Dissecting the Structural Dynamics of the Nuclear Pore Complex.

Cellular processes are largely carried out by macromolecular assemblies, most of which are dynamic, having components that are in constant flux. One such assembly is the nuclear pore complex (NPC), an ∼50 MDa assembly comprised of ∼30 different proteins called Nups that mediates selective macromolecular transport between the nucleus and cytoplasm. We developed a proteomics method to provide a comprehensive picture of the yeast NPC component dynamics. We discovered that, although all Nups display uniformly slow turnover, their exchange rates vary considerably. Surprisingly, this exchange rate was relatively unrelated to each Nup's position, accessibility, or role in transport but correlated with its structural role; scaffold-forming Nups exchange slowly, whereas flexible connector Nups threading throughout the NPC architecture exchange more rapidly. Targeted perturbations in the NPC structure revealed a dynamic resilience to damage. Our approach opens a new window into macromolecular assembly dynamics.

RNF26 binds perinuclear vimentin filaments to integrate ER and endolysosomal responses to proteotoxic stress.

Proteotoxic stress causes profound endoplasmic reticulum (ER) membrane remodeling into a perinuclear quality control compartment (ERQC) for the degradation of misfolded proteins. Subsequent return to homeostasis involves clearance of the ERQC by endolysosomes. However, the factors that control perinuclear ER integrity and dynamics remain unclear. Here, we identify vimentin intermediate filaments as perinuclear anchors for the ER and endolysosomes. We show that perinuclear vimentin filaments engage the ER-embedded RING finger protein 26 (RNF26) at the C-terminus of its RING domain. This restricts RNF26 to perinuclear ER subdomains and enables the corresponding spatial retention of endolysosomes through RNF26-mediated membrane contact sites (MCS). We find that both RNF26 and vimentin are required for the perinuclear coalescence of the ERQC and its juxtaposition with proteolytic compartments, which facilitates efficient recovery from ER stress via the Sec62-mediated ER-phagy pathway. Collectively, our findings reveal a scaffolding mechanism that underpins the spatiotemporal integration of organelles during cellular proteostasis.

The Chromatin Reader ZMYND8 Regulates Igh Enhancers to Promote Immunoglobulin Class Switch Recombination.

Class switch recombination (CSR) is a DNA recombination reaction that diversifies the effector component of antibody responses. CSR is initiated by activation-induced cytidine deaminase (AID), which targets transcriptionally active immunoglobulin heavy chain (Igh) switch donor and acceptor DNA. The 3' Igh super-enhancer, 3' regulatory region (3'RR), is essential for acceptor region transcription, but how this function is regulated is unknown. Here, we identify the chromatin reader ZMYND8 as an essential regulator of the 3'RR. In B cells, ZMYND8 binds promoters and super-enhancers, including the Igh enhancers. ZMYND8 controls the 3'RR activity by modulating the enhancer transcriptional status. In its absence, there is increased 3'RR polymerase loading and decreased acceptor region transcription and CSR. In addition to CSR, ZMYND8 deficiency impairs somatic hypermutation (SHM) of Igh, which is also dependent on the 3'RR. Thus, ZMYND8 controls Igh diversification in mature B lymphocytes by regulating the activity of the 3' Igh super-enhancer.

Bi-allelic NIT1 variants cause a brain small vessel disease characterized by movement disorders, massively dilated perivascular spaces, and intracerebral hemorrhage.

PURPOSE: To describe a recessively inherited cerebral small vessel disease, caused by loss-of-function variants in Nitrilase1 (NIT1). METHODS: We performed exome sequencing, brain magnetic resonance imaging, neuropathology, electron microscopy, western blotting, and transcriptomic and metabolic analyses in 7 NIT1-small vessel disease patients from 5 unrelated pedigrees. RESULTS: The first identified patients were 3 siblings, compound heterozygous for the NIT1 c.727C>T; (p.Arg243Trp) variant and the NIT1 c.198_199del; p.(Ala68∗) variant. The 4 additional patients were single cases from 4 unrelated pedigrees and were all homozygous for the NIT1 c.727C>T; p.(Arg243Trp) variant. Patients presented in mid-adulthood with movement disorders. All patients had striking abnormalities on brain magnetic resonance imaging, with numerous and massively dilated basal ganglia perivascular spaces. Three patients had non-lobar intracerebral hemorrhage between age 45 and 60, which was fatal in 2 cases. Western blotting on patient fibroblasts showed absence of NIT1 protein, and metabolic analysis in urine confirmed loss of NIT1 enzymatic function. Brain autopsy revealed large electron-dense deposits in the vessel walls of small and medium sized cerebral arteries. CONCLUSION: NIT1-small vessel disease is a novel, autosomal recessively inherited cerebral small vessel disease characterized by a triad of movement disorders, massively dilated basal ganglia perivascular spaces, and intracerebral hemorrhage.

