The activated endoplasmic reticulum stress sensor IRE1 oligomerizes into filaments contained in 30 nm membrane tubes of complex topology

The unfolded protein response (UPR) is an intracellular signaling network that adjusts the abundance and protein folding capacity of the endoplasmic reticulum (ER) according to need. The most conversed branch of the UPR is mediated by the ER‐resident transmembrane kinase/endoribonuclease IRE1. It senses unfolded protein accumulation within the ER and transduces the signal via a non‐conventional mRNA splicing mechanism. In response to direct binding of unfolded proteins in the ER lumen, IRE1 activates by oligomerization and accumulates in dynamic foci. IRE1 foci are not autophagosomes as they did not colocalize with the autophagosomal marker LC3. Fluorescence recovery after photobleaching (FRAP) experiments indicate that IRE1 molecules in the foci remain in equilibrium with IRE1 molecules in the surrounding ER network. We determined the structure of IRE1 foci in cells by whole cell correlative light – electron tomography. Our results show that IRE1 oligomers induce membrane deformations, leading to the protrusion of narrow 30 nm ribosome‐free tubes that remain connected to the ER and are twisted into glomeruli of complex topology. The tubes contain two parallel filaments in their lumen, likely representing oligomerized IRE1 ER‐lumenal domains. Taken together, our findings define a previously unrecognized subdomain of the ER membrane and shed new light on the structure and organization of active mammalian IRE1 inside the cell

Experimental study of VEGF immune expression dynamics in the retina using photoinduced BRVO model

Aim. To describe the dynamics of vascular endothelial growth factor (VEGF) immune expression in the retina using the model of photoinduced branch retinal vein occlusion (BRVO) and to establish the terms of neovascularization appearance.Materials and methods. BRVO was modelled on 21 chinchilla rabbits (21 eyes) weighing 1.5‑2 kg (fellow eyes served as controls). Photosensitizer «Fotoditazin» (2.5 mg / kg) was injected intravenously. 15 min later, transpupillary laser irradiation of branch retinal vein near the optic nerve head was performed. Irradiation energy density was 200 J / cm2. Histological analysis and immunohistochemistry of the retina was performed following 30 min, at days 1, 2, 3, 7, 14 and 30.Results. Maximum VEGF accumulation in photoinduced BRVO model was observed on day 2. From day 3, direct neovascularization was confirmed. VEGF levels were stably high throughout the follow-up to the day 30 inclusive.Conclusion. VEGF immune expression in the retina using the model of BRVO induced by photodynamic exposure was explored for the first time. These data can serve as the basis for future studies in order to define optimal anti-VEGF agent, its dosage and terms to manage this condition

Expression and characterization of the bacterial mechanosensitive channel MscS in Xenopus laevis oocytes

We have successfully expressed and characterized mechanosensitive channel of small conductance (MscS) from Escherichia coli in oocytes of the African clawed frog, Xenopus laevis. MscS expressed in oocytes has the same single-channel conductance and voltage dependence as the channel in its native environment. Two hallmarks of MscS activity, the presence of conducting substates at high potentials and reversible adaptation to a sustained stimulus, are also exhibited by oocyte-expressed MscS. In addition to its ease of use, the oocyte system allows the user to work with relatively large patches, which could be an advantage for the visualization of membrane deformation. Furthermore, MscS can now be compared directly to its eukaryotic homologues or to other mechanosensitive channels that are not easily studied in E. coli

The activated endoplasmic reticulum stress sensor IRE1 oligomerizes into filaments contained in 30 nm membrane tubes of complex topology

The unfolded protein response (UPR) is an intracellular signaling network that adjusts the abundance and protein folding capacity of the endoplasmic reticulum (ER) according to need. The most conversed branch of the UPR is mediated by the ER‐resident transmembrane kinase/endoribonuclease IRE1. It senses unfolded protein accumulation within the ER and transduces the signal via a non‐conventional mRNA splicing mechanism. In response to direct binding of unfolded proteins in the ER lumen, IRE1 activates by oligomerization and accumulates in dynamic foci. IRE1 foci are not autophagosomes as they did not colocalize with the autophagosomal marker LC3. Fluorescence recovery after photobleaching (FRAP) experiments indicate that IRE1 molecules in the foci remain in equilibrium with IRE1 molecules in the surrounding ER network. We determined the structure of IRE1 foci in cells by whole cell correlative light – electron tomography. Our results show that IRE1 oligomers induce membrane deformations, leading to the protrusion of narrow 30 nm ribosome‐free tubes that remain connected to the ER and are twisted into glomeruli of complex topology. The tubes contain two parallel filaments in their lumen, likely representing oligomerized IRE1 ER‐lumenal domains. Taken together, our findings define a previously unrecognized subdomain of the ER membrane and shed new light on the structure and organization of active mammalian IRE1 inside the cell

Long-Term Monitoring of Liver Fibrosis and Steatosis in Patients with Chronic Hepatitis C after Achieving a Sustained Virologic Response to Antiviral Therapy

Aim: to analyze the dynamics of fibrosis and steatosis of the liver according to fibroelastometry in patients with chronic hep-atitis C (CHC) after ≥ 6 months from transient elastometry (TE) achieving a sustained virologic response (SVR) to antiviral therapy.Materials and methods. At baseline, a prospective observational study included 628 CHC patients with known stage of liver fibrosis (F) before AVT, some of whom were phased out due to non-compliance with the inclusion criteria. The final analysis included 297 patients who had transient elastometry (TE) data with CAP™ technology on the severity of liver fibrosis (± steatosis) before treatment and after ≥ 6 months after reaching SVR (67 % – interferonfree regimens of therapy). Median follow-up from the moment SVR was confirmed was 3 years [2; 6].Results. At the end of the study, the average age of patients was 49 ± 12 years, of which 53 % were men. In the long-term period after reaching SVR, regression of liver fibrosis was diagnosed in 80 % of cases (including in patients with cirrhosis), and the progression of fibrosis was in 3 % of patient. At the same time, regression of liver steatosis was detected only in 31 % of the patient, worsening of the results was in 23 % (26 % of them had the appearance of steatosis (S) of the liver of 1–3 degrees in persons with no fatty liver before the start of AVT). In the group of patients with liver steatosis, the proportion of men was significantly higher (p = 0.004). Clinically significant stages of fibrosis F3–F4 were significantly more often recorded in patients with hepatic steatosis, both before treatment (46 % S1–S3 and 22 % S0, p < 0.001) and after ≥ 6 months after reaching SVR (19 % S1–S3 and 9 % S0, p = 0.023).Conclusion. In patients with chronic hepatitis C with SVR achieved in the long term, despite a significant regression of liver fibrosis, a high prevalence of hepatic steatosis remains. The data obtained indicate the feasibility of routine diagnosis of both fibrosis and steatosis of the liver in the management of patients with chronic HCV infection before and after successful antiviral therapy