Time-resolved cryo-electron microscopy of vitrified muscular components

Biological objects may be arrested in defined stages of their activity by fast freezing and may then be structurally examined. If the time between the start of activity and freezing is controlled, structural rearrangements due to biological function can be determined. Cryo-electron microscopy shows great potential for the study of such time-dependent phenomena. This study examines the actin polymerization process using cryo-electron microscopy of vitrified specimens. Actin filaments are shown to undergo a structural change during polymerization. In the early stages of the polymerization process (t < 2 min), filaments exhibit a pronounced structural variation and frequently show a central low-density area. In the later stages of the polymerization, F-actin-ADP filaments have a more uniform appearance and rarely display a central low-density area. These findings, analysed on the basis of a previously proposed polymerization model, suggest that polymerization intermediates (F-actin-ATP and more probably F-actin-ADP-P_i) and filaments at steady state (F-actin-ADP) have different structures. To investigate the physiological relevance of these results at the cellular level, the potential of cryo-substitution in preserving the structure of muscular fibre was assessed. Optical diffraction patterns of relaxed and contracted frog cutaneous muscle are similar to the corresponding X-ray diffraction patterns. The resolution of the images extends to about 7 nm. These results show that dynamic study of muscle contraction is possible using cryo-substitution.     

Estimation of diurnal air temperature using MSG SEVIRI data in West Africa

Spatially distributed air temperature data with high temporal resolution are desired for several modeling applications. By exploiting the thermal split window channels in combination with the red and near infrared channels of the geostationary MSG SEVIRI sensor, multiple daily air temperature estimates can be achieved using the contextual temperature-vegetation index method. Air temperature was estimated for 436 image acquisitions during the 2005 rainy season over West Africa and evaluated against in situ data from a field test site in Dahra, Northern Senegal. The methodology was adjusted using data from the test site resulting in RMSE = 2.55 K, MBE = − 0.30 K and R² = 0.63 for the estimated versus observed air temperatures. A spatial validation of the method using 12 synoptic weather stations from Senegal and Mali within the Senegal River basin resulted in overall values of RMSE = 2.96 K, MBE = − 1.11 K and R² = 0.68. The daytime temperature curve is interpolated using a sine function based on the multiple daily air temperature estimates from the SEVIRI data. These estimates (covering the 8:00-20:00 UCT time window) were in good agreement with observed values with RMSE = 2.99 K, MBE = − 0.70 K and R² = 0.64. The temperature-vegetation index method was applied as a moving window technique to produce distributed maps of air temperature with 15 min intervals and 3 km spatial resolution for application in a distributed hydrological model.     

Targeting Fibronectin to Overcome Remyelination Failure in Multiple Sclerosis: The Need for Brain- and Lesion-Targeted Drug Delivery

Multiple sclerosis (MS) is a neuroinflammatory and neurodegenerative disease with unknown etiology that can be characterized by the presence of demyelinated lesions. Prevailing treatment protocols in MS rely on the modulation of the inflammatory process but do not impact disease progression. Remyelination is an essential factor for both axonal survival and functional neurological recovery but is often insufficient. The extracellular matrix protein fibronectin contributes to the inhibitory environment created in MS lesions and likely plays a causative role in remyelination failure. The presence of the blood–brain barrier (BBB) hinders the delivery of remyelination therapeutics to lesions. Therefore, therapeutic interventions to normalize the pathogenic MS lesion environment need to be able to cross the BBB. In this review, we outline the multifaceted roles of fibronectin in MS pathogenesis and discuss promising therapeutic targets and agents to overcome fibronectin-mediated inhibition of remyelination. In addition, to pave the way for clinical use, we reflect on opportunities to deliver MS therapeutics to lesions through the utilization of nanomedicine and discuss strategies to deliver fibronectin-directed therapeutics across the BBB. The use of well-designed nanocarriers with appropriate surface functionalization to cross the BBB and target the lesion sites is recommended.     

