Morphology of Phagophore Precursors by Correlative Light-Electron Microscopy.

Autophagosome biogenesis occurs in the transient subdomains of the endoplasmic reticulum that are called omegasomes, which, in fluorescence microscopy, appear as small puncta, which then grow in diameter and finally shrink and disappear once the autophagosome is complete. Autophagosomes are formed by phagophores, which are membrane cisterns that elongate and close to form the double membrane that limits autophagosomes. Earlier electron-microscopy studies showed that, during elongation, phagophores are lined by the endoplasmic reticulum on both sides. However, the morphology of the very early phagophore precursors has not been studied at the electron-microscopy level. We used live-cell imaging of cells expressing markers of phagophore biogenesis combined with correlative light-electron microscopy, as well as electron tomography of ATG2A/B-double-deficient cells, to reveal the high-resolution morphology of phagophore precursors in three dimensions. We showed that phagophores are closed or nearly closed into autophagosomes already at the stage when the omegasome diameter is still large. We further observed that phagophore precursors emerge next to the endoplasmic reticulum as bud-like highly curved membrane cisterns with a small opening to the cytosol. The phagophore precursors then open to form more flat cisterns that elongate and curve to form the classically described crescent-shaped phagophores.

Native joint infections in Iceland 2003-2017: an increase in postarthroscopic infections.

OBJECTIVES: Nationwide study on the epidemiology, clinical characteristics and outcomes among patients with native joint infection (NJI) in Iceland, 2003-2017. METHODS: All positive synovial fluid culture results in Iceland were identified and medical records reviewed. RESULTS: A total of 299 NJI (40 children and 259 adults) were diagnosed in Iceland in 2003-2017, with a stable incidence of 6.3 cases/100 000/year, but marked gender difference among adults (33% women vs 67% men, p<0.001). The knee joint was most commonly affected, and Staphylococcus aureus was the most common isolate in both adults and children, followed by various streptococcal species in adults and Kingella kingae in children. NJI was iatrogenic in 34% of adults (88/259) but comprised 45% among 18-65 years and a stable incidence. Incidence of infections following arthroscopic procedures in adults increased significantly compared with the previous decade (9/100 000/year in 1990-2002 vs 25/100 000/year in 2003-2017, p<0.01) with no significant increase seen in risk per procedure. The proportion of postarthroscopic NJI was 0.17% overall but 0.24% for knee arthroscopy. Patients with postarthroscopic infection were more likely to undergo subsequent arthroplasty when compared with other patients with NJI (p=0.008). CONCLUSIONS: The incidence of NJI in Iceland has remained stable. The proportion of iatrogenic infections is high, especially among young adults, with an increase seen in postarthroscopic infections when compared with the previous decade. Although rare, NJI following arthroscopy can be a devastating complication, with significant morbidity and these results, therefore, emphasise the need for firm indications when arthroscopic treatment is considered.

Mammalian hybrid pre-autophagosomal structure HyPAS generates autophagosomes.

The biogenesis of mammalian autophagosomes remains to be fully defined. Here, we used cellular and in vitro membrane fusion analyses to show that autophagosomes are formed from a hitherto unappreciated hybrid membrane compartment. The autophagic precursors emerge through fusion of FIP200 vesicles, derived from the cis-Golgi, with endosomally derived ATG16L1 membranes to generate a hybrid pre-autophagosomal structure, HyPAS. A previously unrecognized apparatus defined here controls HyPAS biogenesis and mammalian autophagosomal precursor membranes. HyPAS can be modulated by pharmacological agents whereas its formation is inhibited upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or by expression of SARS-CoV-2 nsp6. These findings reveal the origin of mammalian autophagosomal membranes, which emerge via convergence of secretory and endosomal pathways, and show that this process is targeted by microbial factors such as coronaviral membrane-modulating proteins.

p62/SQSTM1-droplet serves as a platform for autophagosome formation and anti-oxidative stress response.

Autophagy contributes to the selective degradation of liquid droplets, including the P-Granule, Ape1-complex and p62/SQSTM1-body, although the molecular mechanisms and physiological relevance of selective degradation remain unclear. In this report, we describe the properties of endogenous p62-bodies, the effect of autophagosome biogenesis on these bodies, and the in vivo significance of their turnover. p62-bodies are low-liquidity gels containing ubiquitin and core autophagy-related proteins. Multiple autophagosomes form on the p62-gels, and the interaction of autophagosome-localizing Atg8-proteins with p62 directs autophagosome formation toward the p62-gel. Keap1 also reversibly translocates to the p62-gels in a p62-binding dependent fashion to activate the transcription factor Nrf2. Mice deficient for Atg8-interaction-dependent selective autophagy show that impaired turnover of p62-gels leads to Nrf2 hyperactivation in vivo. These results indicate that p62-gels are not simple substrates for autophagy but serve as platforms for both autophagosome formation and anti-oxidative stress.

