Ferritin-based fusion protein shows octameric deadlock state of self-assembly.

Ferritin is a universal protein complex responsible for iron perception in almost all living organisms and has applications from fundamental biophysics to drug delivery and structure-based immunogen design. Different platforms based on ferritin share similar technological challenges limiting their development - control of self-assembling processes of ferritin itself as well as ferritin-based chimeric recombinant protein complexes. In our research, we studied self-assembly processes of ferritin-based protein complexes under different expression conditions. We fused a ferritin subunit with a SMT3 protein tag, a homolog of human Small Ubiquitin-like Modifier (SUMO-tag), which was taken to destabilize ferritin 3-fold channel contacts and increase ferritin-SUMO subunits solubility. We first obtained the octameric protein complex of ferritin-SUMO (8xFer-SUMO) and studied its structural organization by small-angle X-ray scattering (SAXS). Obtained SAXS data correspond well with the high-resolution models predicted by AlphaFold and CORAL software of an octameric assembly around the 4-fold channel of ferritin without formation of 3-fold channels. Interestingly, three copies of 8xFer-SUMO do not assemble into 24-meric globules. Thus, we first obtained and structurally characterized ferritin-based self-assembling oligomers in a deadlock state. Deadlock oligomeric states of ferritin extend the known scheme of its self-assembly process, being new potential tools for a number of applications. Finally, our results might open new directions for various biotechnological platforms utilizing ferritin-based tools.

Methionine gamma lyase fused with S3 domain VGF forms octamers and adheres to tumor cells via binding to EGFR.

Methods for targeting enzymes exhibiting anticancer properties, such as methionine γ-lyase (MGL), have not yet been sufficiently developed. Here, we present the data describing the physico-chemical properties and cytotoxic effect of fusion protein MGL-S3 - MGL from Clostridium sporogenes translationally fused to S3 domain of the viral growth factor of smallpox. MGL-S3 has methioninase activity comparable to native MGL. In solution, MGL-S3 protein primarily forms octamers, whereas native MGL, on the contrary, usually forms tetramers. MGL-S3 binds to the surface of the neuroblastoma SH-SY5Y and epidermoid carcinoma A431 cells and, unlike native MGL, remains there and retains its cytotoxic effect after media removal. In HEK293T cells lacking EGFRs, no adhesion was recorded. Confocal fluorescence microscopy confirms the preferential adhesion of MGL-S3 to tumor cells, while it avoids getting into lysosomes. Both MGL and MGL-S3 arrest cell cycle of SH-SY5Y cells mainly in the G1 phase, while only MGL-S3 retains this ability after washing the cells.

Cholesterol and melatonin regulated membrane fluidity does not affect the membrane breakage triggered by amyloid-beta peptide.

We have studied by means of small angle neutron scattering and diffraction, and molecular dynamics simulations the effect of lipid membrane fluidity on the amyloid-beta peptide interactions with the membrane. These interactions have been discovered previously to trigger the reorganization of model membranes between unilamellar vesicles and planar membranes (bicelle-like structures) during the lipid phase transition. The morphology changes were taking place in rigid membranes prepared of fully saturated lipids and were proposed to play a role in the onset of amyloid related disorders. We show in this study that the replacement of fully saturated lipids by more fluid mono-unsaturated lipids eliminates the mentioned morphology changes, most likely due to the absence of phase transition within the temperature range investigated. We have therefore controlled the membrane rigidity also while ensuring the presence of membrane phase transition within the biologically relevant temperatures. It was done by the addition of melatonin and/or cholesterol to the initial membranes made of saturated lipids. Small angle neutron scattering experiments performed over a range of cholesterol and melatonin concentrations show their distinctive effects on the local membrane structure only. The cholesterol for example affects the membrane curvature such that spontaneously formed unilamellar vesicles are of much larger sizes than those formed by the neat lipid membranes or membranes with melatonin added. The temperature dependent experiments, however, reveal no influence on the previously discovered membrane breakage whether cholesterol or melatonin have been added.

Optogenetic cytosol acidification of mammalian cells using an inward proton-pumping rhodopsin.