Adipokines as Immune Cell Modulators in Multiple Sclerosis

Multiple sclerosis (MS), a chronic inflammatory and demyelinating disease of the central nervous system (CNS), is a major clinical and societal problem, which has a tremendous impact on the life of patients and their proxies. Current immunomodulatory and anti-inflammatory therapies prove to be relatively effective; however, they fail to concomitantly stop ongoing neurological deterioration and do not reverse acquired disability. The proportion to which genetic and environmental factors contribute to the etiology of MS is still incompletely understood; however, a recent association between MS etiology and obesity was shown, with obesity greatly increasing the risk of developing MS. An altered balance of adipokines, which are white adipose tissue (WAT) hormones, plays an important role in the low-grade chronic inflammation during obesity by their pervasive modification of local and systemic inflammation. Vice versa, inflammatory factors secreted by immune cells affect adipokine function. To explore the role of adipokines in MS pathology, we will here review the reciprocal effects of adipokines and immune cells and summarize alterations in adipokine levels in MS patient cohorts. Finally, we will discuss proof-of-concept studies demonstrating the therapeutic potential of adipokines to target both neuroinflammation and neurodegeneration processes in MS.     

Combination of the Glutaminyl Cyclase Inhibitor PQ912 (Varoglutamstat) and the Murine Monoclonal Antibody PBD-C06 (m6) Shows Additive Effects on Brain Aβ Pathology in Transgenic Mice

Compelling evidence suggests that pyroglutamate-modified Aβ (pGlu3-Aβ; AβN3pG) peptides play a pivotal role in the development and progression of Alzheimer’s disease (AD). Approaches targeting pGlu3-Aβ by glutaminyl cyclase (QC) inhibition (Varoglutamstat) or monoclonal antibodies (Donanemab) are currently in clinical development. Here, we aimed at an assessment of combination therapy of Varoglutamstat (PQ912) and a pGlu3-Aβ-specific antibody (m6) in transgenic mice. Whereas the single treatments at subtherapeutic doses show moderate (16–41%) but statistically insignificant reduction of Aβ42 and pGlu-Aβ42 in mice brain, the combination of both treatments resulted in significant reductions of Aβ by 45–65%. Evaluation of these data using the Bliss independence model revealed a combination index of ≈1, which is indicative for an additive effect of the compounds. The data are interpreted in terms of different pathways, in which the two drugs act. While PQ912 prevents the formation of pGlu3-Aβ in different compartments, the antibody is able to clear existing pGlu3-Aβ deposits. The results suggest that combination of the small molecule Varoglutamstat and a pE3Aβ-directed monoclonal antibody may allow a reduction of the individual compound doses while maintaining the therapeutic effect.     

Galactose in the Post-Weaning Diet Programs Improved Circulating Adiponectin Concentrations and Skeletal Muscle Insulin Signaling

Short-term post-weaning nutrition can result in long-lasting effects in later life. Partial replacement of glucose by galactose in the post-weaning diet showed direct effects on liver inflammation. Here, we examined this program on body weight, body composition, and insulin sensitivity at the adult age. Three-week-old female C57BL/6JRccHsd mice were fed a diet with glucose plus galactose (GAL; 16 energy% (en%) each) or a control diet with glucose (GLU; 32 en%) for three weeks, and afterward, both groups were given the same high-fat diet (HFD). After five weeks on a HFD, an oral glucose tolerance test was performed. After nine weeks on a HFD, energy metabolism was assessed by indirect calorimetry, and fasted mice were sacrificed fifteen minutes after a glucose bolus, followed by serum and tissue analyses. Body weight and body composition were not different between the post-weaning dietary groups, during the post-weaning period, or the HFD period. Glucose tolerance and energy metabolism in adulthood were not affected by the post-weaning diet. Serum adiponectin concentrations were significantly higher (p = 0.02) in GAL mice while insulin, leptin, and insulin-like growth factor 1 concentrations were not affected. Expression of Adipoq mRNA was significantly higher in gonadal white adipose tissue (gWAT; p = 0.03), while its receptors in the liver and skeletal muscles remained unaffected. Irs2 expression was significantly lower in skeletal muscles (p = 0.01), but not in gWAT or Irs1 expression (in both tissues). Gene expressions of inflammatory markers in gWAT and the liver were also not affected. Conclusively, galactose in the post-weaning diet significantly improved circulating adiponectin concentrations and reduced skeletal muscle Irs2 expression in adulthood without alterations in fat mass, glucose tolerance, and inflammation.     

