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Numerous lipids are heterogeneously distributed among organelles. Most lipid trafficking between organelles is achieved by a group of lipid transfer proteins (LTPs) that carry lipids using their hydrophobic cavities. The human genome encodes many intracellular LTPs responsible for lipid trafficking and the function of many LTPs in defining cellular lipid levels and distributions is unclear. Here, we created a gene knockout library targeting 90 intracellular LTPs and performed whole-cell lipidomics analysis. This analysis confirmed known lipid disturbances and identified new ones caused by the loss of LTPs. Among these, we found major sphingolipid imbalances in ORP9 and ORP11 knockout cells, two proteins of previously unknown function in sphingolipid metabolism. ORP9 and ORP11 form a heterodimer to localize at the ER-trans-Golgi membrane contact sites, where the dimer exchanges phosphatidylserine (PS) for phosphatidylinositol-4-phosphate (PI(4)P) between the two organelles. Consequently, loss of either protein causes phospholipid imbalances in the Golgi apparatus that result in lowered sphingomyelin synthesis at this organelle. Overall, our LTP knockout library toolbox identifies various proteins in control of cellular lipid levels, including the ORP9-ORP11 heterodimer, which exchanges PS and PI(4)P at the ER-Golgi membrane contact site as a critical step in sphingomyelin synthesis in the Golgi apparatus.
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Retículo Endoplásmico , Esfingomielinas , Esfingomielinas/metabolismo , Esfingomielinas/biosíntesis , Humanos , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Multimerización de Proteína , Receptores de Esteroides/metabolismo , Receptores de Esteroides/genética , Técnicas de Inactivación de Genes , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatos de Fosfatidilinositol/biosíntesisRESUMEN
The terpolymers of N-vinylpyrrolidone (VP) with acrylic acid and triethylene glycol methacrylate were synthesized with more than 90% yield by radical copolymerization in ethanol from monomeric mixtures of different molar composition (98:2:2, 95:5: 2 and 98:2:5) and their monomer composition, absolute molecular masses and hydrodynamic radii in aqueous media were determined. Using the MTT test, these terpolymers were established to be low toxic for non-tumor Vero cells and HeLa tumor cells. Polymer compositions of hydrophobic dye methyl pheophorbide a (MPP) based on studied terpolymers and linear polyvinylpyrrolidone (PVP) were obtained and characterized in water solution. Quantum-chemical modeling of the MPP-copolymer structures was conducted, and the possibility of hydrogen bond formation between terpolymer units and the MPP molecule was shown. Using fluorescence microscopy, the accumulation and distribution of polymer particles in non-tumor (FetMSC) and tumor (HeLa) cells was studied, and an increase in the accumulation of MPP with both types of particles was found.
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Acrilatos , Humanos , Animales , Chlorocebus aethiops , Acrilatos/química , Células Vero , Células HeLa , Sistemas de Liberación de Medicamentos , Pirrolidinonas/química , Metacrilatos/química , Polietilenglicoles/química , Polímeros/química , Polímeros/síntesis química , Supervivencia Celular/efectos de los fármacosRESUMEN
Indigenous animal genetic resources should be preserved because of their well adaptation to the environment, their tolerance to low food availability and their sociocultural importance. The characterization of the quality of the products generated by heritage breeds may bring more arguments to encourage the raising of these animals. This study aimed at evaluating the egg performance and quality of Spanish indigenous Black Castellana (BC) breed as compared with a selected strain (Lohmann LSL-Classic). Four groups of 30 hens were arranged: 1) Lohmann hens fed a control diet; 2) BC hens fed the control diet; 3) Lohmann hens fed a diet including linseed at 70 g/kg (omega-3 diet); 4) BC hens fed the omega-3 diet. Egg production was higher by 12.3% for Lohmann hens but, since BC eggs were heavier by 15.4%, no effect of genetics was found on daily egg mass. Feed intake was higher by 5.0% for BC hens. Nonetheless, no difference was detected for feed conversion ratio. Eggshell was thicker by 6.78% in Lohmann eggs. Haugh units did not differ among freshly laid and stored eggs in Lohmann hens, whereas Haugh units decreased in stored BC eggs (80.5 vs. 76.7 vs. 72.3 at 0, 14, and 30 d of storage). Yolks of BC eggs contained less fat (57.5 vs. 60.8% DM), more protein (32.8 vs. 31.9% DM) and more cholecalciferol (1.25 vs. 1.22 µg/g DM), and showed lower proportion of saturated fatty acids (29.0 vs. 37.0%) and higher proportions of monounsaturated (45.7 vs. 39.6%) and polyunsaturated (25.2 vs. 23.4%) fatty acids. Feeding the omega-3 diet reduced the yolk proportions of saturated (32.5 vs. 33.5%) and monounsaturated (42.0 vs. 43.3%) fatty acids and increased those of polyunsaturated (25.4 vs. 23.2%) and ω-3 (7.05 vs. 2.42%) fatty acids. No effect due to genetics or diet was found on yolk color score or on yolk content in cholesterol, cobalamin, retinol and γ-tocopherol. This study represents the first exhaustive characterization of eggs from Spanish indigenous Black Castellana breed.
