Lipid and Nucleocapsid N-Protein Accumulation in COVID-19 Patient Lung and Infected Cells.

The pandemic of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global outbreak and prompted an enormous research effort. Still, the subcellular localization of the coronavirus in lungs of COVID-19 patients is not well understood. Here, the localization of the SARS-CoV-2 proteins is studied in postmortem lung material of COVID-19 patients and in SARS-CoV-2-infected Vero cells, processed identically. Correlative light and electron microscopy on semithick cryo-sections demonstrated induction of electron-lucent, lipid-filled compartments after SARS-CoV-2 infection in both lung and cell cultures. In lung tissue, the nonstructural protein 4 and the stable nucleocapsid N-protein were detected on these novel lipid-filled compartments. The induction of such lipid-filled compartments and the localization of the viral proteins in lung of patients with fatal COVID-19 may explain the extensive inflammatory response and provide a new hallmark for SARS-CoV-2 infection at the final, fatal stage of infection. IMPORTANCE Visualization of the subcellular localization of SARS-CoV-2 proteins in lung patient material of COVID-19 patients is important for the understanding of this new virus. We detected viral proteins in the context of the ultrastructure of infected cells and tissues and discovered that some viral proteins accumulate in novel, lipid-filled compartments. These structures are induced in Vero cells but, more importantly, also in lung of patients with COVID-19. We have characterized these lipid-filled compartments and determined that this is a novel, virus-induced structure. Immunogold labeling demonstrated that cellular markers, such as CD63 and lipid droplet marker PLIN-2, are absent. Colocalization of lipid-filled compartments with the stable N-protein and nonstructural protein 4 in lung of the last stages of COVID-19 indicates that these compartments play a key role in the devastating immune response that SARS-CoV-2 infections provoke.

Impaired myelin production due to an intrinsic failure of oligodendrocytes in mTORpathies.

AIMS: We aim to evaluate if the myelin pathology observed in epilepsy-associated focal cortical dysplasia type 2B (FCD2B) and-histologically indistinguishable-cortical tubers of tuberous sclerosis complex (TSC) is primarily related to the underlying malformation or constitutes a secondary phenomenon due to the toxic microenvironment created by epileptic seizures. We also aim to investigate the possible beneficial effect of the mTOR pathway regulator everolimus on white matter pathology. METHODS: Primary mixed glial cell cultures derived from epilepsy surgery specimens of one TSC and seven FCD2B patients were grown on polycaprolactone fibre matrices and analysed using immunofluorescence and electron microscopy. Unaffected white matter from three age-matched epilepsy patients with mild malformations of cortical development (mMCD) and one with FCD3D served as controls. Additionally, TSC2 knock-out was performed using an oligodendroglial cell line. Myelination capacities of nanofibre grown cells in an inflammatory environment after mTOR-inhibitor treatment with everolimus were further investigated. RESULTS: Reduced oligodendroglial turnover, directly related to a lower myelin content was found in the patients' primary cells. In our culture model of myelination dynamics, primary cells grown under 'inflammatory condition' showed decreased myelination, that was repaired by treatment with everolimus. CONCLUSIONS: Results obtained in patient-derived primary oligodendroglial and TSC2 knock-out cells suggest that maturation of oligodendroglia and production of a proper myelin sheath seem to be impaired as a result of mTOR pathway disturbance. Hence, oligodendroglial pathology may reflect a more direct effect of the abnormal genetic programme rather than to be an inactive bystander of chronic epilepsy.

GCase and LIMP2 Abnormalities in the Liver of Niemann Pick Type C Mice.

