The mitochondrial UPR: mechanisms, physiological functions and implications in ageing.

Mitochondrial function declines during ageing owing to the accumulation of deleterious mitochondrial genomes and damage resulting from the localized generation of reactive oxygen species, both of which are often exacerbated in diseases such as Parkinson disease. Cells have several mechanisms to assess mitochondrial function and activate a transcriptional response known as the mitochondrial unfolded protein response (UPRmt) when mitochondrial integrity and function are impaired. The UPRmt promotes cell survival and the recovery of the mitochondrial network to ensure optimal cellular function. Recent insights into the regulation, mechanisms and functions of the UPRmt have uncovered important and complex links to ageing and ageing-associated diseases. In this Review, we discuss the signal transduction mechanisms that regulate the UPRmt and the physiological consequences of its activation that affect cellular and organismal health during ageing.

MLKL Requires the Inositol Phosphate Code to Execute Necroptosis.

Necroptosis is an important form of lytic cell death triggered by injury and infection, but whether mixed lineage kinase domain-like (MLKL) is sufficient to execute this pathway is unknown. In a genetic selection for human cell mutants defective for MLKL-dependent necroptosis, we identified mutations in IPMK and ITPK1, which encode inositol phosphate (IP) kinases that regulate the IP code of soluble molecules. We show that IP kinases are essential for necroptosis triggered by death receptor activation, herpesvirus infection, or a pro-necrotic MLKL mutant. In IP kinase mutant cells, MLKL failed to oligomerize and localize to membranes despite proper receptor-interacting protein kinase-3 (RIPK3)-dependent phosphorylation. We demonstrate that necroptosis requires IP-specific kinase activity and that a highly phosphorylated product, but not a lowly phosphorylated precursor, potently displaces the MLKL auto-inhibitory brace region. These observations reveal control of MLKL-mediated necroptosis by a metabolite and identify a key molecular mechanism underlying regulated cell death.

Mitochondrial Sterol Oxidation Marks the Spot.

Maintenance of mitochondrial function during stress conditions is vital for cellular survival. In this issue of Molecular Cell, Nielson et al. (2017) characterize a unique domain within Vms1 that allows this protein quality control component to specifically recognize damaged or stressed compartments within the mitochondrial network.

Metabolism and the UPR(mt).

During mitochondrial dysfunction or the accumulation of unfolded proteins within mitochondria, cells employ a transcriptional response known as the mitochondrial unfolded protein response (UPR(mt)) to promote cell survival along with the repair and recovery of defective mitochondria. Considerable progress has been made in understanding how cells monitor mitochondrial function and activate the response, as well as in identifying scenarios where the UPR(mt) plays a protective role, such as during bacterial infection, hematopoietic stem cell maintenance, or general aging. To date, much of the focus has been on the role of the UPR(mt) in maintaining or re-establishing protein homeostasis within mitochondria by transcriptionally inducing mitochondrial molecular chaperone and protease genes. In this review, we focus on the metabolic adaptations or rewiring mediated by the UPR(mt) and how this may contribute to the resolution of mitochondrial unfolded protein stress and cell-type-specific physiology.

Understanding Public Perception of Naloxone: A Study of FAQs and Answer Source Credibility.

PURPOSE: The most commonly used intervention for opioid overdoses is naloxone. With naloxone soon to be sold over-the-counter in the United States, the goal of this paper is to categorize frequently asked questions (FAQs) and answers about naloxone using internet sources in a cross-sectional fashion. METHODS: Terms "narcan" and "naloxone" were searched on a clean Google Chrome browser using the "People also asked" tab to find FAQs and their answer sources. We classified questions and sources and assessed each website's quality and credibility grading with JAMA benchmark criteria. The Kruskal-Wallis H test was used to determine variance of mean JAMA score by source type and Post-Hoc Dunn's test with Bonferroni corrected alpha of 0.005 used to compare source types. RESULTS: Of the 305 unique questions, 202 (66.2%) were classified as facts, 78 (25.6%) were policy, and 25 (8.2%) were value. Of the 144 unique answer sources, the two most common included 55 (38.2%) which were government entities and 47 (32.6%) which were commercial entities. Ninety-two (of 144, 63.9%) sources met three or more JAMA benchmark criteria. Statistical analysis showed a significant difference between the JAMA benchmark scores by source type H(4) = 12.75, p = 0.0126 and between the mean rank of academic and government sources (p = 0.0036). CONCLUSION: We identified FAQs and their citations about naloxone, highlighting potential lack of understanding and knowledge of this important intervention. We recommend updating websites to accurately reflect current and useful information for those that may require naloxone.

