle control, macrophage infiltration was not observed in the retina of STZ-diabetic mice treated with GSK3 inhibitor. Collectively, the findings support a model wherein diabetes enhances REDD1-dependent activation of GSK3ß to promote canonical NF-κB signaling and the development of retinal inflammation.

SWOG 1609 cohort 48: anti-CTLA-4 and anti-PD-1 for advanced gallbladder cancer.

INTRODUCTION: Most patients with advanced gallbladder cancer are treated with multiagent chemotherapy. Immune checkpoint inhibitors offer the possibility of a durable response with less toxicity. This prospective, multicenter, open-label study was designed to evaluate the anticancer activity of nivolumab plus ipilimumab in patients with advanced gallbladder cancer. METHODS: Nineteen patients with advanced gallbladder cancer refractory to ≥1 previous therapy received nivolumab 240 mg intravenously every 2 weeks and ipilimumab 1 mg/kg intravenously every 6 weeks until disease progression or unacceptable toxicity. The primary end point was confirmed radiographic overall response rate (ORR) (complete response [CR] + partial response [PR] confirmed on subsequent scan); secondary end points included unconfirmed overall response, clinical benefit rate (confirmed and unconfirmed responses + stable disease >6 months), progression-free survival, overall survival, and toxicity. RESULTS: The confirmed ORR was 16% (CR, n = 1 [5%]; PR, n = 2 [11%]); all were microsatellite stable, and the confirmed CR had undetectable programmed death-ligand 1 by immunohistochemistry. The unconfirmed ORR and clinical benefit rates were both 32%. The median duration of response was 14.8 months (range, 4-35.1+ months). The 6-month progression-free survival was 26% (95% CI, 12-55). The median overall survival was 7.0 months (95% CI, 3.9-19.1). The most common toxicities were fatigue (32%), anemia (26%), and anorexia (26%). Aspartate aminotransferase elevation was the most common grade 3/4 toxicity (11%). There was 1 possibly related death (sepsis with attendant hepatic failure). CONCLUSIONS: Ipilimumab plus nivolumab was well tolerated and showed modest efficacy with durable responses in previously treated patients with advanced gallbladder cancer. CLINICAL TRIAL REGISTRATION: NCT02834013 (ClincialTrials.gov). PLAIN LANGUAGE SUMMARY: This prospective study assessed the efficacy and safety of nivolumab plus ipilimumab in 19 patients with advanced gallbladder cancer refractory to previous therapy. The combination demonstrated modest efficacy with a 16% confirmed overall response rate, durable responses, and manageable toxicities, suggesting potential benefits for this challenging patient population.

REDD1 Deletion Suppresses NF-κB Signaling in Cardiomyocytes and Prevents Deficits in Cardiac Function in Diabetic Mice.

Activation of the transcription factor NF-κB in cardiomyocytes has been implicated in the development of cardiac function deficits caused by diabetes. NF-κB controls the expression of an array of pro-inflammatory cytokines and chemokines. We recently discovered that the stress response protein regulated in development and DNA damage response 1 (REDD1) was required for increased pro-inflammatory cytokine expression in the hearts of diabetic mice. The studies herein were designed to extend the prior report by investigating the role of REDD1 in NF-κB signaling in cardiomyocytes. REDD1 genetic deletion suppressed NF-κB signaling and nuclear localization of the transcription factor in human AC16 cardiomyocyte cultures exposed to TNFα or hyperglycemic conditions. A similar suppressive effect on NF-κB activation and pro-inflammatory cytokine expression was also seen in cardiomyocytes by knocking down the expression of GSK3ß. NF-κB activity was restored in REDD1-deficient cardiomyocytes exposed to hyperglycemic conditions by expression of a constitutively active GSK3ß variant. In the hearts of diabetic mice, REDD1 was required for reduced inhibitory phosphorylation of GSK3ß at S9 and upregulation of IL-1ß and CCL2. Diabetic REDD1+/+ mice developed systolic functional deficits evidenced by reduced ejection fraction. By contrast, REDD1-/- mice did not exhibit a diabetes-induced deficit in ejection fraction and left ventricular chamber dilatation was reduced in diabetic REDD1-/- mice, as compared to diabetic REDD1+/+ mice. Overall, the results support a role for REDD1 in promoting GSK3ß-dependent NF-κB signaling in cardiomyocytes and in the development of cardiac function deficits in diabetic mice.

Stress response protein REDD1 promotes diabetes-induced retinal inflammation by sustaining canonical NF-κB signaling.