[First case of COVID-19 treated with tocilizumab in Iceland]. / Fyrsta meðferð með tocilizumab við COVID-19 hérlendis - sjúkratilfelli

A gentleman in his early fifties became ill with flu-like symptoms after vacationing abroad and was diagnosed with COVID-19 after returning to Iceland. A few days later he was admitted to the University Hospital, Landspitali, due to worsening respiratory symptoms and severe fatigue. A computed tomography scan of lthe lungs showed diffuse bilateral consolidations and ground glass changes. He developed respiratory failure and was transferred to the intensive care unit where he received further treatment, including tocilizumab (IL-6 receptor inhibitor). He subsequently showed clinical improvement and did not require endotracheal intubation.

Selective Autophagy of Mitochondria on a Ubiquitin-Endoplasmic-Reticulum Platform.

The dynamics and coordination between autophagy machinery and selective receptors during mitophagy are unknown. Also unknown is whether mitophagy depends on pre-existing membranes or is triggered on the surface of damaged mitochondria. Using a ubiquitin-dependent mitophagy inducer, the lactone ivermectin, we have combined genetic and imaging experiments to address these questions. Ubiquitination of mitochondrial fragments is required the earliest, followed by auto-phosphorylation of TBK1. Next, early essential autophagy proteins FIP200 and ATG13 act at different steps, whereas ULK1 and ULK2 are dispensable. Receptors act temporally and mechanistically upstream of ATG13 but downstream of FIP200. The VPS34 complex functions at the omegasome step. ATG13 and optineurin target mitochondria in a discontinuous oscillatory way, suggesting multiple initiation events. Targeted ubiquitinated mitochondria are cradled by endoplasmic reticulum (ER) strands even without functional autophagy machinery and mitophagy adaptors. We propose that damaged mitochondria are ubiquitinated and dynamically encased in ER strands, providing platforms for formation of the mitophagosomes.

Correlative Light and Electron Microscopy of Autophagosomes.

Live-cell imaging has been widely used to study autophagosome biogenesis and maturation. When combined with correlative electron microscopy, this approach can be extended to reveal ultrastructural details in three dimensions. The resolution of electron microscopy is needed when membrane contact sites and tubular connections between organelles are studied.

Subcellular localization and stability of MITF are modulated by the bHLH-Zip domain.

Microphthalmia-associated transcription factor (MITF) is a member of the basic helix-loop-helix leucine zipper (bHLH-Zip) family and functions as the master regulator of the melanocytic lineage. MITF-M is the predominant isoform expressed in melanocytes and melanoma cells, and, unlike other MITF isoforms, it is constitutively nuclear. Mutational analysis revealed three karyophilic signals in the bHLH-Zip domain of MITF-M, spanning residues 197-206, 214-217, and 255-265. Structural characterization of the MITF protein showed that basic residues within these signals are exposed for interactions in the absence of DNA. Moreover, our data indicate that neither DNA binding nor dimerization of MITF-M are required for its nuclear localization. Finally, dimerization-deficient MITF-M mutants exhibited a significantly reduced stability in melanoma cells when compared to the wild-type protein. Taken together, we have shown that, in addition to its well-established role in DNA binding and dimer formation, the bHLH-Zip domain of MITF modulates the transcription factor's subcellular localization and stability.

Regional differences in mortality, hospital discharges and primary care contacts for cardiovascular disease.

