Basal ryanodine receptor activity suppresses autophagic flux

The inositol 1,4,5-trisphosphate receptors (IP3Rs) and intracellular Ca2+ signaling are critically involved in regulating different steps of autophagy, a lysosomal degradation pathway. The ryanodine receptors (RyR), intracellular Ca2+-release channels mainly expressed in excitable cell types including muscle and neurons, have however not yet been extensively studied in relation to autophagy. Yet, aberrant expression and excessive activity of RyRs in these tissues has been implicated in the onset of several diseases including Alzheimer’s disease, where impaired autophagy regulation contributes to the pathology. In this study, we determined whether pharmacological RyR inhibition could modulate autophagic flux in ectopic RyR-expressing models, like HEK293 cells and in cell types that endogenously express RyRs, like C2C12 myoblasts and primary hippocampal neurons. Importantly, RyR3 overexpression in HEK293 cells impaired the autophagic flux. Conversely, in all cell models tested, pharmacological inhibition of endogenous or ectopically expressed RyRs, using dantrolene or ryanodine, augmented autophagic flux by increasing lysosomal turn-over (number of autophagosomes and autolysosomes measured as mCherry-LC3 punctae/cell increased from 70.37 ± 7.81 in control HEK RyR3 cells to 111.18 ± 7.72 and 98.14 ± 7.31 after dantrolene and ryanodine treatments, respectively). Moreover, in differentiated C2C12 cells, transmission electron microscopy demonstrated that dantrolene treatment decreased the number of early autophagic vacuoles from 5.9 ± 2.97 to 1.8 ± 1.03 per cellular cross section. The modulation of the autophagic flux could be linked to the functional inhibition of RyR channels as both RyR inhibitors efficiently diminished the number of cells showing spontaneous RyR3 activity in the HEK293 cell model (from 41.14% ± 2.12 in control cells to 18.70% ± 2.25 and 9.74% ± 2.67 after dantrolene and ryanodine treatments, respectively). In conclusion, basal RyR-mediated Ca2+-release events suppress autophagic flux at the level of the lysosomes

Removal of the N-glycosylation sequon at position N116 located in p27 of the respiratory syncytial virus fusion protein elicits enhanced antibody responses after DNA immunization

Prevention of severe lower respiratory tract infections in infants caused by the human respiratory syncytial virus (hRSV) remains a major public health priority. Currently, the major focus of vaccine development relies on the RSV fusion (F) protein since it is the main target protein for neutralizing antibodies induced by natural infection. The protein conserves 5 N-glycosylation sites, two of which are located in the F2 subunit (N27 and N70), one in the F1 subunit (N500) and two in the p27 peptide (N116 and N126). To study the influence of the loss of one or more N-glycosylation sites on RSV F immunogenicity, BALB/c mice were immunized with plasmids encoding RSV F glycomutants. In comparison with F WT DNA immunized mice, higher neutralizing titres were observed following immunization with F N116Q. Moreover, RSV A2-K-line19F challenge of mice that had been immunized with mutant F N116Q DNA was associated with lower RSV RNA levels compared with those in challenged WT F DNA immunized animals. Since p27 is assumed to be post-translationally released after cleavage and thus not present on the mature RSV F protein, it remains to be elucidated how deletion of this glycan can contribute to enhanced antibody responses and protection upon challenge. These findings provide new insights to improve the immunogenicity of RSV F in potential vaccine candidates

Behavioral and Neuropathological Phenotyping of the Tau58/2 and Tau58/4 Transgenic Mouse Models for FTDP-17

Background: The Tau58/2 and Tau58/4 mouse lines expressing 0N4R tau with a P301S mutation mimic aspects of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). In a side-by-side comparison, we report the age-dependent development of cognitive, motor, and behavioral deficits in comparison with the spatial-temporal evolution of cellular tau pathology in both models. Methods: We applied the SHIRPA primary screen and specific neuromotor, behavioral, and cognitive paradigms. The spatiotemporal development of tau pathology was investigated immunohistochemically. Levels of sarkosyl-insoluble paired helical filaments were determined via a MesoScale Discovery biomarker assay. Results: Neuromotor impairments developed from age 3 months in both models. On electron microscopy, spinal cord neurofibrillary pathology was visible in mice aged 3 months; however, AT8 immunoreactivity was not yet observed in Tau58/4 mice. Behavioral abnormalities and memory deficits occurred at a later stage (&gt;9 months) when tau pathology was fully disseminated throughout the brain. Spatiotemporally, tau pathology spread from the spinal cord via the midbrain to the frontal cortex, while the hippocampus was relatively spared, thus explaining the late onset of cognitive deficits. Conclusions: Our findings indicate the face and construct validity of both Tau58 models, which may provide new, valuable insights into the pathologic effects of tau species in vivo and may consequently facilitate the development of new therapeutic targets to delay or halt neurodegenerative processes occurring in tauopathies.</p

