Mitochondrial Permeability Uncouples Elevated Autophagy and Lifespan Extension.

Autophagy is required in diverse paradigms of lifespan extension, leading to the prevailing notion that autophagy is beneficial for longevity. However, why autophagy is harmful in certain contexts remains unexplained. Here, we show that mitochondrial permeability defines the impact of autophagy on aging. Elevated autophagy unexpectedly shortens lifespan in C. elegans lacking serum/glucocorticoid regulated kinase-1 (sgk-1) because of increased mitochondrial permeability. In sgk-1 mutants, reducing levels of autophagy or mitochondrial permeability transition pore (mPTP) opening restores normal lifespan. Remarkably, low mitochondrial permeability is required across all paradigms examined of autophagy-dependent lifespan extension. Genetically induced mPTP opening blocks autophagy-dependent lifespan extension resulting from caloric restriction or loss of germline stem cells. Mitochondrial permeability similarly transforms autophagy into a destructive force in mammals, as liver-specific Sgk knockout mice demonstrate marked enhancement of hepatocyte autophagy, mPTP opening, and death with ischemia/reperfusion injury. Targeting mitochondrial permeability may maximize benefits of autophagy in aging.

Publisher Correction: Autophagy as a promoter of longevity: insights from model organisms.

In the original article a Note added in proof was not included. This has now been amended.

Autophagy as a promoter of longevity: insights from model organisms.

Autophagy is a conserved process that catabolizes intracellular components to maintain energy homeostasis and to protect cells against stress. Autophagy has crucial roles during development and disease, and evidence accumulated over the past decade indicates that autophagy also has a direct role in modulating ageing. In particular, elegant studies using yeasts, worms, flies and mice have demonstrated a broad requirement for autophagy-related genes in the lifespan extension observed in a number of conserved longevity paradigms. Moreover, several new and interesting concepts relevant to autophagy and its role in modulating longevity have emerged. First, select tissues may require or benefit from autophagy activation in longevity paradigms, as tissue-specific overexpression of single autophagy genes is sufficient to extend lifespan. Second, selective types of autophagy may be crucial for longevity by specifically targeting dysfunctional cellular components and preventing their accumulation. And third, autophagy can influence organismal health and ageing even non-cell autonomously, and thus, autophagy stimulation in select tissues can have beneficial, systemic effects on lifespan. Understanding these mechanisms will be important for the development of approaches to improve human healthspan that are based on the modulation of autophagy.

A cool way to live long.

In this issue, Xiao et al. challenge the notion that cold temperatures promote longevity solely through thermodynamic effects. They show that low temperatures activate a cold-sensitive cation channel, TRPA-1, which triggers a complex signaling pathway in both neurons and nonneuronal cells to extend the lifespan of Caenorhabditis elegans.

A conserved SREBP-1/phosphatidylcholine feedback circuit regulates lipogenesis in metazoans.

Sterol regulatory element-binding proteins (SREBPs) activate genes involved in the synthesis and trafficking of cholesterol and other lipids and are critical for maintaining lipid homeostasis. Aberrant SREBP activity, however, can contribute to obesity, fatty liver disease, and insulin resistance, hallmarks of metabolic syndrome. Our studies identify a conserved regulatory circuit in which SREBP-1 controls genes in the one-carbon cycle, which produces the methyl donor S-adenosylmethionine (SAMe). Methylation is critical for the synthesis of phosphatidylcholine (PC), a major membrane component, and we find that blocking SAMe or PC synthesis in C. elegans, mouse liver, and human cells causes elevated SREBP-1-dependent transcription and lipid droplet accumulation. Distinct from negative regulation of SREBP-2 by cholesterol, our data suggest a feedback mechanism whereby maturation of nuclear, transcriptionally active SREBP-1 is controlled by levels of PC. Thus, nutritional or genetic conditions limiting SAMe or PC production may activate SREBP-1, contributing to human metabolic disorders.

Beyond Autophagy: The Expanding Roles of ATG8 Proteins.

The ATG8 family proteins are critical players in autophagy, a cytoprotective process that mediates degradation of cytosolic cargo. During autophagy, ATG8s conjugate to autophagosome membranes to facilitate cargo recruitment, autophagosome biogenesis, transport, and fusion with lysosomes, for cargo degradation. In addition to these canonical functions, recent reports demonstrate that ATG8s are also delivered to single-membrane organelles, which leads to highly divergent degradative or secretory fates, vesicle maturation, and cargo specification. The association of ATG8s with different vesicles involves complex regulatory mechanisms still to be fully elucidated. Whether individual ATG8 family members play unique canonical or non-canonical roles, also remains unclear. This review summarizes the many open molecular questions regarding ATG8s that are only beginning to be unraveled.