The lysosomal storage disease Niemann-Pick type C (NPC) is caused by impaired cholesterol efflux from lysosomes, which is accompanied by secondary lysosomal accumulation of sphingomyelin and glucosylceramide (GlcCer). Similar to Gaucher disease (GD), patients deficient in glucocerebrosidase (GCase) degrading GlcCer, NPC patients show an elevated glucosylsphingosine and glucosylated cholesterol. In livers of mice lacking the lysosomal cholesterol efflux transporter NPC1, we investigated the expression of established biomarkers of lipid-laden macrophages of GD patients, their GCase status, and content on the cytosol facing glucosylceramidase GBA2 and lysosomal integral membrane protein type B (LIMP2), a transporter of newly formed GCase to lysosomes. Livers of 80-week-old Npc1-/- mice showed a partially reduced GCase protein and enzymatic activity. In contrast, GBA2 levels tended to be reciprocally increased with the GCase deficiency. In Npc1-/- liver, increased expression of lysosomal enzymes (cathepsin D, acid ceramidase) was observed as well as increased markers of lipid-stressed macrophages (GPNMB and galectin-3). Immunohistochemistry showed that the latter markers are expressed by lipid laden Kupffer cells. Earlier reported increase of LIMP2 in Npc1-/- liver was confirmed. Unexpectedly, immunohistochemistry showed that LIMP2 is particularly overexpressed in the hepatocytes of the Npc1-/- liver. LIMP2 in these hepatocytes seems not to only localize to (endo)lysosomes. The recent recognition that LIMP2 harbors a cholesterol channel prompts the speculation that LIMP2 in Npc1-/- hepatocytes might mediate export of cholesterol into the bile and thus protects the hepatocytes.

Investigating the emergence of sex differences in jealousy responses in a large community sample from an evolutionary perspective.

Sex differences in jealousy responses to sexual and emotional infidelity are robust in samples of heterosexual adults, especially in more gender egalitarian nations. However, investigations of when and how these differences develop have been scant. We applied two forced choice infidelity scenarios in a large community sample of high school students (age 16-19, N = 1266). In line with previous findings on adults using the forced choice paradigm, adolescent males found the sexual aspect of imagined infidelity more distressing than adolescent females did. Nevertheless, there was no effect of age on the jealousy responses, and age did not moderate the sex difference. There were neither any effects of three covariates (having had first sexual intercourse, being in a committed romantic relationship, and sociosexuality), neither as markers of pubertal maturation nor as psychosocial environmental stimuli. Future research needs to investigate even younger samples in order to specify at what age the sex difference in jealousy responses emerges.

Control of septum thickness by the curvature of SepF polymers.

Gram-positive bacteria divide by forming a thick cross wall. How the thickness of this septal wall is controlled is unknown. In this type of bacteria, the key cell division protein FtsZ is anchored to the cell membrane by two proteins, FtsA and/or SepF. We have isolated SepF homologs from different bacterial species and found that they all polymerize into large protein rings with diameters varying from 19 to 44 nm. Interestingly, these values correlated well with the thickness of their septa. To test whether ring diameter determines septal thickness, we tried to construct different SepF chimeras with the purpose to manipulate the diameter of the SepF protein ring. This was indeed possible and confirmed that the conserved core domain of SepF regulates ring diameter. Importantly, when SepF chimeras with different diameters were expressed in the bacterial host Bacillus subtilis, the thickness of its septa changed accordingly. These results strongly support a model in which septal thickness is controlled by curved molecular clamps formed by SepF polymers attached to the leading edge of nascent septa. This also implies that the intrinsic shape of a protein polymer can function as a mold to shape the cell wall.

Platelet inhibition by ticagrelor is protective against diabetic nephropathy in mice.