AIMS: Surveillance of geographical variations in cardiovascular health is important in order to achieve the objectives of reducing regional health disparities. We aimed to explore differences in cardiovascular disease (CVD) mortality and prevalence of CVD diagnoses made in primary and in-patient care, as well as risk factor distribution by geographic regions (urban/rural) in Iceland. METHODS: From nationwide health registers, we obtained data on CVD mortalities ( N = 7113), primary healthcare CVD contacts ( N = 58,246) and hospital CVD discharges ( N = 14,039), as well as data on CVD risk factors from a national health survey ( N = 5909; response rate 60.3%). Age-standardised annual mortality, primary healthcare contact and hospital discharge rates due to CVD were calculated per 100,000 population inside (urban) and outside (rural) the Capital Area (CA). Logistic regression was used to explore regional differences in CVD risk factors. RESULTS: We observed slightly higher total CVD mortality rates among women outside compared to inside the CA (Standardised Rate Ratio (SRR) 1.06 (95% confidence interval (CI) 1.05-1.07)), particularly due to atrial fibrillation (SRR 1.47 (95% CI 1.46-1.48)), heart failure (SRR 1.29 (95% CI 1.27-1.31)) and ischemic heart disease (SRR 1.11 (95% CI 1.10-1.12)), while reduced mortality risk for cerebrovascular disease (SRR 0.81 (95% CI 0.80-0.83)). The rates of hospital discharges and primary care contacts for these diseases, as well as prevalence of several modifiable risk factors, were generally higher outside the CA, particularly among women. CONCLUSIONS: The higher prevalence of modifiable risk factors and CVD in rural areas, especially among women, calls for refined treatment and health-promoting efforts in rural areas.

[Malaria in Iceland, a rare but looming threat for travelers].

INTRODUCTION: Malaria is one of the most common causes of preventable deaths in the developing countries, especially among children. A previous study of imported malaria in Iceland during 1980-1997 identified 15 confirmed cases. The objective of this retrospective study was to update epidemiological data on malaria in Iceland, 1998-2014. MATERIALS AND METHODS: The inclusion criteria were a positive thick or thin blood smear for malaria parasites at the Dept. of Microbiology at Landspitali University Hospital, which serves as a referral laboratory for malaria diagnosis in the country. Medical records of confirmed cases, nationwide sales data for antimalarial agents and international travel of Icelanders were reviewed. RESULTS: Thirty-one cases of malaria were confirmed in Iceland during 1998-2014, 1.8 cases/year on average, a rate of 0.6 cases/100.000 inhabitants/year. The rate was 0.3/100.000 inhabitants/year in the previous study 1980-1997 (p=0.056). Plasmodium falciparum was identified in 71% of cases, P. vivax in 16%, P. ovale and P. malariae in 7% each. Only 2 patients (7%) had used chemoprophylaxis prior to diagnosis. Two patients needed intensive care, but no fatalities were documented. One patient had a relapse. The most common agent used for treatment was atovaquone with proguanil, however annual sales figures plateaued during 2010-2014 despite a significant increase in foreign travel by Icelanders during the same period. CONCLUSION: The detection rate for malaria in Iceland showed a tendency for increase between study periods while a slight decrease was noted in the neighbouring countries at the same time. The importance of antimalarial chemoprophylaxis and other preventive measures among Icelandic travellers to endemic areas needs to be emphasized. KEY WORDS: malaria, epidemiology, complications, treatment, travelers' health. Correspondence: Magnús Gottfredsson, magnusgo@landspitali.is.

Maternal geographic residence, local health service supply and birth outcomes.

OBJECTIVE: To describe pregnancy complications, mode of delivery and neonatal outcomes by mother's residence. DESIGN: Register-based cohort study. SETTING: Geographical regions of Iceland. POPULATION: Live singleton births from 1 January 2000 to 31 December 2009 (n = 40 982) and stillbirths ≥22 weeks or weighing ≥500 g (n = 145). METHODS: Logistic regression was used to explore differences in outcomes by area of residence while controlling for potential confounders. Maternal residence was classified according to distance from Capital Area and availability of local health services. MAIN OUTCOME MEASURES: Preterm birth, low birthweight, perinatal death, gestational diabetes and hypertension. RESULTS: Of the 40 982 infants of the study population 26 255 (64.1%) were born to mothers residing in the Capital Area and 14 727 (35.9%) to mothers living outside the Capital Area. Infants outside the Capital Area were more likely to have been delivered by cesarean section (adjusted odds ratio 1.28; 95% CI 1.21-1.36). A lower prevalence of gestational diabetes (adjusted odds ratio 0.68; 95% CI 0.59-0.78), hypertension (adjusted odds ratio 0.82; 95% CI 0.71-0.94) as well as congenital malformations (adjusted odds ratio 0.55; 95% CI 0.48-0.63) was observed outside the Capital Area. We observed neither differences in mean birthweight, gestation length nor rate of preterm birth or low birthweight across Capital Area and non-Capital Area. The odds of perinatal deaths were significantly higher (adjusted odds ratio 1.87; 95% CI 1.18-2.95) outside the Capital Area in the second half of the study period. CONCLUSION: Lower prevalence of gestational diabetes and hypertension outside the Capital Area may be an indication of underreporting and/or lower diagnostic activity.