Ion gradients are a universal form of energy, information storage and conversion in living cells. Advances in optogenetics inspire the development of novel tools towards control of different cellular processes with light. Rhodopsins are perspective tools for optogenetic manipulation of ion gradients in cells and subcellular compartments, controlling pH of the cytosol and intracellular organelles. The key step of the development of new optogenetic tools is evaluation of their efficiency. Here, we used a high-throughput quantitative method for comparing efficiency of proton-pumping rhodopsins in Escherichia coli cells. This approach allowed us to show that an inward proton pump xenorhodopsin from Nanosalina sp. (NsXeR) is a powerful tool for optogenetic control of pH of mammalian subcellular compartments. Further, we demonstrate that NsXeR can be used for fast optogenetic acidification of the cytosol of mammalian cells. This is the first evidence of optogenetic cytosol acidification by an inward proton pump at physiological pH values. Our approach offers unique opportunities to study cellular metabolism at normal and pathological conditions and might help to understand the role of pH dysregulation in cellular dysfunctions.

Gadolinium-oxide nanoparticles for cryogenic magnetocaloric applications.

The series of advanced nanocomposites consisting of Gd2O3 nanoparticles (NPs) embedded into periodic porous SiO2 matrix have been investigated with respect to their structural and magnetocaloric properties. By means of small angle neutron scattering and transmission electron microscopy, regular nanopores organized in the cubic or hexagonal superlattice have been documented. The pores are occupied by the NPs of progressive concentration within the nanocomposite series. All of the examined systems have exhibited extraordinarily high values of magnetic entropy change (up to 70 J kg-1 K-1) at low temperatures with the absence of thermal hysteresis, indicating their perspective utilization in cryogenic refrigeration. Profound analysis of magnetic entropy change data via scaling laws has been applied to the nanocomposite materials for the very first time. With the aid of scaling analysis, conclusions on magnetic properties and phase transition type have been made, even for the conditions unavailable in the laboratory.

[Surgical prevention of progressive cerebral ischemia after ischemic stroke]. / Khirurgicheskaya profilaktika progressirovaniya ishemii golovnogo mozga u patsientov posle insul'ta.

OBJECTIVE: To evaluate the effectiveness of carotid endarterectomy after ischemic stroke and to analyze postoperative neurological status. MATERIAL AND METHODS: There were 120 patients with carotid artery stenosis complicated by chronic cerebrovascular insufficiency (CVI). Patients with CVI grade I-III were included in the first group (n=70), 50 patients had previous ischemic stroke (the second group). Age of patients was 61-89 years. All patients underwent unilateral carotid endarterectomy. RESULTS: Over 36-month follow-up period, postoperative stroke occurred in 5 (7.2%) and 9 (18.3%) patients in both groups, respectively. The combined end-point (stroke + mortality) was significantly less common in group I compared to group II (10 (14.5%) and 15 (30%) cases, p>0.05). Positive changes in neurological status were found in the 2nd group (relief of focal neurological deficit, symptoms of cerebrovascular insufficiency, no progression of vascular dementia). Barthel score increased from 74.3 to 92.8 after carotid endarterectomy in the 2nd group. Baseline FAB score <11 was observed in 9% of patients in the 1st group and 22% in the 2nd group. No progression of dementia was observed in long-term period. CONCLUSION: Carotid endarterectomy is effective in prevention of primary and recurrent stroke. Moreover, this procedure slows down progression of chronic cerebral ischemia and cognitive impairment.

[Minimally invasive carotid artery surgery]. / Maloinvazivnaya khirurgiya sonnykh arterii.

OBJECTIVE: To determine the criteria for choosing a surgical approach and compare an effectiveness of carotid endarterectomy (CEAE) via 3 approaches. MATERIAL AND METHODS: The study included 120 patients who underwent CEAE via 3 different approaches. Intraoperative skin marking included lower jaw angle, skin fold closest to common carotid artery bifurcation. Carotid artery bifurcation and borders of atherosclerotic plaque were visualized using ultrasound. An effectiveness of each access was evaluated in accordance with the following criteria: neurological complications, cosmetic effect and quality of life after 1 and 12 months. The patients were divided into 2 groups. Group I - 80 patients with CEAE with access through the natural skin fold (NSF); group II - 40 patients with CEAE using the classical longitudinal access. The 1st group was divided into 2 subgroups. Subgroup I A - 39 patients with CEAE using mini-access via NSF; subgroup I B - 41 patients with CEAE using extended access via NSF. RESULTS: There were no strokes and transient ischemic attacks in a month after surgery in both groups. After 12 months, stroke occurred in 2 (%) patients of group II, cranial neuropathy - 8 (21%) patients in the same group. The best cosmic effect was achieved in subgroup I A after 1 and 12 months (37.1±6.7 scores). Mean score of physical health was 51.59±5.9 scores in subgroup I A, 46.03±7.53 scores - in subgroup I B, 38.84±5.28 scores - in group II. Index of mental health was 49.63±6.69, 45.68±5.6, and 48.59±7.29 scores, respectively. CONCLUSION: Considering these data, we developed a personalized computer program ensuring fast choice of optimal surgical approach.