Diabetic nephropathy (DN) is a major complication of diabetes and is associated with high risk for cardiovascular mortality, which is partially related to elevated platelet activity. Platelets are also active players in inflammation and fibrosis. In this study, we examine the effect of ticagrelor-induced platelet inhibition on the development of DN. DN was induced by unilateral nephrectomy followed by streptozotocin injections for 5 days. Mice received ticagrelor (300 mg/kg) or vehicle every other day, for 16 weeks. Experimental groups: non-diabetic control, diabetic control, non-diabetic ticagrelor, and diabetic ticagrelor. Ticagrelor treatment in diabetic mice lowered urinary albumin excretion, it prevented diabetes-induced mesangial matrix expansion, podocyte effacement, and glomerular endothelial cell injury, which includes loss of endothelial fenestrations, ICAM-1 expression, and PECAM expression. In addition, ticagrelor treatment prevented collagen IV deposition and macrophage infiltration in the tubulointerstitium and these diabetic mice showed lower systemic and tubular inflammation and tubular apoptosis. This tubular protection is likely to be a result of protection to the glomerular endothelium by ticagrelor, which reduces albuminuria and albumin toxicity to the tubules and reduced tubular and interstitial inflammation and fibrosis. In conclusion, ticagrelor-induced platelet inhibition protects against renal injury in diabetic mice, likely by protecting the glomerular endothelial cells.

Three-dimensional assessment of facial morphology in children and adolescents with juvenile idiopathic arthritis and moderate to severe TMJ involvement using 3D surface scans.

OBJECTIVE: This study aims to (1) assess the facial morphology in juvenile idiopathic arthritis (JIA) subjects with moderate to severe temporomandibular joint (TMJ) involvement using 3D surface scans and (2) compare the facial morphology in these subjects to that in JIA subjects without TMJ involvement. METHODS: Sixty JIA subjects were included and grouped as follows: group 1 (no involvement group), JIA without TMJ involvement; Group 2 (unilateral group), JIA with moderate to severe unilateral TMJ involvement; and group 3 (bilateral group), JIA with bilateral TMJ involvement. Standard orientation of all surfaces was accomplished. The means and variabilities of facial morphology in groups 2 and 3 were assessed and compared with those of group 1 in three dimensions, respectively. RESULTS: Group 2 (unilateral group) exhibited a more retruded and wider chin, shorter mandibular height, and more prominent cheek (2, 2, 5, and 2 mm, on average, respectively) on the affected side and a more retruded and narrower chin and more prominent malar region (4, 3, and 2 mm, on average, respectively) on the unaffected side compared with group 1 (no involvement group) (p < 0.05). Group 3 (bilateral group) exhibited a more retruded chin, shorter mandibular height, more prominent upper cheeks, and narrower perioral region (5, 5, 3, and 2 mm, respectively) compared with group 1 (no involvement group) (p < 0.05). CONCLUSIONS: In JIA subjects with moderate to severe unilateral or bilateral TMJ involvement, the affected side(s) revealed similar facial dysmorphology with reduced mandibular height, chin retrusion, and prominent upper cheek. CLINICAL RELEVANCE: Three-dimensional surface scans can be a non-ionizing indicator of signs of TMJ involvement in JIA subjects.

Author Correction: Combining streptozotocin and unilateral nephrectomy is an effective method for inducing experimental diabetic nephropathy in the 'resistant' C57Bl/6J mouse strain.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

[Workup and treatment of Eustachian tube dysfunction in adults].

Symptoms of Eustachian tube dysfunction are frequent and multiple. Therefore, clear definitions and diagnostic criteria are important in order to achieve appropriate patient flow. So far, there has been a lack of consensus on this subject, but the proposed definitions and diagnostic criteria in this review may aid to achieve this. Tubomanometry is a diagnostic tool to evaluate Eustachian tube function, and balloon dilation of the Eustachian tube can be a helpful treatment in patients with Eustachian tube dysfunction.

Combining streptozotocin and unilateral nephrectomy is an effective method for inducing experimental diabetic nephropathy in the 'resistant' C57Bl/6J mouse strain.