Inflammation contributes to the progression of retinal pathology caused by diabetes. Here, we investigated a role for the stress response protein regulated in development and DNA damage response 1 (REDD1) in the development of retinal inflammation. Increased REDD1 expression was observed in the retina of mice after 16-weeks of streptozotocin (STZ)-induced diabetes, and REDD1 was essential for diabetes-induced pro-inflammatory cytokine expression. In human retinal MIO-M1 Müller cell cultures, REDD1 deletion prevented increased pro-inflammatory cytokine expression in response to hyperglycemic conditions. REDD1 deletion promoted nuclear factor erythroid-2-related factor 2 (Nrf2) hyperactivation; however, Nrf2 was not required for reduced inflammatory cytokine expression in REDD1-deficient cells. Rather, REDD1 enhanced inflammatory cytokine expression by promoting activation of nuclear transcription factor κB (NF-κB). In WT cells exposed to tumor necrosis factor α (TNFα), inflammatory cytokine expression was increased in coordination with activating transcription factor 4 (ATF4)-dependent REDD1 expression and sustained activation of NF-κB. In both Müller cell cultures exposed to TNFα and in the retina of STZ-diabetic mice, REDD1 deletion promoted inhibitor of κB (IκB) expression and reduced NF-κB DNA-binding activity. We found that REDD1 acted upstream of IκB by enhancing both K63-ubiquitination and auto-phosphorylation of IκB kinase complex. In contrast with STZ-diabetic REDD1+/+ mice, IκB kinase complex autophosphorylation and macrophage infiltration were not observed in the retina of STZ-diabetic REDD1-/- mice. The findings provide new insight into how diabetes promotes retinal inflammation and support a model wherein REDD1 sustains activation of canonical NF-κB signaling.

PERK/ATF4-dependent expression of the stress response protein REDD1 promotes proinflammatory cytokine expression in the heart of obese mice.

Endoplasmic reticulum (ER) stress and inflammation are hallmarks of myocardial impairment. Here, we investigated the role of the stress response protein regulated in development and DNA damage 1 (REDD1) as a molecular link between ER stress and inflammation in cardiomyocytes. In mice fed a high-fat high-sucrose (HFHS, 42% kcal fat, 34% sucrose by weight) diet for 12 wk, REDD1 expression in the heart was increased in coordination with markers of ER stress and inflammation. In human AC16 cardiomyocytes exposed to either hyperglycemic conditions or the saturated fatty acid palmitate, REDD1 expression was increased coincident with ER stress and upregulated expression of the proinflammatory cytokines IL-1ß, IL-6, and TNFα. In cardiomyocytes exposed to hyperglycemic/hyperlipidemic conditions, pharmacological inhibition of the ER kinase protein kinase RNA-like endoplasmic reticulum kinase (PERK) or knockdown of the transcription factor ATF4 prevented the increase in REDD1 expression. REDD1 deletion reduced proinflammatory cytokine expression in both cardiomyocytes exposed to hyperglycemic/hyperlipidemic conditions and in the hearts of obese mice. Overall, the findings support a model wherein HFHS diet contributes to the development of inflammation in cardiomyocytes by promoting REDD1 expression via activation of a PERK/ATF4 signaling axis.NEW & NOTEWORTHY Interplay between endoplasmic reticulum stress and inflammation contributes to cardiovascular disease progression. The studies here identify the stress response protein known as REDD1 as a missing molecular link that connects the development of endoplasmic reticulum stress with increased production of proinflammatory cytokines in the hearts of obese mice.

Evaluating e-Connect: Mitigating Race by Gender Disparities in Behavioral Health Referral Among Youths on Probation, Northeast United States, 2019.

Addressing the behavioral health needs of youths involved in the justice system is key to reducing recidivism risk and preventing long-term system involvement. However, rates of treatment referral and initiation remain low, especially among minoritized youths and boys. The e-Connect System, a digital, clinical decision support system, addresses this problem by increasing rates of behavioral health treatment referral and initiation rates among youths on probation. In this study, we examine whether e-Connect helps improve equity in referral and treatment initiation outcomes. (Am J Public Health. 2023;113(12):1267-1270. https://doi.org/10.2105/AJPH.2023.307417).

Quality of life measurement in vitiligo. Position statement of the European Academy of Dermatology and Venereology Task Force on Quality of Life and Patient Oriented Outcomes with external experts.