Racial Disparities in Treatment and Outcomes of Patients With Hepatitis C Undergoing Elective Total Joint Arthroplasty.

BACKGROUND: African Americans have the highest prevalence of chronic Hepatitis C virus (HCV) infection. Racial disparities in outcome are observed after elective total hip arthroplasty (THA) and total knee arthroplasty (TKA). This study sought to identify if disparities in treatments and outcomes exist between Black and White patients who have HCV prior to elective THA and TKA. METHODS: Patient demographics, comorbidities, HCV characteristics, perioperative variables, in-hospital outcomes, and postoperative complications at 1-year follow-up were collected and compared between the 2 races. Patients who have preoperative positive viral load (PVL) and undetectable viral load were identified. Chi-square and Fisher's exact tests were used to compare categorical variables, while 2-tailed Student's Kruskal-Wallis t-tests were used for continuous variables. A P value of less than .05 was statistically significant. RESULTS: The liver function parameters, including aspartate aminotransferase and model for end-stage liver disease scores, were all higher preoperatively in Black patients undergoing THA (P = .01; P < .001) and TKA (P = .03; P = .003), respectively. Black patients were more likely to undergo THA (65.8% versus 35.6%; P = .002) and TKA (72.1% versus 37.3%; 0.009) without receiving prior treatment for HCV. Consequently, Black patients had higher rates of preoperative PVL compared to White patients in both THA (66% versus 38%, P = .006) and TKA (72% versus 37%, P < .001) groups. Black patients had a longer length of stay for both THA (3.7 versus 3.3; P = .008) and TKA (4.1 versus 3.0; P = .02). CONCLUSIONS: The HCV treatment prior to THA and TKA with undetectable viral load has been shown to be a key factor in mitigating postoperative complications, including joint infection. We noted that Black patients were more likely to undergo joint arthroplasty who did not receive treatment and with a PVL. While PVL rates decreased over time for both races, a significant gap persists for Black patients.

Mitochondrial and nuclear accumulation of the transcription factor ATFS-1 promotes OXPHOS recovery during the UPR(mt).

Mitochondrial diseases and aging are associated with defects in the oxidative phosphorylation machinery (OXPHOS), which are the only complexes composed of proteins encoded by separate genomes. To better understand genome coordination and OXPHOS recovery during mitochondrial dysfunction, we examined ATFS-1, a transcription factor that regulates mitochondria-to-nuclear communication during the mitochondrial UPR, via ChIP-sequencing. Surprisingly, in addition to regulating mitochondrial chaperone, OXPHOS complex assembly factor, and glycolysis genes, ATFS-1 bound directly to OXPHOS gene promoters in both the nuclear and mitochondrial genomes. Interestingly, atfs-1 was required to limit the accumulation of OXPHOS transcripts during mitochondrial stress, which required accumulation of ATFS-1 in the nucleus and mitochondria. Because balanced ATFS-1 accumulation promoted OXPHOS complex assembly and function, our data suggest that ATFS-1 stimulates respiratory recovery by fine-tuning OXPHOS expression to match the capacity of the suboptimal protein-folding environment in stressed mitochondria, while simultaneously increasing proteostasis capacity.

Tyrosinase Inhibitors: A Perspective.

Due to its integral role in the biosynthesis of melanin in all kingdoms of life, tyrosinase has become an extremely important target for inhibition in several sectors of research including agricultural and cosmetic research. Inhibitors of tyrosinase have made it to the market in the cosmetics industry, but their use has been limited due to conflicting efficacy and potential toxicity, which has led to several small molecules being removed from the market. Undaunted, researchers have continued to pursue tyrosinase inhibitors with varying degrees of success. These pursuits have built an impressive and rich library of research. This review is intended to provide a perspective of the past twenty years (2003-2023) of research on tyrosinase inhibitors by highlighting exemplar molecules and developments.