[Advantages of minimally invasive approach for carotid endarterectomy]. / Preimushchestva mini-dostupa pri karotidnoi éndarteréktomii.

OBJECTIVE: To identify the advantages and disadvantages of different approaches for carotid endarterectomy (conventional, longitudinal and transverse incision). MATERIAL AND METHODS: There were 58 patients who underwent carotid endarterectomy. Patients were divided into 2 groups depending on surgical approach. Group 1 (n=37) - minimal skin incision (less than 5 cm). There were subgroup 1A (transverse minimal skin incision along the natural skin wrinkle, n=17) and subgroup 1B (longitudinal minimal skin incision, n=20). Group 2 (n=21) - conventional longitudinal incision. Surgical outcomes were analyzed after 1 month and 1 year. End-points were mortality, stroke, TIA, cranial nerve neuropathy. Cosmetic effect was evaluated using POSAS scale (Patient and Observer Scar Assessment Scale, Draaijers, 2004). RESULTS: Mortality, stroke and TIA were absent after 1 month. Cranial nerve neuropathy was not observed in subgroup 1A and diagnosed in 2 (10%) patients of subgroup 1B and 6 (28.5%) patients of group 2. Cosmetic effect: subgroup 1A - 48.4±9.5 scores, subgroup 1B - 52.4±9.2, group 2 - 63.1±11.1 (p<0.05). The outcomes after 12 months: mortality was absent in subgroups 1A and 1B, 2 patients died in group 2 from AMI. Stroke was absent in subgroups 1A and 1B, group 2 - 1 patient. Cranial nerve neuropathy was absent in 1A and 1B subgroups and diagnosed in 4 (21%) patients of group 2. Cosmetic effect: subgroup 1A - 37.2 scores, subgroup 1B - 40.0 scores, group 2 - 55.1 scores. Physical component of QOL: subgroup 1A - 51.63±6.31 scores, subgroup 1B - 46.01±7.53 scores, group 2 - 38.85±5.33 scores. Psychological component of QOL: subgroup 1A - 49.64±6.72 scores, subgroup 1B - 45.68±5.63 scores, group 2 - 48.6±7.36 scores (p<0.05). CONCLUSION: Transverse minimal skin incision for carotid endarterectomy is a safe alternative to classic longitudinal incision and reduces the risk of postoperative complications with significant cosmetic effect.

[Meta-analysis of the results of eversion carotid endarterectomy and endarterectomy with patch plasty]. / Metaanaliz rezul'tatov éversionnoi karotidnoi éndarteréktomii i éndarteréktomii s plastikoi zaplatoi.

AIM: The study was aimed at comparing the results of eversion carotid endarterectomy and carotid endarterectomy with patch plasty in the immediate and remote postoperative periods. MATERIALS AND METHODS: The literature was retrieved by means of electronic databases, with the dates of publications ranging from 1970 to 2019. According to the inclusion and exclusion criteria we selected the literature making it possible to carry out a meta-analysis in the immediate and remote postoperative periods. The results were obtained with the help of the Stata 14 software package. Eventually, we retrieved and analysed a total of 2139 articles. Of these, ten were included into the study and contained 3568 patients subjected to 3672 operations (eversion carotid endarterectomy - 1718 and carotid endarterectomy with a patch - 1954). The results of the meta-analysis were as follows: the mean time of carotid artery cross-clamping for eversion carotid endarterectomy was shorter than that for carotid endarterectomy with a patch (4.1±2.9 min); the frequency of using intraoperative temporary bypass in eversion carotid endarterectomy turned out to be significantly less compared with carotid endarterectomy with patch plasty - 13.5% (91/672) and 62.0% (492/793), OR=0.183, 95% CI: 0.136-0.254, p<0.001; the incidence rate of ischaemic stroke in the immediate and remote postoperative periods was significantly lower after eversion carotid endarterectomy than that after carotid endarterectomy with patch plasty - OR=0.452, 95% CI: 0258-0.792, p=0.005 and OR=0.300, 95% CI; 0.155-0.579, p=0.000. The development of restenosis in the immediate and remote postoperative periods was observed less often for eversion carotid endarterectomy compared with carotid endarterectomy with patch plasty - OR=0.604, 95% CI: 0.422-0.864, p=0.006. CONCLUSION: Eversion carotid endarterectomy was associated with shorter time of carotid artery cross-clamping, lower frequency of intraoperative temporary bypass, lower number of cases of ischaemic stroke in the immediate and remote postoperative periods, as well as those of restenosis in the long-term postoperative period.