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The pericellular matrix (PCM), with its hallmark proteins collagen type VI (COLVI) and fibronectin (FN), surrounds chondrocytes and is critical in transducing the biomechanical cues. To identify genetic variants that change protein function, exome sequencing is performed in a patient with symptomatic OA at multiple joint sites. A predicted damaging variant in COL6A3 is identified and introduced by CRISPR-Cas9 genome engineering in two established human induced pluripotent stem cell-derived in-vitro neocartilage organoid models. The downstream effects of the COL6A3 variant on the chondrocyte phenotypic state are studied by a multi-omics (mRNA and lncRNA) approach in interaction with hyper-physiological mechanical loading conditions. The damaging variant in COL6A3 results in significantly lower binding between the PCM proteins COLVI and FN and provokes an osteoarthritic chondrocyte state. By subsequently exposing the neocartilage organoids to hyperphysiological mechanical stress, it is demonstrated that the COL6A3 variant in chondrocytes abolishes the characteristic inflammatory signaling response after mechanical loading with PTGS2, PECAM1, and ADAMTS5, as central genes. Finally, by integrating epigenetic regulation, the lncRNA MIR31HG is identified as key regulator of the characteristic inflammatory signaling response to mechanical loading.
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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.
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Lipid nanoparticle (LNP) remains the most advanced platform for messenger RNA (mRNA) delivery. To date, mRNA LNPs synthesis is mostly performed by mixing lipids and mRNA with microfluidics. In this study, a cost-effective microfluidic setup for synthesizing mRNA LNPs is developed. It allows to fine-tune the LNPs characteristics without compromising LNP properties. It is compared with a commercial device (NanoAssemblr) and ethanol injection and the influence of manufacturing conditions on the performance of mRNA LNPs is investigated. LNPs prepared by ethanol injection exhibit broader size distributions and more inhomogeneous internal structure (e.g., bleb-like substructures), while other LNPs show uniform structure with dense cores. Small angel X-ray scattering (SAXS) data indicate a tighter interaction between mRNA and lipids within LNPs synthesized by custom device, compared to LNPs produced by NanoAssemblr. Interestingly, the better transfection efficiency of polysarcosine (pSar)-modified LNPs correlates with a higher surface roughness than that of PEGylated ones. The manufacturing approach, however, shows modest influence on mRNA expression in vivo. In summary, the home-developed cost-effective microfluidic device can synthesize LNPs and represents a potent alternative to NanoAssemblr. The preparation methods show notable effect on LNPs' structure but a minor influence on mRNA delivery in vitro and in vivo.
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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.
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Interacciones Huésped-Parásitos , Caracoles , Trematodos , Animales , Trematodos/fisiología , Trematodos/metabolismo , Caracoles/parasitología , Metaboloma , Metabolómica , Cromatografía de Gases y Espectrometría de MasasRESUMEN
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.
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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.
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Hemorragia Cerebral , Enfermedades de los Pequeños Vasos Cerebrales , Trastornos del Movimiento , Linaje , Humanos , Femenino , Masculino , Enfermedades de los Pequeños Vasos Cerebrales/genética , Enfermedades de los Pequeños Vasos Cerebrales/patología , Enfermedades de los Pequeños Vasos Cerebrales/diagnóstico por imagen , Persona de Mediana Edad , Hemorragia Cerebral/genética , Hemorragia Cerebral/patología , Hemorragia Cerebral/diagnóstico por imagen , Trastornos del Movimiento/genética , Trastornos del Movimiento/patología , Trastornos del Movimiento/diagnóstico por imagen , Imagen por Resonancia Magnética , Alelos , Adulto , Anciano , Sistema Glinfático/patología , Sistema Glinfático/diagnóstico por imagen , Secuenciación del Exoma , Encéfalo/patología , Encéfalo/diagnóstico por imagen , Aminohidrolasas/genéticaRESUMEN
The development of resistance to chemotherapy is one of the main problems for effective cancer treatment. Drug resistance may result from disturbances in two important physiological processes-cell proliferation and cell death. Importantly, both processes characterize alterations in cell metabolism, the level of which is often measured using MTT/MTS assays. To examine resistance to chemotherapy, different cancer cell lines are usually used for the in vitro modulation of developing resistance. However, after the creation of resistant cell lines, researchers often have difficulty in starting investigations of the mechanisms of insensitivity. In the first stage, researchers should address the question of whether the drug resistance results from a depression of cell proliferation or an inhibition of cell death. To simplify the choice of research strategy, we have suggested a combination of different approaches which reveal the actual mechanism. This combination includes rapid and high-throughput methods such as the MTS test, the LIVE/DEAD assay, real-time cell metabolic analysis, and Western blotting. To create chemoresistant tumor cells, we used four different cancer cell lines of various origins and utilized the most clinically relevant pulse-selection approach. Applying a set of methodological approaches, we demonstrated that three of them were more capable of modulating proliferation to avoid the cytostatic effects of anti-cancer drugs. At the same time, one of the studied cell lines developed resistance to cell death, overcoming the cytotoxic action.