Targeting the De Novo Purine Synthesis Pathway Through Adenylosuccinate Lyase Depletion Impairs Liver Cancer Growth by Perturbing Mitochondrial Function.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is among the most common cancer types worldwide, yet patients with HCC have limited treatment options. There is an urgent need to identify drug targets that specifically inhibit the growth of HCC cells. APPROACH AND RESULTS: We used a CRISPR library targeting ~2,000 druggable genes to perform a high-throughput screen and identified adenylosuccinate lyase (ADSL), a key enzyme involved in the de novo purine synthesis pathway, as a potential drug target for HCC. ADSL has been implicated as a potential oncogenic driver in some cancers, but its role in liver cancer progression remains unknown. CRISPR-mediated knockout of ADSL impaired colony formation of liver cancer cells by affecting AMP production. In the absence of ADSL, the growth of liver tumors is retarded in vivo. Mechanistically, we found that ADSL knockout caused S-phase cell cycle arrest not by inducing DNA damage but by impairing mitochondrial function. Using data from patients with HCC, we also revealed that high ADSL expression occurs during tumorigenesis and is linked to poor survival rate. CONCLUSIONS: Our findings uncover the role of ADSL-mediated de novo purine synthesis in fueling mitochondrial ATP production to promote liver cancer cell growth. Targeting ADSL may be a therapeutic approach for patients with HCC.

Maintenance and propagation of a deleterious mitochondrial genome by the mitochondrial unfolded protein response.

Mitochondrial genomes (mitochondrial DNA, mtDNA) encode essential oxidative phosphorylation (OXPHOS) components. Because hundreds of mtDNAs exist per cell, a deletion in a single mtDNA has little impact. However, if the deletion genome is enriched, OXPHOS declines, resulting in cellular dysfunction. For example, Kearns-Sayre syndrome is caused by a single heteroplasmic mtDNA deletion. More broadly, mtDNA deletion accumulation has been observed in individual muscle cells and dopaminergic neurons during ageing. It is unclear how mtDNA deletions are tolerated or how they are propagated in somatic cells. One mechanism by which cells respond to OXPHOS dysfunction is by activating the mitochondrial unfolded protein response (UPR(mt)), a transcriptional response mediated by the transcription factor ATFS-1 that promotes the recovery and regeneration of defective mitochondria. Here we investigate the role of ATFS-1 in the maintenance and propagation of a deleterious mtDNA in a heteroplasmic Caenorhabditis elegans strain that stably expresses wild-type mtDNA and mtDNA with a 3.1-kilobase deletion (∆mtDNA) lacking four essential genes. The heteroplasmic strain, which has 60% ∆mtDNA, displays modest mitochondrial dysfunction and constitutive UPR(mt) activation. ATFS-1 impairment reduced the ∆mtDNA nearly tenfold, decreasing the total percentage to 7%. We propose that in the context of mtDNA heteroplasmy, UPR(mt) activation caused by OXPHOS defects propagates or maintains the deleterious mtDNA in an attempt to recover OXPHOS activity by promoting mitochondrial biogenesis and dynamics.

RIP3 induces apoptosis independent of pronecrotic kinase activity.

Receptor-interacting protein kinase 3 (RIP3 or RIPK3) has emerged as a central player in necroptosis and a potential target to control inflammatory disease. Here, three selective small-molecule compounds are shown to inhibit RIP3 kinase-dependent necroptosis, although their therapeutic value is undermined by a surprising, concentration-dependent induction of apoptosis. These compounds interact with RIP3 to activate caspase 8 (Casp8) via RHIM-driven recruitment of RIP1 (RIPK1) to assemble a Casp8-FADD-cFLIP complex completely independent of pronecrotic kinase activities and MLKL. RIP3 kinase-dead D161N mutant induces spontaneous apoptosis independent of compound, whereas D161G, D143N, and K51A mutants, like wild-type, only trigger apoptosis when compound is present. Accordingly, RIP3-K51A mutant mice (Rip3(K51A/K51A)) are viable and fertile, in stark contrast to the perinatal lethality of Rip3(D161N/D161N) mice. RIP3 therefore holds both necroptosis and apoptosis in balance through a Ripoptosome-like platform. This work highlights a common mechanism unveiling RHIM-driven apoptosis by therapeutic or genetic perturbation of RIP3.

Mitochondrial UPR repression during Pseudomonas aeruginosa infection requires the bZIP protein ZIP-3.