Autophagy in major human diseases.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

Post-translational modifications of ATG8 proteins - an emerging mechanism of autophagy control.

Autophagy is a recycling mechanism involved in cellular homeostasis with key implications for health and disease. The conjugation of the ATG8 family proteins, which includes LC3B (also known as MAP1LC3B), to autophagosome membranes, constitutes a hallmark of the canonical autophagy process. After ATG8 proteins are conjugated to the autophagosome membranes via lipidation, they orchestrate a plethora of protein-protein interactions that support key steps of the autophagy process. These include binding to cargo receptors to allow cargo recruitment, association with proteins implicated in autophagosome transport and autophagosome-lysosome fusion. How these diverse and critical protein-protein interactions are regulated is still not well understood. Recent reports have highlighted crucial roles for post-translational modifications of ATG8 proteins in the regulation of ATG8 functions and the autophagy process. This Review summarizes the main post-translational regulatory events discovered to date to influence the autophagy process, mostly described in mammalian cells, including ubiquitylation, acetylation, lipidation and phosphorylation, as well as their known contributions to the autophagy process, physiology and disease.

Intestine-to-neuronal signaling alters risk-taking behaviors in food-deprived Caenorhabditis elegans.

Animals integrate changes in external and internal environments to generate behavior. While neural circuits detecting external cues have been mapped, less is known about how internal states like hunger are integrated into behavioral outputs. Here, we use the nematode C. elegans to examine how changes in internal nutritional status affect chemosensory behaviors. We show that acute food deprivation leads to a reversible decline in repellent, but not attractant, sensitivity. This behavioral change requires two conserved transcription factors MML-1 (MondoA) and HLH-30 (TFEB), both of which translocate from the intestinal nuclei to the cytoplasm during food deprivation. Next, we identify the insulin-like peptide INS-31 as a candidate ligand relaying food-status signals from the intestine to other tissues. Further, we show that neurons likely use the DAF-2 insulin receptor and AGE-1/PI-3 Kinase, but not DAF-16/FOXO to integrate these intestine-released peptides. Altogether, our study shows how internal food status signals are integrated by transcription factors and intestine-neuron signaling to generate flexible behaviors via the gut-brain axis.

Effects of anti-CD20 therapy on circulating and intrathecal follicular helper T cell subsets in multiple sclerosis.

Follicular helper T (Tfh) cells and their interplay with B cells likely contribute to the pathogenesis of relapsing-remitting multiple sclerosis (RRMS). Tfh cells are enriched in cerebrospinal fluid (CSF) in RRMS, but effects of anti-CD20 therapy are unknown. We investigated Tfh cells in controls, untreated and anti-CD20-treated patients with RRMS using flow cytometry. CSF Tfh cells were increased in untreated patients. Compared to paired blood samples, CD25- Tfh cells were enriched in CSF in RRMS, but not in controls. Contrast-enhancing brain MRI lesions and IgG index correlated with CSF CD25- Tfh cell frequency in untreated patients with RRMS. Anti-CD20 therapy reduced the numbers of circulating PD1+ Tfh cells and CD25- Tfh cells, and the frequency of CSF CD25- Tfh cells. The study suggests that CD25- Tfh cells are recruited to the CSF in RRMS, associated with focal inflammation, and are reduced by anti-CD20 therapy.

When are You Recovered? Defining Cutoff Points Using the Acute Respiratory Tract Infection Questionnaire.

OBJECTIVES: Acute respiratory tract infections (ARTIs) are a heterogenous group of diseases. Often, it is difficult to obtain a precise diagnosis in general practice but also difficult to determine when the patient is recovered. The lack of a precise definition of recovery after ARTI complicates scientific research aiming to optimize diagnostics and compare treatments. The study aimed to define cutoff points to determine the end of an ARTI as a proxy for recovery in patients diagnosed with ARTI in general practice using a validated patient-reported outcome instrument; The ARTI Questionnaire (ARTIQ). METHODS: A total of 259 participants was divided in 2 groups-1 with ARTI and 1 without. Histograms and area under the curve were calculated for each of the 5 dimensions within the ARTIQ to evaluate the discriminative effect. For the most discriminative dimensions receiver operating comparison curves were performed to determine relevant cutoff points for having or not having ARTI symptoms and serve as a proxy for recovery in clinical research. RESULTS: The highest discriminative effect was found in 2 dimensions: "physical-upper airways" and "physical-lower airways." When combining these dimensions, the area under the curve was 0.97. Sensitivity, specificity, and predictive values were calculated for selected cutoff points. CONCLUSION: Cutoff points serving as proxy for recovery from ARTI using a patient-reported outcome were identified. The specific cutoff point for a certain research project must be selected considering the specific clinical situation of interest.