Members of the European Academy of Dermatology and Venereology (EADV) Task Force on Quality of Life (QoL) and Patient Oriented Outcomes reviewed the instruments available for health-related (HR) QoL assessment in vitiligo and together with external vitiligo experts (including representatives of the EADV Vitiligo Task Force) have made practical recommendations concerning the assessment of QoL in vitiligo patients. The Dermatology Life Quality Index (DLQI) was the most frequently used HRQoL instrument, making comparison of results between different countries possible. Several vitiligo-specific instruments were identified. The vitiligo Impact Scale (VIS) is an extensively validated vitiligo-specific HRQoL instrument with proposed minimal important change and clinical interpretation for VIS-22 scores. VIS-22 was developed for use in India, where there are some specific cultural beliefs concerning vitiligo. The EADV Task Force on QoL and Patient Oriented Outcomes recommends use of the DLQI and the Children's Dermatology Life Quality Index (CDLQI) as dermatology-specific instruments in vitiligo. There is a strong need for a valid (including cross-cultural validation) vitiligo-specific instrument that can be either a new instrument or the improvement of existing instruments. This validation must include the proof of responsiveness.

Immune Checkpoint Inhibitor Rechallenge After Prior Immune Toxicity.

OPINION STATEMENT: Immune checkpoint inhibitors (ICIs) have become an essential part of treatment for many cancer types. These monoclonal antibodies remove a critical negative regulatory signal that allows the immune system to recognize and destroy malignant cells that were previously undetectable. Unfortunately, their use has ushered in a whole new form of drug toxicity whereby the immune system attacks normal tissues in the body, referred to hereafter as immune-related adverse events (irAEs). irAEs are common and can result in treatment discontinuation, hospitalization, and death. When alternative modes of treatment are limited, or considered less efficacious, there may be a desire to resume treatment with ICIs after an irAE. Rechallenge with ICIs carries with it a heightened risk of subsequent toxicity, but with careful consideration and appropriate patient selection, this can be considered a reasonable approach.

The comparative effectiveness of Core versus Core+Enhanced implementation strategies in a randomized controlled trial to improve substance use treatment receipt among justice-involved youth.

BACKGROUND: Most justice-involved youth are supervised in community settings, where assessment and linkage to substance use (SU) treatment services are inconsistent and fragmented. Only 1/3 of youth with an identified SU need receive a treatment referral and even fewer initiate services. Thus, improving identification and linkage to treatment requires coordination across juvenile justice (JJ) and behavioral health (BH) agencies. The current study examines the comparative effectiveness of two bundled implementation intervention strategies for improving SU treatment initiation, engagement, and continuing care among justice-involved youth supervised in community settings. Exploration, Preparation, Implementation, Sustainment (EPIS) served as the conceptual framework for study design and selection/timing of implementation intervention components, and the BH Services Cascade served as the conceptual and measurement framework for identifying and addressing gaps in service receipt. METHODS: Part of a larger Juvenile-Justice Translational Research on Interventions for Adolescents in the Legal System (JJ-TRIALS) Cooperative, this study involved a multisite, cluster-randomized control trial where sites were paired then randomly assigned to receive Core (training teams on the BH Services Cascade and data-driven decision making; supporting goal selection) or Core+Enhanced (external facilitation of implementation teams) intervention components. Youth service records were collected from 20 JJ community supervision agencies (in five states) across five study phases (baseline, pre-randomization, early experiment, late experiment, maintenance). Implementation teams comprised of JJ and BH staff collaboratively identified goals along the BH Cascade and used data-driven decision-making to implement change. RESULTS: Results suggest that Core intervention components were effective at increasing service receipt over time relative to baseline, but differences between Core and Core+Enhanced conditions were non-significant. Time to service initiation was shorter among Core+Enhanced sites, and deeper Cascade penetration occurred when external facilitation (of implementation teams) was provided. Wide variation existed in the degree and nature of change across service systems. CONCLUSIONS: Findings demonstrate the criticality of early EPIS phases, demonstrating that strategies provided during the formative exploration and preparation phases produced some improvement in service receipt, whereas implementation-focused activities produced incremental improvement in moving youth farther along the Cascade.

The stress response protein REDD1 promotes diabetes-induced oxidative stress in the retina by Keap1-independent Nrf2 degradation.