Mitochondria generate most cellular energy and are targeted by multiple pathogens during infection. In turn, metazoans employ surveillance mechanisms such as the mitochondrial unfolded protein response (UPRmt) to detect and respond to mitochondrial dysfunction as an indicator of infection. The UPRmt is an adaptive transcriptional program regulated by the transcription factor ATFS-1, which induces genes that promote mitochondrial recovery and innate immunity. The bacterial pathogen Pseudomonas aeruginosa produces toxins that disrupt oxidative phosphorylation (OXPHOS), resulting in UPRmt activation. Here, we demonstrate that Pseudomonas aeruginosa exploits an intrinsic negative regulatory mechanism mediated by the Caenorhabditis elegans bZIP protein ZIP-3 to repress UPRmt activation. Strikingly, worms lacking zip-3 were impervious to Pseudomonas aeruginosa-mediated UPRmt repression and resistant to infection. Pathogen-secreted phenazines perturbed mitochondrial function and were the primary cause of UPRmt activation, consistent with these molecules being electron shuttles and virulence determinants. Surprisingly, Pseudomonas aeruginosa unable to produce phenazines and thus elicit UPRmt activation were hypertoxic in zip-3-deletion worms. These data emphasize the significance of virulence-mediated UPRmt repression and the potency of the UPRmt as an antibacterial response.

The nuclear hormone receptor NHR-86 controls anti-pathogen responses in C. elegans.

Nuclear hormone receptors (NHRs) are ligand-gated transcription factors that control adaptive host responses following recognition of specific endogenous or exogenous ligands. Although NHRs have expanded dramatically in C. elegans compared to other metazoans, the biological function of only a few of these genes has been characterized in detail. Here, we demonstrate that an NHR can activate an anti-pathogen transcriptional program. Using genetic epistasis experiments, transcriptome profiling analyses and chromatin immunoprecipitation-sequencing, we show that, in the presence of an immunostimulatory small molecule, NHR-86 binds to the promoters of immune effectors to activate their transcription. NHR-86 is not required for resistance to the bacterial pathogen Pseudomonas aeruginosa at baseline, but activation of NHR-86 by this compound drives a transcriptional program that provides protection against this pathogen. Interestingly, NHR-86 targets immune effectors whose basal regulation requires the canonical p38 MAPK PMK-1 immune pathway. However, NHR-86 functions independently of PMK-1 and modulates the transcription of these infection response genes directly. These findings characterize a new transcriptional regulator in C. elegans that can induce a protective host response towards a bacterial pathogen.

Redirection of SKN-1 abates the negative metabolic outcomes of a perceived pathogen infection.

Early host responses toward pathogens are essential for defense against infection. In Caenorhabditis elegans, the transcription factor, SKN-1, regulates cellular defenses during xenobiotic intoxication and bacterial infection. However, constitutive activation of SKN-1 results in pleiotropic outcomes, including a redistribution of somatic lipids to the germline, which impairs health and shortens lifespan. Here, we show that exposing C. elegans to Pseudomonas aeruginosa similarly drives the rapid depletion of somatic, but not germline, lipid stores. Modulating the epigenetic landscape refines SKN-1 activity away from innate immunity targets, which alleviates negative metabolic outcomes. Similarly, exposure to oxidative stress redirects SKN-1 activity away from pathogen response genes while restoring somatic lipid distribution. In addition, activating p38/MAPK signaling in the absence of pathogens, is sufficient to drive SKN-1-dependent loss of somatic fat. These data define a SKN-1- and p38-dependent axis for coordinating pathogen responses, lipid homeostasis, and survival and identify transcriptional redirection, rather than inactivation, as a mechanism for counteracting the pleiotropic consequences of aberrant transcriptional activity.

Leveraging Partnerships to Reduce Insulin Needlestick Injuries: Nurse-Led System-Wide Quality Improvement Project.

BACKGROUND: Percutaneous injuries from needlesticks are a major occupational hazard for nurses. LOCAL PROBLEM: Reducing subcutaneous insulin-related needlestick injuries was part of a nurse-led comprehensive sharps injury-reduction program at an integrated, not-for-profit health system. METHODS: The incident rate of needlestick injuries was compared between 1 year before and 1 year after introducing this quality improvement project. INTERVENTIONS: A system-wide educational program instituting changes in subcutaneous insulin administration practices was combined with supply chain standardization using a single type of safety-engineered insulin syringe. RESULTS: The average monthly incidence of needlestick injuries per 10 000 subcutaneous insulin injections fell significantly from year to year (incidence rate ratio, 0.49; 95% CI, 0.30-0.80; Poisson regression P = .004). One-year cost savings for supplies totaled $3500; additional annual median savings were $24 875 (2019 US dollars) in estimated costs of needlestick injuries averted. CONCLUSIONS: The effectiveness of this multifaceted project provides a practical template to reduce subcutaneous insulin-related needlestick injuries.