Removal from the wild endangers the once widespread long-tailed macaque.

In 2022, long-tailed macaques (Macaca fascicularis), a once ubiquitous primate species, was elevated to Endangered on the International Union for Conservation of Nature (IUCN) Red List of Threatened Species. In 2023, recognizing that the long-tailed macaque is threatened by multiple factors: (1) declining native habitats across Southeast Asia; (2) overutilization for scientific, commercial, and recreational purposes; (3) inadequate regulatory mechanisms; and (4) culling due to human-macaque conflicts, a petition for rulemaking was submitted to the United States Fish and Wildlife Service to add the species to the US Endangered Species Act, the nation's most effective law to protect at risk species. The long-tailed macaque remains unprotected across much of its geographical range despite the documented continual decline of the species and related sub-species and the recent IUCN reassessment. This commentary presents a review of the factors that have contributed to the dramatic decline of this keystone species and makes a case for raising the level of protection they receive.

Exploring general practice staff perspectives on a teaching concept based on instruction videos for diabetic retinopathy screening - an interview study.

OBJECTIVE: The aim of this study is to explore general practice staff perspectives regarding a teaching concept based on instructional videos for conducting DR screenings. Furthermore, this study aims to investigate the competencies acquired by the staff through this teaching concept. DESIGN AND SETTING: Qualitative cross-sectional study conducted in general practice clinics in the North Denmark Region. METHOD: A teaching concept was developed based on instruction videos to teach general practice staff to conduct diabetic retinopathy screenings with automated grading through artificial intelligence. Semi-structured interviews were performed with 16 staff members to investigate their perspectives on the concept and acquired competencies. RESULTS: This study found no substantial resistance to the teaching concept from staff; however, participants' satisfaction with the methods employed in the instruction session, the progression of learning curves, screening competencies, and their acceptance of a known knowledge gap during screenings varied slightly among the participants. CONCLUSION: This study showed that the teaching concept can be used to teach general practice staff to conduct diabetic retinopathy screenings. Staffs' perspectives on the teaching concept and acquired competencies varied, and this study suggest few adjustments to the concept to accommodate staff's preferences and establish more consistent competencies.

Cluster headache polygenetic risk and known functional variants of CYP3A4 are not associated with treatment response.

BACKGROUND AND PURPOSE: The response to cluster headache treatments has a high interindividual variation. To date, treatment response has only been assessed by a candidate gene approach and no investigations into metabolic pathways have been performed. Our aim was to investigate the association between the polygenetic risk of cluster headache and treatment response to first-line cluster headache treatments as well as known functional variants of CYP3A4 and the response to verapamil. Further, it was aimed to replicate previous single nucleotide polymorphisms found to be associated with treatment response in cluster headache and/or migraine. METHODS: In, 508 cluster headache patients diagnosed according to the International Classification of Headache Disorders were genotyped and participated in a semi-structured interview to evaluate treatment response. Polygenetic risk scores were calculated by the effect retrieved from a meta-analysis of the latest two genome-wide association studies on cluster headache. RESULTS: Inferior treatment response to oxygen, triptans and verapamil is associated with chronicity of cluster headache were confirmed but no evidence was found that a response could be predicted by a high genetic risk of cluster headache. Likewise, verapamil response was not associated with functional variants of CYP3A4. No support of the genetic variants previously reported to be associated with treatment response to triptans or verapamil was found. CONCLUSION: The clinically relevant variation in treatment response for cluster headache was not influenced by genetic factors in the present study.

Red blood cell parameters in early childhood: a prospective cohort study.

OBJECTIVES: Red blood cell parameters are frequently used biomarkers when assessing clinical status in newborns and in early childhood. Cell counts, amounts, and concentrations of these parameters change through gestation and after birth. Robust age-specific reference intervals are needed to optimize clinical decision making. METHODS: The Copenhagen Baby Heart Study (CBHS) and the COMPARE study are prospective cohort studies including red blood cell parameters from 7,938 umbilical cord blood samples and 295 parallel venous blood samples from newborns with follow-up at two and at 14-16 months after birth. RESULTS: For venous blood at birth, reference intervals for hemoglobin, erythrocytes, and hematocrit were 145-224 g/L, 4.1-6.4 × 1012/L, and 0.44-0.64, respectively. Hemoglobin, erythrocytes, and hematocrit were lower at birth in children delivered by prelabor cesarean section compared to vaginal delivery. Conversion algorithms based on term newborns were: venous hemoglobin=(umbilical cord hemoglobin-86.4)/0.39; venous erythrocytes=(umbilical cord erythrocytes-2.20)/0.44; and venous hematocrit=(umbilical cord hematocrit-0.24)/0.45. CONCLUSIONS: This study presents new reference intervals for red blood cell parameters in early childhood, describes the impact of delivery mode, and provide exact functions for converting umbilical cord to venous blood measurements for term newborns. These findings may improve clinical decision making within neonatology and infancy and enhance our clinical understanding of red blood cell parameters for health and diseases in early life.