Herpes simplex encephalitis in Iceland 1987-2011.

Herpes simplex encephalitis (HSE) is a serious disease with 10-20% mortality and high rate of neuropsychiatric sequelae. This study is a long-term, nationwide study in a single country, Iceland. Clinical data were obtained from patient records and from DNA PCR and antibody assays of CSF. Diagnosis of HSE was classified as definite, possible or rejected based on symptoms, as well as virological, laboratory and brain imaging criteria. A total of 30 definite cases of HSE were identified during the 25 year period 1987-2011 corresponding to incidence of 4.3 cases/106 inhabitants/year. Males were 57% of all patients, median age 50 years (range, 0-85). Fever (97%), cognitive deficits (79%), impaired consciousness (79% with GCS < 13), headache (55%) and seizures (55%) were the most common symptoms. Brain lesions were found in 24 patients (80%) by MRI or CT. All patients received intravenous acyclovir for a mean duration of 20 days. Three patients (10%) died within one year and 21/28 pts (75%) had a Karnofsky performance score of <70% with memory loss (59%), dysphasia (44%), frontal symptoms (44%) and seizures (30%) as the most frequent sequelae. Mean delay from onset of symptoms to treatment was 6 days; this was associated with adverse outcome. In conclusion, the incidence of `HSE is higher than recently reported in a national registry study from Sweden. Despite advances in rapid diagnosis and availability of treatment of HSE, approximately three of every four patients die or are left with serious neurological impairment.

Poorer self-rated health in residential areas with limited healthcare supply.

AIMS: The aim of this study was to explore differences in self-rated health and physician-diagnosed disease across geographical regions in Iceland to better understand regional requirements for health services. METHODS: Data on self-rated health and diagnosed disease from a 2007 national health survey (n=5909; response rate 60.3%) across geographic regions were analysed. Area of residence was classified according to distance from the Capital Area (CA) and availability of local health services. We used regression models to calculate crude and multivariable adjusted odds ratios (aOR) and corresponding 95% confidence intervals (95% CI) of self-rated health and diagnosed diseases by area of residence. Models were adjusted for age, gender, education, civil status, and income. RESULTS: Residents in rural areas with no local health service supply rated their physical health worse than residents of areas with diverse supply of specialised services (aOR 1.40, 95% CI 1.21-1.61). Residents outside the CA rate both their physical (aOR 1.35, 95% CI 1.23-1.50) and mental (aOR 1.17, 95% CI 1.06-1.30) health worse than residents in the CA. In contrast, we observed a lower prevalence of several diagnosed chronic diseases, including cancers (aOR 0.78, 95% CI 0.60-0.99) and cardiovascular disease (aOR 0.77, 95% CI 0.62-0.95) outside the CA. CONCLUSIONS: These findings from a national survey of almost 6000 Icelanders indicate that self-rated health is related to regional healthcare supply. The findings have implications for national planning of health services aiming at equality both in health and access to health services.

Bacterial meningitis in adults in Iceland, 1995-2010.

INTRODUCTION: Bacterial meningitis is a serious disease with a mortality rate of 15-20% in adults. We conducted a population-based study of bacterial meningitis in adults (≥ 16 y) in Iceland, 1995-2010. METHODS: Cases were identified based on positive bacterial cultures from cerebrospinal fluid (CSF) and/or the ICD codes for bacterial meningitis. Medical charts were reviewed and outcomes were assessed using the national population registry. The study period was divided into 2 equal parts, 1995-2002 and 2003-2010, before and after implementation of routine childhood vaccination against serogroup C meningococci, respectively. RESULTS: In total, 111 episodes occurred in 110 individuals. The most common causative organisms were Neisseria meningitidis (41%) and Streptococcus pneumoniae (30%). Only 30% of the patients presented with the classical symptom triad of fever, neck stiffness, and an altered mental status. The overall incidence was 3.2/100,000 inhabitants/y, and dropped significantly between the first and second halves of the study (p = 0.03). This drop was due to a reduced incidence of N. meningitidis meningitis: 34 and 12 cases in the first and second periods, respectively (p = 0.006). The incidence of meningitis caused by S. pneumoniae remained unchanged. The case fatality rates were 18% and 13% in the first and second halves of the study, respectively (difference not significant). CONCLUSIONS: The incidence of bacterial meningitis has decreased since the implementation of meningococcal C vaccination in 2002. However, the case fatality rate has remained unchanged.