The transcription factor nuclear factor erythroid-2-related factor 2 (Nrf2) plays a critical role in reducing oxidative stress by promoting the expression of antioxidant genes. Both individuals with diabetes and preclinical diabetes models exhibit evidence of a defect in retinal Nrf2 activation. We recently demonstrated that increased expression of the stress response protein regulated in development and DNA damage 1 (REDD1) is necessary for the development of oxidative stress in the retina of streptozotocin-induced diabetic mice. In the present study, we tested the hypothesis that REDD1 suppresses the retinal antioxidant response to diabetes by repressing Nrf2 function. We found that REDD1 ablation enhances Nrf2 DNA-binding activity in the retina and that the suppressive effect of diabetes on Nrf2 activity is absent in the retina of REDD1-deficient mice compared with WT. In human MIO-M1 Müller cell cultures, REDD1 deletion prevented oxidative stress in response to hyperglycemic conditions, and this protective effect required Nrf2. REDD1 suppressed Nrf2 stability by promoting its proteasomal degradation independently of Nrf2's interaction with Kelch-like ECH-associated protein 1 (Keap1), but REDD1-mediated Nrf2 degradation required glycogen synthase kinase 3 (GSK3) activity and Ser-351/Ser-356 of Nrf2. Diabetes diminished inhibitory phosphorylation of glycogen synthase kinase 3ß (GSK3ß) at Ser-9 in the retina of WT mice but not in REDD1-deficient mice. Pharmacological inhibition of GSK3 enhanced Nrf2 activity and prevented oxidative stress in the retina of diabetic mice. The findings support a model wherein hyperglycemia-induced REDD1 blunts the Nrf2 antioxidant response to diabetes by activating GSK3, which, in turn, phosphorylates Nrf2 to promote its degradation.

Diabetes enhances translation of Cd40 mRNA in murine retinal Müller glia via a 4E-BP1/2-dependent mechanism.

Activation of the immune costimulatory molecule cluster of differentiation 40 (CD40) in Müller glia has been implicated in the initiation of diabetes-induced retinal inflammation. Results from previous studies support that CD40 protein expression is elevated in Müller glia of diabetic mice; however, the mechanisms responsible for this increase have not been explored. Here, we evaluated the hypothesis that diabetes augments translation of the Cd40 mRNA. Mice receiving thiamet G (TMG), an inhibitor of the O-GlcNAc hydrolase O-GlcNAcase, exhibited enhanced retinal protein O-GlcNAcylation and increased Cd40 mRNA translation. TMG administration also promoted Cd40 mRNA association with Müller cell-specific ribosomes isolated from the retina of RiboTag mice. Similar effects on O-GlcNAcylation and Cd40 mRNA translation were also observed in the retina of a mouse model of type 1 diabetes. In cultured cells, TMG promoted sequestration of the cap-binding protein eIF4E (eukaryotic translation in initiation factor 4E) by 4E-BP1 (eIF4E-binding protein 1) and enhanced cap-independent Cd40 mRNA translation as assessed by a bicistronic reporter that contained the 5'-UTR of the Cd40 mRNA. Ablation of 4E-BP1/2 prevented the increase in Cd40 mRNA translation in TMG-exposed cells, and expression of a 4E-BP1 variant that constitutively sequesters eIF4E promoted reporter activity. Extending on the cell culture results, we found that in contrast to WT mice, diabetic 4E-BP1/2-deficient mice did not exhibit enhanced retinal Cd40 mRNA translation and failed to up-regulate expression of the inflammatory marker nitric-oxide synthase 2. These findings support a model wherein diabetes-induced O-GlcNAcylation of 4E-BP1 promotes Cd40 mRNA translation in Müller glia.

Retinol-binding protein 4 mRNA translation in hepatocytes is enhanced by activation of mTORC1.

Increased expression of the peptide hormone retinol-binding protein 4 (RBP4) has been implicated in the development of insulin resistance, type 2 diabetes, and visual dysfunction. Prior investigations of the mechanisms that influence RBP4 synthesis have focused solely on changes in mRNA abundance. Yet, the production of many secreted proteins is controlled at the level of mRNA translation, as it allows for a rapid and reversible change in expression. Herein, we evaluated Rbp4 mRNA translation using sucrose density gradient centrifugation. In the liver of fasted rodents, Rbp4 mRNA translation was low. In response to refeeding, Rbp4 mRNA translation was enhanced and RBP4 levels in serum were increased. In H4IIE cells, refreshing culture medium promoted Rbp4 mRNA translation and expression of the protein. Rbp4 mRNA abundance was not increased by either experimental manipulation. Enhanced Rbp4 mRNA translation was associated with activation of the kinase mechanistic target of rapamycin in complex 1 (mTORC1) and enhanced phosphorylation of the translational repressor eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). In H4IIE cells, expression of a 4E-BP1 variant that is unable to be phosphorylated by mTORC1 or suppression of mTORC1 with rapamycin attenuated activity of a luciferase reporter encoding the Rbp4 mRNA 5'-untranslated region (UTR). Purine substitutions to disrupt a terminal oligopyrimidine (TOP)-like sequence in the Rbp4 5'-UTR prevented the suppressive effect of rapamycin on reporter activity. Rapamycin also prevented upregulation of Rbp4 mRNA translation in the liver and reduced serum levels of RBP4 in response to feeding. Overall, the findings support a model in which nutrient-induced activation of mTORC1 upregulates Rbp4 mRNA translation to promote RBP4 synthesis.NEW & NOTEWORTHY RBP4 plays a critical role in metabolic disease, yet relatively little is known about the mechanisms that regulate its production. Herein, we provide evidence for translational control of RBP4 synthesis. We demonstrate that activation of the nutrient-sensitive kinase mTORC1 promotes hepatic Rbp4 mRNA translation. The findings support the possibility that targeting Rbp4 mRNA translation represents an alternative to current therapeutic interventions that lower serum RBP4 concentration by promoting urinary excretion of the protein.