Littorina snails and Microphallus trematodes: Diverse consequences of the trematode-induced metabolic shifts.

The intricate relationships between parasites and hosts encompass a wide range of levels, from molecular interactions to population dynamics. Parasites influence not only the physiological processes in the host organism, but also the entire ecosystem, affecting mortality of individuals, the number of offspring through parasitic castration, and matter and energy cycles. Understanding the molecular mechanisms that govern host-parasite relationships and their impact on host physiology and environment remains challenging. In this study, we analyzed how infection with Microphallus trematodes affects the metabolome of two Littorina snail species inhabiting different intertidal zone shore levels. We applied non-targeted GC-MS-based metabolomics to analyze biochemical shifts induced by trematode infection in a host organism. We have identified changes in energy, amino acid, sugar, and lipid metabolism. In particular, we observed intensified amino acid catabolism and nitrogenous catabolites (glutamine, urea) production. These changes primarily correlated with infection and interspecies differences of the hosts rather than shore level. The changes detected in the host metabolism indicate that other aspects of life may have been affected, both within the host organism and at a supra-organismal level. Therefore, we explored changes in microbiota composition, deviations in the host molluscs behavior, and acetylcholinesterase activity (ACE, an enzyme involved in neuromuscular transmission) in relation to infection. Infected snails displayed changes in their microbiome composition. Decreased ACE activity in snails was associated with reduced mobility, but whether it is associated with trematode infection remains unclear. The authors suggest a connection between the identified biochemical changes and the deformation of the shell of molluscs, changes in their behavior, and the associated microbiome. The role of parasitic systems formed by microphallid trematodes and Littorina snails in the nitrogen cycle at the ecosystem level is also assumed.

Instability Compensation of Recording Interferometer in Phase-Sensitive OTDR.

In the paper, a new method of phase measurement error suppression in a phase-sensitive optical time domain reflectometer is proposed and experimentally proved. The main causes of phase measurement errors are identified and considered, such as the influence of the recording interferometer instabilities and laser wavelength instability, which can cause inaccuracies in phase unwrapping. The use of a Mach-Zender interferometer made by 3 × 3 fiber couplers is proposed and tested to provide insensitivity to the recording interferometer and laser source instabilities. It is shown that using all three available photodetectors of the interferometer, instead of just one pair, achieves significantly better accuracy in the phase unwrapping. A novel compensation scheme for accurate phase measurements in a phase-sensitive optical time domain reflectometer is proposed, and a comparison of the measurement signals with or without such compensation is shown and discussed. The proposed method, using three photodetectors, allows for very good compensation of the phase measurement errors arising from common-mode noise from the interferometer and laser source, providing a significant improvement in signal detection. In addition, the method allows the tracking of slow temperature changes in the monitored fiber/object, which is not obtainable when using a simple low-pass filter for phase unwrapping error reduction, as is customary in several systems of this kind.

Detection and Recognition of Voice Commands by a Distributed Acoustic Sensor Based on Phase-Sensitive OTDR in the Smart Home Concept.

In recent years, attention to the realization of a distributed fiber-optic microphone for the detection and recognition of the human voice has increased, whereby the most popular schemes are based on φ-OTDR. Many issues related to the selection of optimal system parameters and the recognition of registered signals, however, are still unresolved. In this research, we conducted theoretical studies of these issues based on the φ-OTDR mathematical model and verified them with experiments. We designed an algorithm for fiber sensor signal processing, applied a testing kit, and designed a method for the quantitative evaluation of our obtained results. We also proposed a new setup model for lab tests of φ-OTDR single coordinate sensors, which allows for the quick variation of their parameters. As a result, it was possible to define requirements for the best quality of speech recognition; estimation using the percentage of recognized words yielded a value of 96.3%, and estimation with Levenshtein distance provided a value of 15.