Macrophages Release Extracellular Vesicles of Different Properties and Composition Following Exposure to Nanoparticles

Extracellular vesicles are membrane-bound carriers with complex cargoes, which play a major role in intercellular communication, for instance, in the context of the immune response. Macrophages are known to release extracellular vesicles in response to different stimuli, and changes in their size, number, and composition may provide important insights into the responses induced. Macrophages are also known to be highly efficient in clearing nanoparticles, when in contact with them, and in triggering the immune system. However, little is known about how the nature and composition of the vesicles released by these cells may vary upon nanoparticle exposure. In order to study this, in this work, alveolar-like macrophages were exposed to a panel of nanoparticles with varying surface and composition, including amino-modified and carboxylated polystyrene and plain silica. We previously showed that these nanoparticles induced very different responses in these cells. Here, experimental conditions were carefully tuned in order to separate the extracellular vesicles released by the macrophages several hours after exposure to sub-toxic concentrations of the same nanoparticles. After separation, different methods, including high-sensitivity flow cytometry, TEM imaging, Western blotting, and nanoparticle tracking analysis, were combined in order to characterize the extracellular vesicles. Finally, proteomics was used to determine their composition and how it varied upon exposure to the different nanoparticles. Our results show that depending on the nanoparticles’ properties. The macrophages produced extracellular vesicles of varying number, size, and protein composition. This indicates that macrophages release specific signals in response to nanoparticles and overall suggests that extracellular vesicles can reflect subtle responses to nanoparticles and nanoparticle impact on intercellular communication.     

Intravesicular Genomic DNA Enriched by Size Exclusion Chromatography Can Enhance Lung Cancer Oncogene Mutation Detection Sensitivity

Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RNA and DNA as potential templates for molecular diagnostics, e.g., in cancer genotyping. While it has been established that DNA templates appear on the outside of EVs, no consensus exists on which nucleic acid species inside small EVs (<200 nm, sEVs) are sufficiently abundant and accessible for developing genotyping protocols. We investigated this by extracting total intravesicular nucleic acid content from sEVs isolated from the conditioned cell medium of the human NCI-H1975 cell line containing the epidermal growth factor (EGFR) gene mutation T790M as a model system for non-small cell lung cancer. We observed that mainly short genomic DNA (<35–100 bp) present in the sEVs served as a template. Using qEV size exclusion chromatography (SEC), significantly lower yield and higher purity of isolated sEV fractions were obtained as compared to exoEasy membrane affinity purification and ultracentrifugation. Nevertheless, we detected the EGFR T790M mutation in the sEVs’ lumen with similar sensitivity using digital PCR. When applying SEC-based sEV separation prior to cell-free DNA extraction on spiked human plasma samples, we found significantly higher mutant allele frequencies as compared to standard cell-free DNA extraction, which in part was due to co-purification of circulating tumor DNA. We conclude that intravesicular genomic DNA can be exploited next to ctDNA to enhance EGFR T790M mutation detection sensitivity by adding a fast and easy-to-use sEV separation method, such as SEC, upstream of standard clinical cell-free DNA workflows.     

Outcome of Nissen fundoplication using intraoperative manometry in children

BACKGROUND: Intraoperative manometry is useful in performing Nissen fundoplication (NF) in children. Long-term clinical outcome information after use of this method is lacking. METHODS: A retrospective review of the outcomes of 62 consecutive NFs using intraoperative manometry was performed. The follow-up period was 3.4 years. Approximately half of the patients were neurologically normal (NN) and half were neurologically impaired (NI). All patients with gastroesophageal reflux disease (GERD) did not respond to an adequate trial of medical treatment. RESULTS: The NF was tailored to result in a twofold increase in the lower esophageal sphincter pressure (LESP) and a 75% increase in the LES length (LESL). An accelerated growth rate in 40% of "failure to thrive" (FTT) patients was demonstrated. Eighty-four percent of caregivers reported improved quality of life after NF. There was a twofold reduction in the number of hospital admissions and a sixfold reduction in total inpatient days for both NI and NN children. The early and late mortality rate was 13%, and the complication rate was similar to other series reported in the literature, with more complications occurring in NI patients. There was a 2% incidence of wrap herniation. An improvement in long-term outcomes after NF was seen in 89% of NN children and over half of NI patients. CONCLUSIONS: Intraoperative manometry is useful in standardizing the tightness of the wrap in NF. There was a low incidence of complications, dysphagia, recurrent emesis, and GERD in this series. Long-term outcomes using this technique were deemed very good based on caregivers' responses.