Dry Powder Mixing Is Feasible in Continuous Twin Screw Extruder: Towards Lean Extrusion Process for Oral Solid Dosage Manufacturing.

Using discrete element method (DEM) modeling and near-infrared (NIR) spectroscopy, the feasibility of powder mixing in the initial pre-melting zones of a twin screw extruder using two independent feeders was studied. Previous work in the pharmaceutical and food industry has focused on mixing when materials are melted or on material homogeneity at the extruder's output. Depending on the formulation, ensuring a fully blended formulation prior to melting may be desired. Experiments were conducted using a Coperion ZSK-18 extruder to evaluate if blend uniformity can be achieved by exploring screw configuration, screw speed, and powder feed rate. As powder exited the extruder and deposited on a conveyor belt, an in-line NIR spectrophotometer measured spectra of material. Chemometric-based models predicted unknown concentrations to evaluate if blend uniformity was achieved. Using the EDEM software, Hertz-Mindlin contact model, and dimensions of the extruder, DEM simulations complemented the experimental work. The DEM computational models provided understanding of mixing patterns inside the extruder at particle scale and helped select the screw configuration before doing experimentation. The simulations showed good axial mixing for all the screw configurations studied, while good cross (radial) mixing was only observed for the screw configuration with 90-degree kneading elements. Therefore, the screw configuration with two 90-degree kneading elements was chosen for the experimental study. The RTD profiles when using a screw configuration with only conveying screw elements are comparable to a plug flow reactor (PFR), while the profiles when using kneading elements are more comparable to an ideal continuous stirred tank reactor (CSTR). For the screw configuration with 90 degrees kneading elements, the mean residence time (MRT) decreases with an increase in the screw speed. Experimental NIR spectra showed that concentrations can be predicted with an error of 2%. It was demonstrated that the twin screw extruder can provide proper dry powder mixing of two powder feed streams based on a unit dose scale, enabling continuous powder mixing prior to the melting zone in the extruder for the formulation studied with a cohesive API. This setup may also work for other types of formulations. These studies can help in developing lean hot melt as well as wet extrusion/granulation processes using twin screw extruders for the continuous manufacturing of oral solid dosage products.

O-GlcNAcylation alters the selection of mRNAs for translation and promotes 4E-BP1-dependent mitochondrial dysfunction in the retina.

Diabetes promotes the posttranslational modification of proteins by O-linked addition of GlcNAc (O-GlcNAcylation) to Ser/Thr residues of proteins and thereby contributes to diabetic complications. In the retina of diabetic mice, the repressor of mRNA translation, eIF4E-binding protein 1 (4E-BP1), is O-GlcNAcylated, and sequestration of the cap-binding protein eukaryotic translation initiation factor (eIF4E) is enhanced. O-GlcNAcylation has also been detected on several eukaryotic translation initiation factors and ribosomal proteins. However, the functional consequence of this modification is unknown. Here, using ribosome profiling, we evaluated the effect of enhanced O-GlcNAcylation on retinal gene expression. Mice receiving thiamet G (TMG), an inhibitor of the O-GlcNAc hydrolase O-GlcNAcase, exhibited enhanced retinal protein O-GlcNAcylation. The principal effect of TMG on retinal gene expression was observed in ribosome-associated mRNAs (i.e. mRNAs undergoing translation), as less than 1% of mRNAs exhibited changes in abundance. Remarkably, ∼19% of the transcriptome exhibited TMG-induced changes in ribosome occupancy, with 1912 mRNAs having reduced and 1683 mRNAs having increased translational rates. In the retina, the effect of O-GlcNAcase inhibition on translation of specific mitochondrial proteins, including superoxide dismutase 2 (SOD2), depended on 4E-BP1/2. O-GlcNAcylation enhanced cellular respiration and promoted mitochondrial superoxide levels in WT cells, and 4E-BP1/2 deletion prevented O-GlcNAcylation-induced mitochondrial superoxide in cells in culture and in the retina. The retina of diabetic WT mice exhibited increased reactive oxygen species levels, an effect not observed in diabetic 4E-BP1/2-deficient mice. These findings provide evidence for a mechanism whereby diabetes-induced O-GlcNAcylation promotes oxidative stress in the retina by altering the selection of mRNAs for translation.