Unusual features of the c-ring of F1FO ATP synthases.

Membrane integral ATP synthases produce adenosine triphosphate, the universal "energy currency" of most organisms. However, important details of proton driven energy conversion are still unknown. We present the first high-resolution structure (2.3 Å) of the in meso crystallized c-ring of 14 subunits from spinach chloroplasts. The structure reveals molecular mechanisms of intersubunit contacts in the c14-ring, and it shows additional electron densities inside the c-ring which form circles parallel to the membrane plane. Similar densities were found in all known high-resolution structures of c-rings of F1FO ATP synthases from archaea and bacteria to eukaryotes. The densities might originate from isoprenoid quinones (such as coenzyme Q in mitochondria and plastoquinone in chloroplasts) that is consistent with differential UV-Vis spectroscopy of the c-ring samples, unusually large distance between polar/apolar interfaces inside the c-ring and universality among different species. Although additional experiments are required to verify this hypothesis, coenzyme Q and its analogues known as electron carriers of bioenergetic chains may be universal cofactors of ATP synthases, stabilizing c-ring and prevent ion leakage through it.

[Early carotid endarterectomy in patients after endured acute cerebral circulation impairment]. / Ranniaia karotidnaia éndarteréktomiia u patsientov, perenesshikh ostroe narushenie mozgovogo krovoobrashcheniia.

Recent years have witnessed a series of studies dedicated to assessment of optimal terms of performing carotid endarterectomy (CEA) after sustained acute cerebral circulatory impairment (ACCI). However, there is no commonly accepted opinion concerning feasibility and safety of early CEA in 'symptomatic' patients. The 2015 Guidelines of the American Heart Association suggest that surgical intervention should be performed within the shortest terms or during 2 weeks after a neurological event (class IIb, level B evidence). In the presented herein review of literature most authors demonstrated safety and efficacy of performing early CEA after endured ACCI. However, further prospective, randomized studies are needed in order to work out new standards of examination and to determine optimal surgical policy.

Heavy Metals in Bottom Sediments of Lake Kenon (The Trans-Baikal Territory, Russia).

Concentrations of heavy metals in bottom sediments of Lake Kenon in descending order are distributed as follows: Mn > Zn > Pb > Mo > Cd. Spatial distribution of metals in bottom sediments of Lake Kenon depends on composition of bottom deposits (sands in shallow water and sapropel silt in the deep part), location of contamination sources (thermal power station, residential area), as well as density and duration of growth of aquatic plants. The greatest pollution of bottom sediments was observed in the area of TPP-1. Due to the intense and all-year-round process of aquatic vegetation growth in the area of TPP-1 contaminants are being accumulated in the bottom sediments of this part of the lake. However, plants that absorb metals in excess amounts and are passively moved by currents through of the system become a source of contamination of bottom sediments in relatively clean parts of the water reservoirs.

[An efficacy of carotid arteries repair for tortuosity combined with stenosis]. / Éffektivnost' rekonstruktivnykh operatsii na sonnykh arteriiakh pri sochetanii izvitosti i stenoza.

AIM: To assess an efficacy of carotid arteries reconstruction in patients with internal carotid artery stenosis combined with tortuosity. MATERIAL AND METHODS: 86 patients with ICA tortuosity and stenosis were enrolled. All patients were divided into groups depending on type of surgery: group I - open carotid endarterectomy (CEA) followed by obligatory repair with synthetic patch (31 (36%) patients); group II - eversion CEA with ICA resection, redressation and reimplantation into own ostium (35 (40.7%) patients); group III - ICA replacement (20 (23.3%) patients). Synthetic prosthesis and autovein were used in 13 (65%) and 7 (35%) patients respectively. The study included patients with ICA stenosis ≥60% (any type of plaque) and any degree of cerebrovascular insufficiency or ICA stenosis <60% (plaque type I-III) with CVI grade II-IV combined with S- or C-tortuosity, bend or loop with blood flow velocity over 110 cm/s and its turbulence. Only 6 (7.0%) out of 86 patients had no clinical signs of CVI/previous stroke. Asymptomatic/symptomatic patients ratio was following in all groups: group I - 12 (38.7%)/19 (61.3%); group II - 29 (82.9%)/6 (17.1%); group III - 10 (50%)/10 (50%). RESULTS: Within 6-month follow-up 22 (70.9%) out of 31 patients were asymptomatic in group I, 30 (85.7%) (p=0.9475) out of 35 - in group II, 9 (45%) (p=0.9511) out of 20 patients - in group III and 1 (5%) patient developed thrombosis of the reconstruction zone followed by ischemic stroke. After 12 months following patients were asymptomatic: 22 (70.9%) in group I, 30 (85.7%) (p=0.9475) in group II and 9 (45%) patients (p=0.9511) in group III. After 1 year 4 (33.3%) out of 12 patients with CVI grade IV had partial regression of focal neurological symptoms. CONCLUSION: Surgery for ICA tortuosity combined with stenosis confirmed its efficacy and safety for both asymptomatic and symptomatic patients. Significantly better results were observed in eversion CEA compared with conventional procedure and ICA replacement.