Studies of the transition between amplified spontaneous emission and optical lasing in ultrahigh-Q polymeric micro-pedestals.

In this work, we demonstrate the properties of Rhodamine B-doped polymeric cylindrical microlasers to perform either as gain amplification devices through amplified spontaneous emission (ASE) or as optical lasing gain devices. A study based on different %wt concentrations of microcavity families with distinct geometrical features demonstrates the characteristic dependence on either gain amplification phenomena. Principal component analysis (PCA) discriminates the relationship between the main ASE and lasing properties and the geometrical aspects of the cavity families. ASE and optical lasing thresholds were found, respectively, as low as 0.2 µJcm-2 and 0.1 µJcm-2 passing the best-reported microlaser performances in literature for cylindrical cavities, even in comparison with those based on 2D patterns. Moreover, our microlasers showed ultrahigh Q-factor of ∼3 × 106, and for the first time, to the best of our knowledge, a visible emission comb constituted by above a hundred peaks at 40 µJcm-2 with a registered free spectral range (FSR) of 0.25 nm corroborated through the whispery gallery mode (WGM) theory.

Fiber-Optic Telecommunication Network Wells Monitoring by Phase-Sensitive Optical Time-Domain Reflectometer with Disturbance Recognition.

The paper presents the application of a phase-sensitive optical time-domain reflectometer (phi-OTDR) in the field of urban infrastructure monitoring. In particular, the branched structure of the urban network of telecommunication wells. The encountered tasks and difficulties are described. The possibilities of usage are substantiated, and the numerical values of the event quality classification algorithms applied to experimental data are calculated using machine learning methods. Among the considered methods, the best results were shown by convolutional neural networks, with a probability of correct classification as high as 98.55%.

Physical and Functional Characterization of PLGA Nanoparticles Containing the Antimicrobial Peptide SAAP-148.

Synthetic antimicrobial and antibiofilm peptide (SAAP-148) commits significant antimicrobial activities against antimicrobial resistant (AMR) planktonic bacteria and biofilms. However, SAAP-148 is limited by its low selectivity index, i.e., ratio between cytotoxicity and antimicrobial activity, as well as its bioavailability at infection sites. We hypothesized that formulation of SAAP-148 in PLGA nanoparticles (SAAP-148 NPs) improves the selectivity index due to the sustained local release of the peptide. The aim of this study was to investigate the physical and functional characteristics of SAAP-148 NPs and to compare the selectivity index of the formulated peptide with that of the peptide in solution. SAAP-148 NPs displayed favorable physiochemical properties [size = 94.1 ± 23 nm, polydispersity index (PDI) = 0.08 ± 0.1, surface charge = 1.65 ± 0.1 mV, and encapsulation efficiency (EE) = 86.7 ± 0.3%] and sustained release of peptide for up to 21 days in PBS at 37 °C. The antibacterial and cytotoxicity studies showed that the selectivity index for SAAP-148 NPs was drastically increased, by 10-fold, regarding AMR Staphylococcus aureus and 20-fold regarding AMR Acinetobacter baumannii after 4 h. Interestingly, the antibiofilm activity of SAAP-148 NPs against AMR S. aureus and A. baumannii gradually increased overtime, suggesting a dose-effect relationship based on the peptide's in vitro release profile. Using 3D human skin equivalents (HSEs), dual drug SAAP-148 NPs and the novel antibiotic halicin NPs provided a stronger antibacterial response against planktonic and cell-associated bacteria than SAAP-148 NPs but not halicin NPs after 24 h. Confocal laser scanning microscopy revealed the presence of SAAP-148 NPs on the top layers of the skin models in close proximity to AMR S. aureus at 24 h. Overall, SAAP-148 NPs present a promising yet challenging approach for further development as treatment against bacterial infections.

Versatile Reactivity of Half-Sandwich Rhodium(III) Iminophosphonamide Complexes.