Glucagon-Dependent Suppression of mTORC1 is Associated with Upregulation of Hepatic FGF21 mRNA Translation.

Fibroblast growth factor 21 (FGF21) is a peptide hormone that acts to enhance insulin sensitivity and reverse many of the metabolic defects associated with consumption of a high-fat diet. Recent studies show that the liver is the primary source of FGF21 in the blood, and that hepatic FGF21 expression is upregulated by glucagon. Interestingly, glucagon acts to upregulate FGF21 production by primary cultures of rat hepatocytes and H4IIE and HepG2 hepatocarcinoma cells independent of changes in FGF21 mRNA abundance, suggesting that FGF21 protein expression is regulated post-transcriptionally. Based on these observations, the goal of the present study was to assess whether or not FGF21 mRNA is translationally regulated. The results show that FGF21 mRNA translation and secretion of the hormone are significantly upregulated in H4IIE cells exposed to 25 nM glucagon, independent of changes in FGF21 mRNA abundance. Furthermore, the glucagon-induced upregulation of FGF21 mRNA translation is associated with suppressed activity of the mechanistic target of rapamycin in complex 1 (mTORC1). Similarly, the results show that rapamycin-induced suppression of mTORC1 leads to upregulation of FGF21 mRNA translation with no change in FGF21 mRNA abundance. In contrast, activation of mTORC1 by refreshing the culture medium leads to downregulation of FGF21 mRNA translation. Notably, re-feeding fasted rats also leads to downregulation of FGF21 mRNA translation concomitantly with activation of mTORC1 in the liver. Overall, the findings support a model in which glucagon acts to upregulate FGF21 production by hepatocytes through suppression of mTORC1 and subsequent upregulation of FGF21 mRNA translation.

ATF4-Mediated Upregulation of REDD1 and Sestrin2 Suppresses mTORC1 Activity during Prolonged Leucine Deprivation.

BACKGROUND: The protein kinase target of rapamycin (mTOR) in complex 1 (mTORC1) is activated by amino acids and in turn upregulates anabolic processes. Under nutrient-deficient conditions, e.g., amino acid insufficiency, mTORC1 activity is suppressed and autophagy is activated. Intralysosomal amino acids generated by autophagy reactivate mTORC1. However, sustained mTORC1 activation during periods of nutrient insufficiency would likely be detrimental to cellular homeostasis. Thus, mechanisms must exist to prevent amino acids released by autophagy from reactivating the kinase. OBJECTIVE: The objective of the present study was to test whether mTORC1 activity is inhibited during prolonged leucine deprivation through ATF4-dependent upregulation of the mTORC1 suppressors regulated in development and DNA damage response 1 (REDD1) and Sestrin2. METHODS: Mice (8 wk old; C57Bl/6 × 129SvEV) were food deprived (FD) overnight and one-half were refed the next morning. Mouse embryo fibroblasts (MEFs) deficient in ATF4, REDD1, and/or Sestrin2 were deprived of leucine for 0-16 h. mTORC1 activity and ATF4, REDD1, and Sestrin2 expression were assessed in liver and cell lysates. RESULTS: Refeeding FD mice resulted in activation of mTORC1 in association with suppressed expression of both REDD1 and Sestrin2 in the liver. In cells in culture, mTORC1 exhibited a triphasic response to leucine deprivation, with an initial suppression followed by a transient reactivation from 2 to 4 h and a subsequent resuppression after 8 h. Resuppression occurred concomitantly with upregulated expression of ATF4, REDD1, and Sestrin2. However, in cells lacking ATF4, neither REDD1 nor Sestrin2 expression was upregulated by leucine deprivation, and resuppression of mTORC1 was absent. Moreover, in cells lacking either REDD1 or Sestrin2, mTORC1 resuppression was attenuated, and in cells lacking both proteins resuppression was further blunted. CONCLUSIONS: The results suggest that leucine deprivation upregulates expression of both REDD1 and Sestrin2 in an ATF4-dependent manner, and that upregulated expression of both proteins is involved in resuppression of mTORC1 during prolonged leucine deprivation.

Contacts with primary and secondary healthcare prior to suicide: case-control whole-population-based study using person-level linked routine data in Wales, UK, 2000-2017.