Diabetic nephropathy (DN) is the leading cause of chronic kidney disease. Animal models are essential tools for designing new strategies to prevent DN. C57Bl/6 (B6) mice are widely used for transgenic mouse models, but are relatively resistant to DN. This study aims to identify the most effective method to induce DN in a type 1 (T1D) and a type 2 diabetes (T2D) model in B6 mice. For T1D-induced DN, mice were fed a control diet, and randomised to streptozotocin (STZ) alone, STZ+unilateral nephrectomy (UNx), or vehicle/sham. For T2D-induced DN, mice were fed a western (high fat) diet, and randomised to either STZ alone, STZ+UNx, UNx alone, or vehicle/sham. Mice subjected to a control diet with STZ +UNx developed albuminuria, glomerular lesions, thickening of the glomerular basement membrane, and tubular injury. Mice on control diet and STZ developed only mild renal lesions. Furthermore, kidneys from mice on a western diet were hardly affected by diabetes, UNx or the combination. We conclude that STZ combined with UNx is the most effective model to induce T1D-induced DN in B6 mice. In our hands, combining western diet and STZ treatment with or without UNx did not result in a T2D-induced DN model in B6 mice.

Deletion of NLRX1 increases fatty acid metabolism and prevents diet-induced hepatic steatosis and metabolic syndrome.

NOD-like receptor (NLR)X1 (NLRX1) is an ubiquitously expressed inflammasome-independent NLR that is uniquely localized in mitochondria with as yet unknown effects on metabolic diseases. Here, we report that NLRX1 is essential in regulating cellular metabolism in non-immune parenchymal hepatocytes by decreasing mitochondrial fatty acid-dependent oxidative phosphorylation (OXPHOS) and promoting glycolysis. NLRX1 loss in mice has a profound impact on the prevention of diet-induced metabolic syndrome parameters, non-alcoholic fatty liver disease (NAFLD) progression, and renal dysfunction. Despite enhanced caloric intake, NLRX1 deletion in mice fed a western diet (WD) results in protection from liver steatosis, hepatic fibrosis, obesity, insulin resistance, glycosuria and kidney dysfunction parameters independent from inflammation. While mitochondrial content was equal, NLRX1 loss in hepatocytes leads to increased fatty acid oxidation and decreased steatosis. In contrast, glycolysis was decreased in NLRX1-deficient cells versus controls. Thus, although first implicated in immune regulation, we show that NLRX1 function extends to the control of hepatocyte energy metabolism via the restriction of mitochondrial fatty acid-dependent OXPHOS and enhancement of glycolysis. As such NLRX1 may be an attractive novel therapeutic target for NAFLD and metabolic syndrome.

Children's Doctor Games and Nudity at Danish Childcare Institutions.

This article presents the first Danish study of the acceptance of children's nudity and sexuality at Danish childcare institutions. The study revealed an important cultural shift in the attitude toward children's nudity and sexual games, the so-called doctor games. Although these were quite accepted at Danish childcare institutions until the beginning of this century, the study showed that new, pervasive regulations had been established to control the child's body and its sexuality. A new discourse revealed that fear of child sexual abuse, in particular, had influenced views of children's sexual games and nudity and that, at times, the child itself was viewed as a potential threat to other children. This marks a new development in Denmark, internationally known for its broadmindedness, and this article discusses the background to this cultural shift in the institutions, and possible implications for the children.

Accuracy and precision of manual segmentation of the maxillary sinus in MR images-a method study.

OBJECTIVE: To assess the accuracy and precision of segmentation of the maxillary sinus in MR images to evaluate the potential usefulness of this modality in longitudinal studies of sinus development. METHODS: A total of 15 healthy subjects who had been both craniofacial CT and MR scanned were included and the 30 maxillary sinus volumes were evaluated using segmentation. Two of the authors did segmentation of MRI and one of these authors did double segmentation. Agreement in results between CT and MRI as well as inter- and intraexaminer errors were evaluated by statistical and three-dimensional analysis. RESULTS: The intraclass correlation coefficient for volume measurements for both method error, inter- and intraexaminer agreement were > 0.9 [maximal 95% confidence interval of 0.989-0.997, p < 0.001] and the limit of agreement for all parameters were < 5.1%. Segmentation errors were quantified in terms of overlap [Dice Coefficient (DICE) > 0.9 = excellent agreement] and border distance [95% percentile Hausdorff Distance (HD) < 2 mm = acceptable agreement]. The results were replicable and not influenced by systematic errors. CONCLUSION: We found a high accuracy and precision of manual segmentation of the maxillary sinus in MR images. The largest mean errors were found close to the orbit and the teeth. Advances in knowledge: MRI can be used for 3D models of the paranasal sinuses with equally good results as CT and allows longitudinal follow-up of sinus development.