Federal Regulation

Introduction Infected wounds are difficult to treat and there are no standardized protocols. Presentation of case We report a case of infected postoperative wound and entero-cutaneous fistula in a 83 years-old woman. An innovative treatment protocol for Human amniotic membrane (HAM)-assisted dressing of infected wound as the Idea Stage following the IDEAL recommendations is presented. The development of amnion preparation and the involved treatment steps are described. No adverse events and no graft rejection have been detected. Discussion Favorable results confirm the technical simplicity, safety and efficacy of this procedure. HAM has been shown to promote wound healing and to have antibacterial characteristics, which was supported by the presented case. Conclusion We are able to report a successful treatment of an infected wound caused by entero-cutaneous fistula with HAM dressing. Following the IDEAL recommendations, consecutive prospective cohort trials are justified

Comparison of Two Inoculation Methods of Endophytic Bacteria to Enhance Phytodegradation Efficacy of an Aged Petroleum Hydrocarbons Polluted Soil

Endophyte-enhanced phytodegradation is a promising technology to clean up polluted soils. To improve the success rate of this nature-based remediation approach, it is important to advance the inoculation method as this has been shown to strongly affect the final outcome. However, studies evaluating inoculation strategies and their effect on hydrocarbon degradation are limited. This study aims to investigate two different manners of endophyte inoculation in Lolium perenne growing in an aged petroleum hydrocarbon polluted soil: (1) direct soil inoculation (SI), and (2) pre-inoculation of the caryopses followed by soil inoculation (PI). Different endophytic bacterial strains, Rhodococcus erythropolis 5WK and Rhizobium sp. 10WK, were applied individually as well as in combination. Depending on the method of inoculation, the petroleum hydrocarbon (PHC) degradation potential was significantly different. The highest PHC removal was achieved after pre-inoculation of ryegrass caryopses with a consortium of both bacterial strains. Moreover, both strains established in the aged-polluted soil and could also colonize the roots and shoots of L. perenne. Importantly, used endophytes showed the selective colonization of the environment compartments. Our findings show that the method of inoculation determines the effciency of the phytodegradation process, especially the rate of PHC degradation. This study provides valuable information for choosing the most cost-effective and beneficial means to optimize phytodegradation

Loss-of-function mutations in the X-linked biglycan gene cause a severe syndromic form of thoracic aortic aneurysms and dissections.

Thoracic aortic aneurysm and dissection (TAAD) is typically inherited in an autosomal dominant manner, but rare X-linked families have been described. So far, the only known X-linked gene is FLNA, which is associated with the periventricular nodular heterotopia type of Ehlers-Danlos syndrome. However, mutations in this gene explain only a small number of X-linked TAAD families. We performed targeted resequencing of 368 candidate genes in a cohort of 11 molecularly unexplained Marfan probands. Subsequently, Sanger sequencing of BGN in 360 male and 155 female molecularly unexplained TAAD probands was performed. We found five individuals with loss-of-function mutations in BGN encoding the small leucine-rich proteoglycan biglycan. The clinical phenotype is characterized by early-onset aortic aneurysm and dissection. Other recurrent findings include hypertelorism, pectus deformity, joint hypermobility, contractures, and mild skeletal dysplasia. Fluorescent staining revealed an increase in TGF-β signaling, evidenced by an increase in nuclear pSMAD2 in the aortic wall. Our results are in line with those of prior reports demonstrating that Bgn-deficient male BALB/cA mice die from aortic rupture. In conclusion, BGN gene defects in humans cause an X-linked syndromic form of severe TAAD that is associated with preservation of elastic fibers and increased TGF-β signaling.Genet Med 19 4, 386-395

Differential effects of high fat diet-induced obesity on oocyte mitochondrial functions in inbred and outbred mice

Maternal obesity can cause reduced oocyte quality and subfertility. Mitochondrial dysfunction plays a central role here, and most often inbred mouse models are used to study these pathways. We hypothesized that the mouse genetic background can influence the impact of high fat diet (HFD)-induced obesity on oocyte quality. We compared the inbred C57BL/6 (B6) and the outbred Swiss strains after feeding a HFD for 13w. HFD-mice had increased body weight gain, hypercholesterolemia, and increased oocyte lipid droplet (LD) accumulation in both strains. LD distribution was strain-dependent. In Swiss mouse oocytes, HFD significantly increased mitochondrial inner membrane potential (MMP), reactive oxygen species concentrations, mitochondrial ultrastructural abnormalities (by 46.4%), and endoplasmic reticulum (ER) swelling, and decreased mtDNA copy numbers compared with Swiss controls (P0.1). Interestingly, mtDNA in B6-HFD oocytes was increased suggesting defective mitophagy. In conclusion, we show evidence that the genetic background or inbreeding can affect mitochondrial functions in oocytes and may influence the impact of HFD on oocyte quality. These results should create awareness when choosing and interpreting data obtained from different mouse models before extrapolating to human applications