BACKGROUND: Longitudinal studies of patterns of healthcare contacts in those who die by suicide to identify those at risk are scarce. AIMS: To examine type and timing of healthcare contacts in those who die by suicide. METHOD: A population-based electronic case-control study of all who died by suicide in Wales, 2001-2017, linking individuals' electronic healthcare records from general practices, emergency departments and hospitals. We used conditional logistic regression to calculate odds ratios, adjusted for deprivation. We performed a retrospective continuous longitudinal analysis comparing cases' and controls' contacts with health services. RESULTS: We matched 5130 cases with 25 650 controls (5 per case). A representative cohort of 1721 cases (8605 controls) were eligible for the fully linked analysis. In the week before their death, 31.4% of cases and 15.6% of controls contacted health services. The last point of contact was most commonly associated with mental health and most often occurred in general practices. In the month before their death, 16.6 and 13.0% of cases had an emergency department contact and a hospital admission respectively, compared with 5.5 and 4.2% of controls. At any week in the year before their death, cases were more likely to contact healthcare services than controls. Self-harm, mental health and substance misuse contacts were strongly linked with suicide risk, more so when they occurred in emergency departments or as emergency admissions. CONCLUSIONS: Help-seeking occurs in those at risk of suicide and escalates in the weeks before their death. There is an opportunity to identify and intervene through these contacts.

Risk Factors for Development of Hypocalcemia in Patients With Cancer Treated With Bone-Modifying Agents.

BACKGROUND: This retrospective analysis describes the prevalence of and risk factors associated with the development of hypocalcemia in patients with cancer receiving bone-modifying agents (BMAs) as supportive care. PATIENTS AND METHODS: Patients with cancer treated with an intravenous or subcutaneous BMA, including pamidronate, zoledronic acid, or denosumab, at a tertiary care/safety net hospital in 2005 through 2015 were included in this retrospective review. We reviewed the medical records for predictive clinical and laboratory parameters and for patient outcomes. RESULTS: A total of 835 patients with cancer received at least one dose of a BMA during the specified time frame; 205 patients (25%) developed hypocalcemia of CTCAE grade ≥1 within 8 weeks of BMA initiation, 18 of whom (8.8%) had grade ≥3, and 3 patients died as a result. Multivariate analysis showed that patients with hematologic malignancy (odds ratio [OR], 1.956; P=.025), bone metastases (OR, 2.443; P=.017), inpatient status (OR, 2.592; P<.001), and deficient baseline vitamin D levels (OR, 2.546; P<.023) were more likely to develop hypocalcemia. Hypercalcemia before BMA administration (OR, 0.474; P=.032) was protective. CONCLUSIONS: Certain patient populations, including those with hematologic malignancies and/or bone metastases, warrant closer monitoring of calcium levels while receiving BMAs because of the high rate of hypocalcemia. Low pretreatment vitamin D levels are associated with the development of hypocalcemia. These data support close monitoring of calcium levels in patients with cancer receiving BMAs, in addition to adequate repletion of vitamin D before initiation of BMAs when possible.

Means restriction for the prevention of suicide on roads.

BACKGROUND: Road traffic suicides are common. However, due to the difficulty in distinguishing between motor vehicle crash fatalities and actual suicides, no official figures exist for this method of suicide. Restricting access to means is an important universal or population-based approach to suicide prevention with clear evidence of its effectiveness. However, the evidence with respect to means restriction for the prevention of suicide on roads is not well established. We conducted a systematic review to assess the impact of restrictions on the availability of, or access to, means of suicide on roads. OBJECTIVES: To evaluate the effectiveness of interventions to restrict the availability of, or access to, means of suicide on roads. SEARCH METHODS: We searched the Cochrane Library, MEDLINE, Embase, PsycINFO, and the Transport Research International Documentation (TRID) Database from the date of database inception to March 2020. We conducted searches of the World Health Organization International Clinical Trials Registry Platform (ICTRP) and ClinicalTrials.gov to identify unpublished and ongoing studies. We applied no date, language, or publication status restrictions to these searches. SELECTION CRITERIA: Eligible studies were randomised or quasi-randomised controlled trials, controlled intervention studies without randomisation, before-after studies, or studies using interrupted time series designs, which evaluated interventions to restrict the availability of, or access to, means of suicide on roads. DATA COLLECTION AND ANALYSIS: Two review authors screened abstracts and full-text publications against the inclusion criteria. Two review authors planned to independently extract data and assess risk of bias of included studies. However, we identified no studies eligible for inclusion. MAIN RESULTS: We identified no studies that met the inclusion criteria for this review. AUTHORS' CONCLUSIONS: This systematic review highlights the paucity of research around road traffic suicides and the need for future robust studies that aim to investigate the effectiveness of interventions to prevent suicide on roads. Suicide ascertainment is a key issue; therefore, clear objective criteria are necessary in order to scale up and study this method more accurately. In the absence of any substantial evidence, we advocate for more awareness on road traffic suicides and its inclusion in future government suicide prevention policies. Further research exploring effective measures, particularly those that do not require driver compliance, are also needed.