NLRX1 dampens oxidative stress and apoptosis in tissue injury via control of mitochondrial activity.

Mitochondrial dysfunction is the most prominent source of oxidative stress in acute and chronic kidney disease. NLRX1 is a receptor of the innate immune system that is ubiquitously expressed and localized in mitochondria. We investigated whether NLRX1 may act at the interface of metabolism and innate immunity in a model of oxidative stress. Using a chimeric mouse model for renal ischemia-reperfusion injury, we found that NLRX1 protects against mortality, mitochondrial damage, and epithelial cell apoptosis in an oxidative stress-dependent fashion. We found that NLRX1 regulates oxidative phosphorylation and cell integrity, whereas loss of NLRX1 results in increased oxygen consumption, oxidative stress, and subsequently apoptosis in epithelial cells during ischemia-reperfusion injury. In line, we found that NLRX1 expression in human kidneys decreased during acute renal ischemic injury and acute cellular rejection. Although first implicated in immune regulation, we propose that NLRX1 function extends to the control of mitochondrial activity and prevention of oxidative stress and apoptosis in tissue injury.

Facial morphology in children and adolescents with juvenile idiopathic arthritis and moderate to severe temporomandibular joint involvement.

INTRODUCTION: The aims of this study were to (1) assess lateral facial morphology in children and adolescents with juvenile idiopathic arthritis and moderate to severe temporomandibular joint (TMJ) involvement, (2) compare the lateral facial morphology of these subjects with and without TMJ involvement using cephalograms and 3-dimensional (3D) facial photographs, and (3) compare and correlate the results of the 3D photographic and cephalometric analyses. METHODS: Sixty patients with juvenile idiopathic arthritis were included and grouped as follows: group 1, juvenile idiopathic arthritis patients without TMJ involvement; group 2, juvenile idiopathic arthritis patients with moderate to severe unilateral TMJ involvement; and group 3, juvenile idiopathic arthritis patients with moderate to severe bilateral TMJ involvement. Lateral cephalograms were used to assess and compare lateral facial morphologies between the groups. Lateral projections of oriented 3D photographs were superimposed on the lateral cephalograms. The results of the lateral 3D photographic analysis were correlated with those of lateral cephalometric analysis. RESULTS: Group 3 showed the most severe growth disturbances, including more retrognathic mandible and retruded chin, steep occlusal and mandibular planes, and more hyperdivergent type (P <0.01). Group 2 showed similar growth disturbances, but to a lesser extent than did group 3. Photographic variables were significantly correlated with the soft tissue and skeletal variables of cephalograms (0.5 < r < 0.9; P <0.001). CONCLUSIONS: Subjects with juvenile idiopathic arthritis and unilateral or bilateral moderate to severe TMJ involvement had significant growth disturbances. Early intervention is recommended for these patients to prevent unfavorable facial development. Furthermore, with proper orientation, 3D photographs can be used as an alternative to conventional lateral cephalograms and 2-dimensional photographs.

Facial Asymmetry in Children with Unicoronal Synostosis Who Have Undergone Craniofacial Reconstruction in Infancy.