Novel 18e̅ and 16e̅ pentamethylcyclopentadienyl rhodium(III) complexes [(η5-C5Me5)RhX(NPN)] (1a,b, X = Cl; 2a-c, X = PF6, BAr4F) with chelating zwitterionic iminophosphonamide (NPN) ligands (Ph2P(NR)(NR'); a, R = R' = p-Tol; b, R = p-Tol, R' = Me; c, R = R' = Me) were synthesized and characterized by single-crystal X-ray diffraction. In the 16e̅ complexes 2, the rhodium (Rh) atom is efficiently stabilized by π-donation of unshared N electrons, thus hampering coordination of the external ligands and rendering the 18e̅ complexes labile. Due to low coordination enthalpy, the cationic 18e̅ monocarbonyl and pyridine adducts 2a·L are stable only at low temperatures. At room temperature, 2·CO adducts readily give stable carbonyl-carbamoyl complexes [(η5-C5Me5)Rh(CO){(CO(NR')Ph2P(NR)}]+ (4) formed as a result of CO insertion into the Rh-N bond, thus showing high nucleophilicity of the N atoms in 18e̅ complexes. High basicity of the Na+NPN- precursors caused side deprotonation of the η5-C5Me5 ligand during the synthesis of 1 that yields unstable fulvene Rh(I) complexes [(η4-C5Me4CH2)Rh{Ph2P(NR)(NR')2}] (3a,b). Complex 3a undergoes a facile reaction with isoprene to yield an unusual [(η5:η1-C5Me4(CH2)C(Me)═CHCH2)Rh(NPN)] complex─the first example of intermolecular 1,4-metallacycloaddition of diene to the Rh-fulvene complex.

Highly accurate HF dimer ab initio potential energy surface.

A highly accurate, (HF)2 potential energy surface (PES) is constructed based on ab initio calculations performed at the coupled-cluster single double triple level of theory with an aug-cc-pVQZ-F12 basis set at about 152 000 points. A higher correlation correction is computed at coupled-cluster single double triple quadruple level for 2000 points and is considered alongside other more minor corrections due to relativity, core-valence correlation, and Born-Oppenheimer failure. The analytical surface constructed uses 500 constants to reproduce the ab initio points with a standard deviation of 0.3 cm-1. Vibration-rotation-inversion energy levels of the HF dimer are computed for this PES by variational solution of the nuclear-motion Schrödinger equation using the program WAVR4. Calculations over an extended range of rotationally excited states show very good agreement with the experimental data. In particular, the known empirical rotational constants B for the ground vibrational states are predicted to better than about 2 MHz. B constants for excited vibrational states are reproduced several times more accurately than by previous calculations. This level of accuracy is shown to extend to higher excited inter-molecular vibrational states v and higher excited rotational quantum numbers (J, Ka).

M1-derived extracellular vesicles enhance photodynamic therapy and promote immunological memory in preclinical models of colon cancer.

Extracellular vesicles (EVs) are promising drug carriers of photosensitizers for photodynamic therapy (PDT) in cancer treatment, due to their ability to circulate in blood and enter cells efficiently. The therapeutic potential of EVs has been suggested to depend on the type and physiological state of their cell of origin. However, the effects of deriving EVs from various cells in different physiological states on their antitumor capacity are rarely evaluated. In the present study, we compared the antitumor efficacy of EV-mediated PDT by incorporating the photosensitizer Zinc Phthalocyanine (ZnPc) into EVs from multiple cells sources. ZnPc was incorporated by a direct incubation strategy into EVs derived from immune cells (M1-like macrophages and M2-like macrophages), cancer cells (B16F10 melanoma cancer cells) and external sources (milk). Our data show that all EVs are suitable carriers for ZnPc and enable efficient PDT in vitro in co-culture models and in vivo. We observed that EV-mediated PDT initiates immunogenic cell death through the release and exposure of damage associated molecular patterns (DAMPs) on cancer cells, which subsequently induced dendritic cell (DC) maturation. Importantly, of all ZnPc-EVs tested, in absence of light only M1-ZnPc displayed toxicity to MC38, but not to DC, in monoculture and in co-culture, indicating specificity for cancer over immune cells. In MC38 tumor-bearing mice, only M1-ZnPc induced a tumor growth delay compared to control in absence of light. Interestingly, M1- but not M2-mediated PDT, induced complete responses against MC38 tumors in murine models (100% versus 38% of cases, respectively), with survival of all animals up to at least 60 days post inoculation. Finally, we show that all cured animals are protected from a rechallenge with MC38 cells, suggesting the induction of immunological memory after EV-mediated PDT. Together, our data show the importance of the cell type from which the EVs are obtained and highlight the impact of the immunological state of these cells on the antitumor efficacy of EV-mediated PDT.