Means restriction for the prevention of suicide by jumping.

BACKGROUND: Jumping from a height is an uncommon but lethal means of suicide. Restricting access to means is an important universal or population-based approach to suicide prevention with clear evidence of its effectiveness. However, the evidence with respect to means restriction for the prevention of suicide by jumping is not well established. OBJECTIVES: To evaluate the effectiveness of interventions to restrict the availability of, or access to, means of suicide by jumping. These include the use of physical barriers, fencing or safety nets at frequently-used jumping sites, or restriction of access to these sites, such as by way of road closures. SEARCH METHODS: We searched the Cochrane Library, Embase, MEDLINE, PsycINFO, and Web of Science to May 2019. We conducted additional searches of the international trial registries including the World Health Organization International Clinical Trials Registry Platform (ICTRP) and ClinicalTrials.gov, to identify relevant unpublished and ongoing studies. We searched the reference lists of all included studies and relevant systematic reviews to identify additional studies and contacted authors and subject experts for information on unpublished or ongoing studies. We applied no restrictions on date, language or publication status to the searches. Two review authors independently assessed all citations from the searches and identified relevant titles and abstracts. Our main outcomes of interest were suicide, attempted suicide or self-harm, and cost-effectiveness of interventions. SELECTION CRITERIA: Eligible studies were randomised or quasi-randomised controlled trials, controlled intervention studies without randomisation, before-and-after studies, or studies using interrupted time series designs, which evaluated interventions to restrict the availability of, or access to, means of suicide by jumping. DATA COLLECTION AND ANALYSIS: Two review authors independently selected studies for inclusion and three review authors extracted study data. We pooled studies that evaluated similar interventions and outcomes using a random-effects meta-analysis, and we synthesised data from other studies in a narrative summary. We summarised the quality of the evidence included in this review using the GRADE approach. MAIN RESULTS: We included 14 studies in this review. Thirteen were before-and-after studies and one was a cost-effectiveness analysis. Three studies each took place in Switzerland and the USA, while two studies each were from the UK, Canada, New Zealand, and Australia respectively. The majority of studies (10/14) assessed jumping means restriction interventions delivered in isolation, half of which were at bridges. Due to the observational nature of included studies, none compared comparator interventions or control conditions. During the pre- and postintervention period among the 13 before-and-after studies, a total of 742.3 suicides (5.5 suicides per year) occurred during the pre-intervention period (134.5 study years), while 70.6 suicides (0.8 suicides per year) occurred during the postintervention period (92.4 study years) - a 91% reduction in suicides. A meta-analysis of all studies assessing jumping means restriction interventions (delivered in isolation or in combination with other interventions) showed a directionality of effect in favour of the interventions, as evidenced by a reduction in the number of suicides at intervention sites (12 studies; incidence rate ratio (IRR) = 0.09, 95% confidence interval (CI) 0.03 to 0.27; P < 0.001; I2 = 88.40%). Similar findings were demonstrated for studies assessing jumping means restriction interventions delivered in isolation (9 studies; IRR = 0.05, 95% CI 0.01 to 0.16; P < 0.001; I2 = 73.67%), studies assessing jumping means restriction interventions delivered in combination with other interventions (3 studies; IRR = 0.54, 95% CI 0.31 to 0.93; P = 0.03; I2 = 40.8%), studies assessing the effectiveness of physical barriers (7 studies; IRR = 0.07, 95% CI 0.02 to 0.24; P < 0.001; I2 = 84.07%), and studies assessing the effectiveness of safety nets (2 studies; IRR = 0.09, 95% CI 0.01 to 1.30; P = 0.07; I2 = 29.3%). Data on suicide attempts were limited and none of the studies used self-harm as an outcome. There was considerable heterogeneity between studies for the primary outcome (suicide) in the majority of the analyses except those relating to jumping means restriction delivered in combination with other interventions, and safety nets. Nevertheless, every study included in the forest plots showed the same directional effects in favour of jumping means restriction. Due to methodological limitations of the included studies, we rated the quality of the evidence from these studies as low. A cost-effectiveness analysis suggested that the construction of a physical barrier on a bridge would be a highly cost-effective project in the long term as a result of overall reduced suicide mortality. AUTHORS' CONCLUSIONS: The findings from this review suggest that jumping means restriction interventions are capable of reducing the frequency of suicides by jumping. However, due to methodological limitations of included studies, this finding is based on low-quality evidence. Therefore, further well-designed high-quality studies are required to further evaluate the effectiveness of these interventions, as well as other measures at jumping sites. In addition, further research is required to investigate the potential for suicide method substitution and displacement effects in populations exposed to interventions to prevent suicide by jumping.