Immunocytochemical characterization of ex vivo cultured conjunctival explants; marker validation for the identification of squamous epithelial cells and goblet cells

Tissue-engineered products are at the cutting edge of innovation considering their potential to functionally and structurally repair various tissue defects when the body’s own regenerative capacity is exhausted. At the ocular surface, the wound healing response to extensive conjunctival damage results in tissue repair with structural alterations or permanent scar formation rather than regeneration of the physiological conjunctiva. Conjunctival tissue engineering therefore represents a promising therapeutic option to reconstruct the ocular surface in severe cicatrizing pathologies. During the rapid race to be a pioneer, it seems that one of the fundamental steps of tissue engineering has been neglected; a proper cellular characterization of the tissue-engineered equivalents, both morphologically and functionally. Currently, no consensus has been reached on an identification strategy and/or markers for the characterization of cultured squamous epithelial and goblet cells. This study therefore evaluated the accuracy of promising markers to identify differentiated conjunctival-derived cells in human primary explant cultures through immunocytochemistry, including keratins (i.e., K7, K13, and K19) and mucins (i.e., MUC1, MUC5AC, and PAS-positivity). Comparison of the in vivo and in vitro cellular profiles revealed that the widely used goblet cell marker K7 does not function adequately in an in vitro setting. The other investigated markers offer a powerful tool to distinguish cultured squamous epithelial cells (i.e., MUC1 and K13), goblet cells (i.e., MUC5AC and PAS-staining), and conjunctival-derived cells in general (i.e., K19). In conclusion, this study emphasizes the power alongside potential pitfalls of conjunctival markers to assess the clinical safety and efficacy of conjunctival tissue-engineered products

Responses of the Endophytic Bacterial Communities of Juncus acutus to Pollution With Metals, Emerging Organic Pollutants and to Bioaugmentation With Indigenous Strains

Plants and their associated bacteria play a crucial role in constructed wetlands. In this study, the impact of different levels of pollution and bioaugmentation with indigenous strains individually or in consortia was investigated on the composition of the endophytic microbial communities of Juncus acutus. Five treatments were examined and compared in where the wetland plant was exposed to increasing levels of metal pollution (Zn, Ni, Cd) and emerging pollutants (BPA, SMX, CIP), enriched with different combinations of single or mixed endophytic strains. High levels of mixed pollution had a negative effect on alpha diversity indices of the root communities; moreover, the diversity indices were negatively correlated with the increasing metal concentrations. It was demonstrated that the root communities were separated depending on the level of mixed pollution, while the family Sphingomonadaceae exhibited the higher relative abundance within the root endophytic communities from high and low polluted treatments. This study highlights the effects of pollution and inoculation on phytoremediation efficiency based on a better understanding of the plant microbiome community composition

Biophysical analysis of an oligomerization-attenuated variant of the Leishmania donovani dynamin-1-like protein

Chemotherapy is a cornerstone in the battle against leishmaniasis, a neglected tropical disease caused by Leishmania parasites that affects millions worldwide. An alarming number of reports are describing treatment failure with currently available drugs, thereby explaining the dire need for the discovery of novel compounds, preferably with yet unexplored modes of action. In this respect L. donovani dynamin-1 like protein (LdoDLP1) is of interest as mutations in LdoDLP1 were recently shown to confer resistance to a new antileishmanial compound, suggesting it to be a potential drug target. Through a combination of biochemical, structural, and biophysical methods, we were able to show that wild-type LdoDLP1 has a strong inherent propensity to self-assemble into higher-order oligomers. Guided by structural modeling, a selection of nine point mutations (including resistance markers) were screened for oligomerization behavior to generate self-assembly impaired LdoDLP1 mutants that would occur in solution as dimers and/or tetramers. This led to the identification of a double mutant (G354D/R357S) that exhibits significantly altered and reduced, yet not completely abolished, oligomerization behavior. Further characterization of the LdoDLP1 G354D/R357S double mutant using small-angle X-ray scattering (SAXS) revealed that a fraction of the protein population occurs as a dimer in solution. Additionally, SAXS analysis experimentally confirmed that LdoDLP1, like other dynamin-like proteins, lacks the structurally defined pleckstrin homology (PH) domain of classical dynamins but instead possesses an intrinsically disordered B insert, grouping it among the dynamin-like proteins that play key roles in processes such as mitochondrial fission