EARLY METABOLIC DISORDERS AND MORPHOLOGICAL CHANGES OF INTERNAL ORGANS AFTER GASTRIC BYPASS WITH ONE ANASTOMOSIS. EXPERIMENTAL STUDY.

OBJECTIVE: The aim: To investigate morphofunctional changes in bone tissue, mucous membranes of different parts of the intestine, parathyroid glands, as well as changes in blood biochemical parameters depending on the length of the common loop in modeling gastric bypass with one anastomosis within 60 days. PATIENTS AND METHODS: Materials and methods: Modeling of obesity in males of white outbred rats (n = 50) which were induced high-calorie diet with high animal fat content. Weight was measured before the induced diet, 1,2,3 months after the start of the induced diet and 2 months after surgery to assess the dynamics of excess weight loss. Rats were divided into three groups: control group - 5 rats, 1st group - 10 rats, 2nd group - 10 rats, 25 rats were removed from the experiment. RESULTS: Results: The average weight of rats before introduction into the diet was 180.5 grams, after 3 months of induced diet reached 256.7 grams, the average weight gain was 76.2 grams (42.2%). 1 group of rats had a weight loss of -16.2% (41.7 grams), Second group had a weight loss of -20.6% (53.2 grams) 60 days after gastric bypass surgery. CONCLUSION: Conclusions: The average statistical indicators of blood tests of the second group of animals in comparison with the first group show lower levels of iron phosphorus?magne¬sium, total protein and albumin. The average body weight loss of the first group was -16.2% (41.7 grams), the second -20.6% (53.2 grams) with a difference of 11.7 grams, which is not significant.

Diffractive Sensor Elements for Registration of Long-Term Instability at Writing of Computer-Generated Holograms.

The research and development of methods using of the specialized diffractive microstructure sensors embedded in the pattern of computer-generated holograms (CGH) manufactured on circular and X-Y laser writing systems is discussed. These microstructures consist of two parts: one of which is written before the CGH in the field of future hologram and the second one is written during the long-term writing of the CGH. The shift between the first and second part of the microstructure is the trace of the writing errors and allows one to determine and calculate the error of CGH fabrication along both orthogonal coordinates. The developed method is based on the principle of diffraction-based overlay with 1D and 2D built-in diffractive microstructure-sensors. Mathematical modeling and results of experimental test writings of such diffractive microstructure sensors are described. The efficiency of using these types of build-in sensors for the writing errors estimation for CGHs is demonstrated.

Sensitivity Improvement of Phi-OTDR by Fiber Cable Coils.

We present a theoretical and experimental study in which we increased the sensitivity of a phase-sensitive optical time-domain reflectometer (phi-OTDR). This was achieved by constructing coils in the sensor cable, which increased the total amplitude of the impact on the fiber. We demonstrate this theoretically using the example of a phase-sensitive reflectometer model and practically in testing grounds with a buried nearby conventional sensor and a sensor with coils. The sensitivity increased 2.2 times. We detected 95% of events when using coils, versus 20% when using a straight cable. The suggested method does not require any modifications to the device.

THE INVALIDITY OF CONTRACTS IN THE FIELD OF MEDICAL SERVICES AS A WAY TO PROTECT THE RIGHTS OF THE PATIENT.

OBJECTIVE: The aim of this article is to reveal the essential features of contracts providing medical services. The author also focused on the grounds for the invalidity of such contracts - entering into medical services contract without license or permission, prohibition of some medical services or methods of treatment, the imposing of unnecessary medical services, a contradiction to corporate regulations, fraud. A significant part of the work is devoted to the consequences of the invalidity of the contract - the restitution of the money received under the contract and compensation of harm. PATIENTS AND METHODS: Materials and methods: The study is based on the statutory acts of European Union countries, the USA, and others. The author also uses acts of international law in the field of medical services and cases of court practice. CONCLUSION: Conclusions: Although the invalidity of contracts in the field of medical services is not one of the most common ways to protect a patient's rights, it is in many cases essential to the reliable delivery of medical services. The patient has the right to initiate a legal dispute regarding the invalidity of the contract. The restitution of the money paid by the patient, as well as compensation for the harm caused, can be applied not only within the framework of tort law but also under the responsibility for the breach of the contract.