OBJECTIVE: Quantitatively assess 3D spatially detailed soft-tissue facial asymmetry in children who had undergone craniofacial reconstruction for Unicoronal Synostosis (UCS), and compare the facial asymmetry to control patients. It was hypothesized that there would be no significant differences in the facial asymmetry between the groups. DESIGN: Clinical, retrospective follow-up study. Methodological study. SETTING: Primary care center. PATIENTS/PARTICIPANTS: Twenty-two children with UCS were selected after review of records. INCLUSION CRITERIA: isolated UCS; surgically treated for UCS within the first 19 months of life, without secondary reconstruction; and DNA analysis for the Muenke mutation. An age- and sex-matched control group was employed. INTERVENTIONS: The UCS group had undergone bilateral craniotomy of the frontal bone with unilateral supraorbital rim advancement. MAIN OUTCOME MEASURE(S): Using 3D surface scanning, a detailed map of 3D asymmetry presenting the amount of asymmetry in the sagittal, vertical, and transverse directions was calculated for six facial subregions. RESULTS: The facial asymmetry in the UCS group was significantly larger than in the control group for all regions, to the largest extent in the sagittal direction (level of significance: 5%). The regions with the most pronounced asymmetry were cheeks (mean: 5.45 mm; SD: 1.83 mm), forehead (mean: 5.00 mm; SD: 1.57 mm), and eyes (mean: 4.26 mm; SD: 1.44 mm). CONCLUSIONS: Ninety percent of the UCS patients in the study had significant facial asymmetry throughout the facial area. The study demonstrates a methodology of facial asymmetry quantification well suited for soft-tissue surgical outcome evaluations and long-term follow-up studies in patients with craniofacial anomalies.

The effects of acoustical refurbishment of classrooms on teachers' perceived noise exposure and noise-related health symptoms.

OBJECTIVES: To investigate whether acoustical refurbishment of classrooms for elementary and lower secondary grade pupils affected teachers' perceived noise exposure during teaching and noise-related health symptoms. METHODS: Two schools (A and B) with a total of 102 teachers were subjected to an acoustical intervention. Accordingly, 36 classrooms (20 and 16 in school A and school B, respectively) were acoustically refurbished and 31 classrooms (16 and 15 in school A and school B, respectively) were not changed. Thirteen classrooms in school A were interim "sham" refurbished. Control measurements of RT and activity sound levels were measured before and after refurbishment. Data on perceived noise exposure, disturbance attributed to different noise sources, voice symptoms, and fatigue after work were collected over a year in a total of six consecutive questionnaires. RESULTS: Refurbished classrooms were associated with lower perceived noise exposure and lower ratings of disturbance attributed to noise from equipment in the class compared with unrefurbished classrooms. No associations between the classroom refurbishment and health symptoms were observed. Before acoustical refurbishment, the mean classroom reverberation time was 0.68 (school A) and 0.57 (school B) and 0.55 s in sham refurbished classrooms. After refurbishment, the RT was approximately 0.4 s in both schools. Activity sound level measurements confirmed that the intervention had reduced the equivalent sound levels during lessons with circa 2 dB(A) in both schools. CONCLUSION: The acoustical refurbishment was associated with a reduction in classroom reverberation time and activity sound levels in both schools. The acoustical refurbishment was associated with a reduction in the teachers' perceived noise exposure, and reports of disturbance from equipment in the classroom decreased. There was no significant effect of the refurbishment on the teachers' voice symptoms or fatigue after work.

Hepatic NAD salvage pathway is enhanced in mice on a high-fat diet.

Nicotinamide phosphoribosyltransferase (Nampt) is the rate-limiting enzyme for NAD salvage and the abundance of Nampt has been shown to be altered in non-alcoholic fatty liver disease. It is, however, unknown how hepatic Nampt is regulated in response to accumulation of lipids in the liver of mice fed a high-fat diet (HFD). HFD mice gained more weight, stored more hepatic lipids and had an impaired glucose tolerance compared with control mice. NAD levels as well as Nampt mRNA expression, protein abundance and activity were significantly increased in HFD mice. Enhanced NAD levels were associated with deacetylation of p53 and Nfκb indicating increased activation of Sirt1. Despite impaired glucose tolerance and increased hepatic lipid levels in HFD mice, NAD metabolism was significantly enhanced. Thus, improved NAD metabolism may be a compensatory mechanism to protect against negative impact of hepatic lipid accumulation.