Phosphorylation of LC3 by the Hippo kinases STK3/STK4 is essential for autophagy.

The protein LC3 is indispensible for the cellular recycling process of autophagy and plays critical roles during cargo recruitment, autophagosome biogenesis, and completion. Here, we report that LC3 is phosphorylated at threonine 50 (Thr(50)) by the mammalian Sterile-20 kinases STK3 and STK4. Loss of phosphorylation at this site blocks autophagy by impairing fusion of autophagosomes with lysosomes, and compromises the ability of cells to clear intracellular bacteria, an established cargo for autophagy. Strikingly, mutation of LC3 mimicking constitutive phosphorylation at Thr(50) reverses the autophagy block in STK3/STK4-deficient cells and restores their capacity to clear bacteria. Loss of STK3/STK4 impairs autophagy in diverse species, indicating that these kinases are conserved autophagy regulators. We conclude that phosphorylation of LC3 by STK3/STK4 is an essential step in the autophagy process. Since several pathological conditions, including bacterial infections, display aberrant autophagy, we propose that pharmacological agents targeting this regulatory circuit hold therapeutic potential.

Development over time in point-of-care test use in Danish daytime and out-of-hours general practice: a register-based study.

OBJECTIVE: To describe the development over time of the use of C-reactive protein (CRP) and rapid streptococcal detection test (RADT) point-of-care tests (POCT) in Danish general practice and to explore associations between patient characteristics and POCT use (i.e. CRP and RADT). DESIGN AND SETTINGS: A register-based study including all general practice clinic consultations in daytime and out-of-hours (OOH) settings in Denmark between 2003 and 2018. SUBJECTS: All citizens who had at least one clinic consultation in daytime or OOH general practice within the study period. MAIN OUTCOME MEASURES: We estimated the total and relative use of CRP and RADT POCTs and described the development over time. Crude and adjusted proportion ratios (PRs) were calculated to explore associations between patient characteristics and POCT use. RESULTS: Overall, the relative use of CRP POCTs increased. At OOH, a steep increase was noticed around 2012. The relative use of RADT decreased. Patient age 40-59 years and existing comorbidity were significantly associated with a higher use of CRP testing in both settings. A significantly lower use of CRP testing was found for patients with higher educational level. We found a significantly higher use of RADT testing for patients aged 0-19 years and with higher household educational level, whereas comorbidity was associated with a lower use of RADT testing. CONCLUSION: The use of CRP POCT increased over time, whereas the use of RADT POCT decreased. Perhaps the success of implementing CRP as a tool for reducing antibiotic use has reached it limit. Future studies should focus on how and when POCT are used most optimal.Key pointsCRP POC tests and RADT POCTs are frequently used diagnostic tools in general practice, both in daytime and in the out-of-hours setting.There was an increased use of CRP POCTs, particularly in out-of-hours general practice, whereas the use of RADT POCTs declined between 2003 and 2018.CRP POCTs were associated with age of 40-59 years and co-morbidity, while the use of RADT was mostly associated with younger age.

Increased demand of urine cultures from Danish general practice: a five-year register-based study.

OBJECTIVE: To characterise and explore the development in the number and content of urine samples sent from general practice in the North Denmark Region to the Department of Clinical Microbiology (DCM) at Aalborg University Hospital during a five-year period. DESIGN: A register-based study. SETTING: General practice. SUBJECTS: Urine samples received at DCM, Aalborg University Hospital from general practice between 2017 and 2022. MAIN OUTCOME MEASURES: Number and content of urine samples. RESULTS: A total of 255,271 urine samples from general practice were received at DCM, with 76.1% being from female patients. Uropathogens were identified in 43.0% of the samples. During the five-year period, a 23.0% increase in the number of urine samples per person (incidence rate ratio (IRR) 1.23, 95% CI 1.21-1.25) was observed. A slight increase in the proportion of positive cultures (risk ratio (RR) 1.03, 95% CI 1.01-1.05) was seen. No notable change in the patient population (age, gender) was observed. Overall, Escherichia coli was the most identified uropathogen (60.4%) followed by Klebsiella spp. (8.7%) and Enterococcus spp. (7.7%). Distribution of the various uropathogens differed slightly depending on patient gender and age, importantly E. coli was less frequently observed in males aged >65 years. CONCLUSION: During the past five years an increasing amount of urine cultures have been requested at DCM from general practice. Importantly, the cause(s) of this increasing demand needs to be explored further in future studies.