[Surgical treatment of patients with haemodynamically significant stenosis of the internal carotid artery and contralateral occlusion]. / Khirurgicheskoe lechenie bol'nykh s gemodinamicheski znachimym stenozom vnutrennei sonnoi arterii i kontrlateral'noi okkliuziei.

Presented herein is a review of the literature dedicated to the problem regarding surgical management of patients with haemodynamically significant stenosis of the internal carotid artery and collateral occlusion. Special consideration is given to the studies comparing perioperative and early postoperative outcomes of carotid endarterectomy in patients with the nosology concerned. The literature was retrieved by means of the following databases Scopus, Web of Science, Medline, and the Russian Science Citation Index.

Solution structure and excitation energy transfer in phycobiliproteins of Acaryochloris marina investigated by small angle scattering.

The structure of phycobiliproteins of the cyanobacterium Acaryochloris marina was investigated in buffer solution at physiological temperatures, i.e. under the same conditions applied in spectroscopic experiments, using small angle neutron scattering. The scattering data of intact phycobiliproteins in buffer solution containing phosphate can be well described using a cylindrical shape with a length of about 225Å and a diameter of approximately 100Å. This finding is qualitatively consistent with earlier electron microscopy studies reporting a rod-like shape of the phycobiliproteins with a length of about 250 (M. Chen et al., FEBS Letters 583, 2009, 2535) or 300Å (J. Marquart et al., FEBS Letters 410, 1997, 428). In contrast, phycobiliproteins dissolved in buffer lacking phosphate revealed a splitting of the rods into cylindrical subunits with a height of 28Å only, but also a pronounced sample aggregation. Complementary small angle neutron and X-ray scattering experiments on phycocyanin suggest that the cylindrical subunits may represent either trimeric phycocyanin or trimeric allophycocyanin. Our findings are in agreement with the assumption that a phycobiliprotein rod with a total height of about 225Å can accommodate seven trimeric phycocyanin subunits and one trimeric allophycocyanin subunit, each of which having a height of about 28Å. The structural information obtained by small angle neutron and X-ray scattering can be used to interpret variations in the low-energy region of the 4.5K absorption spectra of phycobiliproteins dissolved in buffer solutions containing and lacking phosphate, respectively.

[Ocular blood flow and carotid artery malfunction].

AIM: to evaluate ocular hemodynamics and informativity of estimated individual normal range of intraocular pressure (IOP). MATERIALS AND METHODS: A total of 12 patients (22 eyes) with carotid artery malfunction were examined. Ocular blood flow (OBF) and IOP were measured with Ocular Blood Flow Analyzer. Actual OBF was then compared with what is considered normal for a given axial length (AL). Individual normal range of IOP was calculated according to an original formula (described in previous publications). Doppler imaging of ocular vessels enabled blood flow velocity measurement. Morphological parameters and functional status of the retina and optic nerve were judged on automated perimetry (Octopus 900) and optical coherence tomography (Cirrus HD-OCT) findings. Statistical analyses were performed using Statistica 10 software. RESULTS: Generally, OBF showed no correlation with the grade of carotid artery stenosis (p < 0.05), however, was significantly reduced as compared to its AL-dependent norm in patients with greater than 85% narrowing of the internal carotid artery, which can cause misestimating of their individual normal range of IOP. A negative relationship was established between the blood flow velocity in short posterior ciliary arteries and the grade of internal carotid artery stenosis (p < 0.005). Ocular blood flow deficit relative to the AL-dependent norm correlated with ophthalmic artery resistance index. CONCLUSION: OBF-based estimation of individual normal range of IOP is inexpedient in patients with greater than 80% carotid artery stenosis due to its possible influence on ocular hemodynamics. In most cases of less than 80% carotid artery stenosis OBF is adequate or slightly reduced as compared to its AL-dependent norm and thus, has no significant impact on estimated individual normal range of IOP.