ANATOMO-FUNCTIONAL CLASSIFICATION OF THE HAND POST-TRAUMATIC SOFT TISSUE DEFECTS.

OBJECTIVE: The aim: Develop a classification of post-traumatic soft tissue defects of the hand to standardize the care of this group of patients. And in order to simplify the choice of plastic technic for post-traumatic soft tissue defects of the hand by surgeons and traumatologists. PATIENTS AND METHODS: Materials and methods: We analyzed the treatment of 54 patients with hand injury and PTSTD. 14 physicians used AFC in the choice of surgical method for the closure of PTSTDH. The practical application of AFC and effectiveness of providing medical care to patients with PTSTDH was performed on a ten-point scale by anonymous questioning immediately at the time of discharge from the hospital and 6 months after the injury. RESULTS: Results: The results of the practical application analysis of AFC by physicians were as follows: «convenience¼ - 8.7 ± 0.6; «usefulness¼ is 9.4 ± 0.2. The results of treatment for 54 patients were: hands «functionality¼ - 8,3 ± 0,4, «aesthetics¼ - 7,6 ± 1,7. The results in the retrospective group, in which the AFC was not applied, were as follows: «functionality¼ - 6.8 ± 0.7, and «aesthetics¼ - 5.6 ± 1.1. CONCLUSION: Conclusions: The proposed AFC of PTSTDH help to choose the proper method of surgical management and get good results.

Yeast Sup35 Prion Structure: Two Types, Four Parts, Many Variants.

The yeast [PSI+] prion, formed by the Sup35 (eRF3) protein, has multiple structural variants differing in the strength of nonsense suppressor phenotype. Structure of [PSI+] and its variation are characterized poorly. Here, we mapped Sup35 amyloid cores of 26 [PSI+] ex vivo prions of different origin using proteinase K digestion and mass spectrometric identification of resistant peptides. In all [PSI+] variants the Sup35 amino acid residues 2-32 were fully resistant and the region up to residue 72 was partially resistant. Proteinase K-resistant structures were also found within regions 73-124, 125-153, and 154-221, but their presence differed between [PSI+] isolates. Two distinct digestion patterns were observed for region 2-72, which always correlated with the "strong" and "weak" [PSI+] nonsense suppressor phenotypes. Also, all [PSI+] with a weak pattern were eliminated by multicopy HSP104 gene and were not toxic when combined with multicopy SUP35. [PSI+] with a strong pattern showed opposite properties, being resistant to multicopy HSP104 and lethal with multicopy SUP35. Thus, Sup35 prion cores can be composed of up to four elements. [PSI+] variants can be divided into two classes reliably distinguishable basing on structure of the first element and the described assays.

WNT3a and WNT5a Transported by Exosomes Activate WNT Signaling Pathways in Human Cardiac Fibroblasts.

WNT signaling plays an important role in fibrotic processes in the heart. Recently, exosomes have been proposed as novel extracellular transporters for WNT proteins. In this study, we analyzed whether WNT3a and WNT5a carried by exosomes could activate downstream molecular pathways in human cardiac fibroblasts. Exosomes were isolated from conditioned medium of control, WNT3a- and WNT5a-producing L cells by differential ultracentrifugations. Obtained exosomes showed size ranging between 20⁻150 nm and expressed exosomal markers ALG-2-interacting protein X (ALIX) and CD63. Treatment with WNT3a-rich exosomes inhibited activity of glycogen synthase kinase 3ß (GSK3ß), induced nuclear translocation of ß-catenin, and activated T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factors as well as expression of WNT/ß-catenin responsive genes in cardiac fibroblasts, but did not coactivate extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and activator protein 1 (AP-1) signaling pathways. In contrast, exosomes produced by WNT5a-producing L cells failed to activate ß-catenin-dependent response, but successfully triggered phosphorylation of ERK1/2 and JNK and stimulated IL-6 production. In conclusion, exosomes containing WNT proteins can functionally contribute to cardiac fibrosis by activating profibrotic WNT pathways on cardiac fibroblasts and may represent a novel mechanism of spreading profibrotic signals in the heart.