Appropriate diagnostics of urinary tract infections can reduce the use of antibiotics in general practice.From 2017 to 2022 a 23% increase per person in requested urine cultures from general practice was observed.A slight increase in positive cultures was found, but no notable change in the patient population (age, gender) was seen.E. coli was the most identified uropathogen independent of gender and age, however, the proportion differed within the various groups.

Coagulation parameters in the newborn and infant - the Copenhagen Baby Heart and COMPARE studies.

OBJECTIVES: The coagulation system is not fully developed at birth and matures during the first months of infancy, complicating clinical decision making within hemostasis. This study evaluates coagulation parameters at birth and two months after birth, and tests whether cord blood can be used as a proxy for neonatal venous blood measurements. METHODS: The Copenhagen Baby Heart Study (CBHS) and the COMPARE study comprise 13,237 cord blood samples and 444 parallel neonatal venous blood samples, with a two month follow-up in 362 children. RESULTS: Because coagulation parameters differed according to gestational age (GA), all analyses were stratified by GA. For neonatal venous blood, reference intervals for activated partial thromboplastin time (APTT) and prothrombin time (PT) were 28-43 s and 33-61% for GA 37-39 and 24-38 s and 30-65% for GA 40-42. Reference intervals for international normalized ratio (INR) and thrombocyte count were 1.1-1.7 and 194-409 × 109/L for GA 37-39 and 1.2-1.8 and 188-433 × 109/L for GA 40-42. Correlation coefficients between umbilical cord and neonatal venous blood for APTT, PT, INR, and thrombocyte count were 0.68, 0.72, 0.69, and 0.77 respectively, and the distributions of the parameters did not differ between the two types of blood (all p-values>0.05). CONCLUSIONS: This study describes new GA dependent reference intervals for common coagulation parameters in newborns and suggests that cord blood may serve as a proxy for neonatal venous blood for these traits. Such data will likely improve clinical decision making within hemostasis among newborn and infant children.

Use and quality of point-of-care microscopy, urine culture and susceptibility testing for urinalysis in general practice.

Objective: To describe the use and quality of point-of-care (POC) microscopy, urine culture and susceptibility testing performed in general practice in Northern Denmark from 2013 to 2018.Design: Descriptive studySetting: General practices receiving a fee for examining urine samples.Subjects: Simulated urine samples containing uropathogenic bacteria distributed by the organisation for improvement of microbiological quality (MIKAP).Main outcome measures: Percentage of use and correct answers for microscopy, culture and susceptibility testing.Results: A total of 5361 samples were analysed by the use of microscopy (39.7%), culture (66.0%) and/or susceptibility testing (76.5%). For culture, Flexicult SSI urinary kittm (87.6%) demonstrated the highest percentage of correct answers followed by chromogenic agar (85.1%) and 2-plate dipslide (85.2%). Mueller Hinton agar with tablets had the highest percentage of correct answers for susceptibility testing of most bacterial strains (84.6%), followed by Flexicult (77.2%). Furthermore, susceptibility testing with tablets (range: 76.1-84.6%) was found to be more accurate than discs (range: 72.9-75.5%). Overall, the highest percentage of correct answers was obtained when examining urine samples containing Escherichia coli: Microscopy (78.3%), culture (87.0%) and susceptibility testing (range: 84.3-90.7%).Conclusion: The quality of POC testing in general practice was high when examining urine samples containing the most common uropathogen E. coli. Surprisingly, susceptibility testing was more frequently used than culture. This approach may compromise the treatment decision as only cultures contribute with information about the flora composition and bacterial quantification. Interestingly, microscopy was the least used method even though the result may be reached within a few minutes.Key pointsThe quality of POC tests (microscopy, urine culture, susceptibility testing) performed in general practice was high when examining urine containing E. coli, whereas difficulties were observed for samples including S. saprophyticus or K. pneumoniae.Susceptibility testing was more often performed than urine culture, which indicates a problem as only urine cultures contribute with information about the flora composition and bacterial quantification.