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Antineoplásicos , Neoplasias , Antineoplásicos/farmacología , Resistencia a Antineoplásicos , Apoptosis , Muerte Celular , Proliferación CelularRESUMEN
Reliable reconstruction of AFM tip geometry is not an easy task. The shape of the tip can be (partially) reconstructed from the AFM image of a calibration sample using a blind reconstruction method. The result is a range of the tip shapes (from unrealistically sharp to blunt). A criterion for selecting the true shape is proposed. It is based on reconstruction of the calibration relief. Further refining the probe geometry by indenting an elastic polymer is discussed. The choice of the test sample is another problem. In practice, the researcher has a small number of commercially available test gratings. Their limitations are highlighted. An approximation of tip geometry by a body of revolution is required in indentation experiments. A hyperboloid was found to be the closest approximation to the conventional probes. The contact area was obtained for the hyperboloids with different parameters indenting an elastic material. RESEARCH HIGHLIGHTS: Criteria for blind reconstruction algorithm of AFM tip shape. Pros and cons of tip shape calibration samples. Contact area of the elastic indentation by hyperbolic indenter.
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Metal-Organic CVD method (MOCVD) allows for deposition of ultrathin 2D transition metal dichalcogenides (TMD) films of electronic quality onto wafer-scale substrates. In this work, the effect of temperature on structure, chemical states, and electronic qualities of the MOCVD MoS2 films were investigated. The results demonstrate that the temperature increase in the range of 650 °C to 950 °C results in non-monotonic average crystallite size variation. Atomic force microscopy (AFM), transmission electron microscopy (TEM), and Raman spectroscopy investigation has established the film crystal structure improvement with temperature increase in this range. At the same time, X-Ray photoelectron spectroscopy (XPS) method allowed to reveal non-stoichiometric phase fraction increase, corresponding to increased sulfur vacancies (VS) concentration from approximately 0.9 at.% to 3.6 at.%. Established dependency between the crystallite domains size and VS concentration suggests that these vacancies are form predominantly at the grain boundaries. The results suggest that an increased Vs concentration and enhanced charge carriers scattering at the grains' boundaries should be the primary reasons of films' resistivity increase from 4 kΩ·cm to 39 kΩ·cm.
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Cancers evade T-cell immunity by several mechanisms such as secretion of anti-inflammatory cytokines, down regulation of antigen presentation machinery, upregulation of immune checkpoint molecules, and exclusion of T cells from tumor tissues. The distribution and function of immune checkpoint molecules on tumor cells and tumor-infiltrating leukocytes is well established, but less is known about their impact on intratumoral endothelial cells. Here, we demonstrated that V-domain Ig suppressor of T-cell activation (VISTA), a PD-L1 homolog, was highly expressed on endothelial cells in synovial sarcoma, subsets of different carcinomas, and immune-privileged tissues. We created an ex vivo model of the human vasculature and demonstrated that expression of VISTA on endothelial cells selectively prevented T-cell transmigration over endothelial layers under physiologic flow conditions, whereas it does not affect migration of other immune cell types. Furthermore, endothelial VISTA correlated with reduced infiltration of T cells and poor prognosis in metastatic synovial sarcoma. In endothelial cells, we detected VISTA on the plasma membrane and in recycling endosomes, and its expression was upregulated by cancer cell-secreted factors in a VEGF-A-dependent manner. Our study reveals that endothelial VISTA is upregulated by cancer-secreted factors and that it regulates T-cell accessibility to cancer and healthy tissues. This newly identified mechanism should be considered when using immunotherapeutic approaches aimed at unleashing T cell-mediated cancer immunity.