PER2 regulation of mammary gland development.

The molecular clock plays key roles in daily physiological functions, development and cancer. Period 2 (PER2) is a repressive element, which inhibits transcription activated by positive clock elements, resulting in diurnal cycling of genes. However, there are gaps in our understanding of the role of the clock in normal development outside of its time-keeping function. Here, we show that PER2 has a noncircadian function that is crucial to mammalian mammary gland development. Virgin Per2-deficient mice, Per2-/- , have underdeveloped glands, containing fewer bifurcations and terminal ducts than glands of wild-type mice. Using a transplantation model, we show that these changes are intrinsic to the gland and further identify changes in cell fate commitment. Per2-/- mouse mammary glands have a dual luminal/basal phenotypic character in cells of the ductal epithelium. We identified colocalization of E-cadherin and keratin 14 in luminal cells. Similar results were demonstrated using MCF10A and shPER2 MCF10A human cell lines. Collectively this study reveals a crucial noncircadian function of PER2 in mammalian mammary gland development, validates the Per2-/- model, and describes a potential role for PER2 in breast cancer.

Pre-existing influenza-specific nasal IgA or nasal viral infection does not affect live attenuated influenza vaccine immunogenicity in children.

The United Kingdom has a national immunization programme which includes annual influenza vaccination in school-aged children, using live attenuated influenza vaccine (LAIV). LAIV is given annually, and it is unclear whether repeat administration can affect immunogenicity. Because LAIV is delivered intranasally, pre-existing local antibody might be important. In this study, we analysed banked samples from a study performed during the 2017/18 influenza season to investigate the role of pre-existing influenza-specific nasal immunoglobulin (Ig)A in children aged 6-14 years. Nasopharyngeal swabs were collected prior to LAIV immunization to measure pre-existing IgA levels and test for concurrent upper respiratory tract viral infections (URTI). Oral fluid samples were taken at baseline and 21-28 days after LAIV to measure IgG as a surrogate of immunogenicity. Antibody levels at baseline were compared with a pre-existing data set of LAIV shedding from the same individuals, measured by reverse transcription-polymerase chain reaction. There was detectable nasal IgA specific to all four strains in the vaccine at baseline. However, baseline nasal IgA did not correlate with the fold change in IgG response to the vaccine. Baseline nasal IgA also did not have an impact upon whether vaccine virus RNA was detectable after immunization. There was no difference in fold change of antibody between individuals with and without an URTI at the time of immunization. Overall, we observed no effect of pre-existing influenza-specific nasal antibody levels on immunogenicity, supporting annual immunization with LAIV in children.

Readmission, Complication, and Disposition Calculators in Total Joint Arthroplasty: A Systemic Review.

BACKGROUND: Predictive tools are useful adjuncts in surgical planning. They help guide patient selection, candidacy for inpatient vs outpatient surgery, and discharge disposition as well as predict the probability of readmissions and complications after total joint arthroplasty (TJA). Surgeons may find it difficult due to significant variation among risk calculators to decide which tool is best suited for a specific patient for optimal decision-based care. Our aim is to perform a systematic review of the literature to determine the existing post-TJA readmission calculators and compare the specific elements that comprise their formula. Second, we intend to evaluate the pros and cons of each calculator. METHODS: Using a Preferred Reporting Items for Systematic Reviews and Meta-Analysis Protocols protocol, we conducted a systematic search through 3 major databases for publications addressing TJA risk stratification tools for readmission, discharge disposition, and early complications. We excluded those manuscripts that were not comprehensive for hips and knees, did not list discharge, readmission or complication as the primary outcome, or were published outside the North America. RESULTS: Ten publications met our criteria and were compared on their sourced data, variable types, and overall algorithm quality. Seven of these were generated with single institution data and 3 from large administrative datasets. Three tools determined readmission risk, 5 calculated discharge disposition, and 2 predicted early complications. Only 4 prediction tools were validated by external studies. Seven studies utilized preoperative data points in their risk equations while 3 utilized intraoperative or postsurgical data to delineate risk. CONCLUSION: The extensive variation among TJA risk calculators underscores the need for tools with more individualized stratification capabilities and verification. The transition to outpatient and same-day discharge TJA may preclude or change the need for many of these calculators. Further studies are needed to develop more streamlined risk calculator tools that predict readmission and surgical complications.