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Antígenos B7 , Sarcoma Sinovial , Humanos , Células Endoteliales/metabolismo , Endotelio/metabolismo , Proteínas de Punto de Control Inmunitario , Linfocitos TRESUMEN
Diabetes is one of the significant risk factors for ischemic stroke. Hyperglycemia exacerbates the pathogenesis of stroke, leading to more extensive cerebral damage and, as a result, to more severe consequences. However, the mechanism whereby the hyperglycemic status in diabetes affects biochemical processes during the development of ischemic injury is still not fully understood. In the present work, we record for the first time the real-time dynamics of H2O2 in the matrix of neuronal mitochondria in vitro in culture and in vivo in the brain tissues of rats during development of ischemic stroke under conditions of hyperglycemia and normal glucose levels. To accomplish this, we used a highly sensitive HyPer7 biosensor and a fiber-optic interface technology. We demonstrated that a high glycemic status does not affect the generation of H2O2 in the tissues of the ischemic core, while significantly exacerbating the consequences of pathogenesis. For the first time using Raman microspectroscopy approach, we have shown how a sharp increase in the blood glucose level increases the relative amount of reduced cytochromes in the mitochondrial electron transport chain in neurons under normal conditions in awake mice.
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Isquemia Encefálica , Diabetes Mellitus , Hiperglucemia , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Ratas , Ratones , Animales , Peróxido de Hidrógeno , Accidente Cerebrovascular/patología , Hiperglucemia/patología , Isquemia Encefálica/patologíaRESUMEN
Gametogenesis is a complex and sex-specific multistep process during which the gonadal somatic niche plays an essential regulatory role. One of the most crucial steps during human female gametogenesis is the formation of primordial follicles, the functional unit of the ovary that constitutes the pool of follicles available at birth during the entire reproductive life. However, the relation between human fetal germ cells (hFGCs) and gonadal somatic cells during the formation of the primordial follicles remains largely unexplored. We have discovered that hFGCs can form multinucleated syncytia, some connected via interconnecting intercellular bridges, and that not all nuclei in hFGC-syncytia were synchronous regarding meiotic stage. As hFGCs progressed in development, pre-granulosa cells formed protrusions that seemed to progressively constrict individual hFGCs, perhaps contributing to separate them from the multinucleated syncytia. Our findings highlighted the cell-cell interaction and molecular dynamics between hFGCs and (pre)granulosa cells during the formation of primordial follicles in humans. Knowledge on how the pool of primordial follicle is formed is important to understand human infertility.
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Comunicación Celular , Ovario , Recién Nacido , Masculino , Humanos , Femenino , Núcleo Celular , Gametogénesis , Células GerminativasRESUMEN
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%.
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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.
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Filamentos Intermedios , Estrés Proteotóxico , Filamentos Intermedios/metabolismo , Vimentina/genética , Vimentina/metabolismo , Estrés del Retículo Endoplásmico , Retículo Endoplásmico/metabolismo , AutofagiaRESUMEN
Heterogeneous nanostructures composed of metastable tetragonal 1T-MoS2 and stable hexagonal 2H-MoS2 phases are highly promising for a wide range of applications, including catalysis and ion batteries, due to the high electrical conductivity and catalytic activity of the 1T phase. However, a controllable synthesis of stabilized 1T-MoS2 films over the wafer-scale area is challenging. In this work, a metal-organic chemical vapor deposition process allowing us to obtain ultrathin MoS2 films containing both 1T and 2H phases and control their ratio through rhenium doping was suggested. As a result, Mo1-xRexS2 films with a 1T-MoS2 fraction up to ≈30% were obtained, which were relatively stable under normal conditions for a long time. X-ray photoelectron spectroscopy and Raman spectroscopy also indicated that the 1T-MoS2 phase fraction increased with rhenium concentration increase saturating at Re concentrations above 5 at. %. Also, its concentration was found to significantly affect the film resistivity. Thus, the resistivity of the film containing approximately 30% of the 1T phase was about 130 times lower than that of the film without the 1T phase.
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Sternorrhyncha representatives are serious pests of agriculture and forestry all over the world, primarily causing damage to woody plants. Sternorrhyncha members are vectors for the transfer of a large number of viral diseases, and subsequently, the host plant weakens. Additionally, many are inherent in the release of honeydew, on which fungal diseases develop. Today, an innovative approach is needed to create new and effective ways to control the number of these insects based on environmentally friendly insecticides. Of particular relevance to such developments is the need to take into account the large number of organisms living together with insect pests in this group, including beneficial insects. Practically without changing their location on their host plant, they adopted to be more invisible and protected due to their small size, symbiosis with ants, the ability to camouflage with a leaf, and moderately deplete plants and others, rarely leading them to death but still causing substantial economic loss in the subtropics and tropics. Due to the lack of presence in the literature, this review fills in this pesky spot by examining (on the example of distinct species from four superfamilies) the characteristic adaptations for this suborder and the chemical methods of combating these insects that allow them to survive in various environmental conditions, suggesting new and highly promising ways of using olinscides for plant protection against Sternorrhyncha members.