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The safety of titanium dioxide (TiO2), widely used in foods and personal care products, has been of ongoing concern. Significant toxicity of TiO2 has been reported, suggesting a risk to human health. To evaluate its potential epigenotoxicity, the effect of exposure to a TiO2 product to which humans could be exposed on DNA methylation, a primary epigenetic mechanism, was investigated using two human cell lines (Caco-2 (colorectal) and HepG2 (liver)) relevant to human exposure. Global methylation was determined by enzyme-linked immunosorbent assay-based immunochemical analysis. Gene promoter methylation was evaluated using EpiTect Methyl II Signature PCR System Array technology. Expression of DNA methyltransferases, MBD2, and URHF1 was quantified by qRT-PCR. A decrease in global DNA methylation was observed in both cell lines. Across the cell lines, seven genes (BNIP3, DNAJC15, GADD45G, GDF15, INSIG1, SCARA3, and TP53) were identified in which promoters were methylated. Changes in promoter methylation were associated with gene expression. Results also revealed aberrant expression of regulatory genes, DNA methyltransferases, MBD2, and UHRF1. Findings from the study clearly demonstrate the impact of TiO2 exposure on DNA methylation in two cell types, supporting the potential involvement of this epigenetic mechanism in its biological responses. Hence, epigenetic studies are critical for complete assessment of potential risk from exposure. [ABSTRACT FROM AUTHOR]

Трёхмерные клеточные модели активно используются в качестве объекта исследований в данное время. Клеточные сфероиды являются особо привлекательным объектом в связи с универсальностью, дешевизной и простотой применения. Определение размера трёхмерных клеточных культур является существенным аспектом при изучении характеристик и поведения трёхмерных клеточных моделей. Ручное определение морфологических характеристик является долгим и трудоёмким процессом. Цель исследования – анализ размера трёхмерных клеточных моделей с помощью технологий компьютерного зрения. Данный способ реализован на языке программирования Python с использованием библиотеки OpenCV. Предлагаемый способ был применён для анализа изображений трёхмерных клеточных моделей, полученных из разных клеточных линий и клеточных концентраций.

Although fossil fuels are widely used to meet energy needs, intensive research has been carried out in recent years on hydrogen production from renewable sources due to their decrease over time and environmental pollution concerns. Biofuel cell technology is one of the promising current technologies. It has been proven that various microorganisms produce energy through their natural metabolism, and that energy production is produced in biofuel cells by exoelectrogenic microorganisms that can transfer electrons to an electrode surface. Although it has been stated that employing human cells to generate energy is feasible, it is unknown whether doing so would enable the production of hydrogen. Within the scope of this perspective article, the issue of hydrogen production in bioelectrolysis cells using human cells will be discussed for the first time. Optimizing hydrogen production in bioelectrolysis cells using human cells is important in terms of contributing to hydrogen technologies. Within the scope of the article, promising human cell lines for hydrogen production are emphasized and hydrogen production potentials in bioelectrolysis cells using these cell lines are discussed. In conclusion, some human cells can be used for hydrogen gas production in bioelectrolysis cells due to their bioelectrochemical and metabolic properties. [ABSTRACT FROM AUTHOR]

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19 and responsible for the global coronavirus pandemic which started in 2019. Despite exhaustive efforts to trace its origins, including potential links with pangolins and bats, the precise origins of the virus remain unclear. Bats have been recognized as natural hosts for various coronaviruses, including the Middle East respiratory coronavirus (MERS-CoV) and the SARS-CoV. This study presents a comparative analysis of the SARS-CoV-2 nucleocapsid protein (N) interactome in human and bat cell lines. We identified approximately 168 cellular proteins as interacting partners of SARS-CoV-2 N in human cells and 196 cellular proteins as interacting partners with this protein in bat cells. The results highlight pathways and events that are both common and unique to either bat or human cells. Understanding these interactions is crucial to comprehend the reasons behind the remarkable resilience of bats to viral infections. This study provides a foundation for a deeper understanding of host–virus interactions in different reservoirs. [ABSTRACT FROM AUTHOR]

Capillary zone electrophoresis-mass spectrometry (CZE-MS) has been recognized as a valuable technique for the proteomics of mass-limited biological samples (i.e., single cells). However, its broad adoption for single cell proteomics (SCP) of human cells has been impeded by the low sample loading capacity of CZE, only allowing us to use less than 5% of the available peptide material for each measurement. Here we present a reversed-phase-based solid-phase microextraction (RP-SPME)-CZE-MS platform to solve the issue, paving the way for SCP of human cells using CZE-MS. The RP-SPME-CZE system was constructed in one fused silica capillary with zero dead volume for connection via in situ synthesis of a frit, followed by packing C8 beads into the capillary to form a roughly 2 mm long SPME section. Peptides captured by SPME were eluted with a buffer containing 30% (v/v) acetonitrile and 50 mM ammonium acetate (pH 6.5), followed by dynamic pH junction-based CZE-MS. The SPME-CZE-MS enabled the injection of nearly 40% of the available peptide sample for each measurement. The system identified 257 ± 24 proteins and 523 ± 69 peptides (N = 2) using a Q-Exactive HF mass spectrometer when only 0.25 ng of a commercial HeLa cell digest was available in the sample vial and 0.1 ng of the sample was injected. The amount of available peptide is equivalent to the protein mass of one HeLa cell. The data indicate that SPME-CZE-MS is ready for SCP of human cells. [ABSTRACT FROM AUTHOR]

Microplastics and nanoplastics (MNPs) are becoming an increasingly severe global problem due to their widespread distribution and complex impact on living organisms. Apart from their environmental impact, the effects of MNPs on living organisms have also continued to attract attention. The harmful impact of MNPs has been extensively documented in marine invertebrates and larger marine vertebrates like fish. However, the research on the toxicity of these particles on mammals is still limited, and their possible effects on humans are poorly understood. Considering that MNPs are commonly found in food or food packaging, humans are primarily exposed to them through ingestion. It would be valuable to investigate the potential harmful effects of these particles on gut health. This review focuses on recent research exploring the toxicological impacts of micro- and nanoplastics on the gut, as observed in human cell lines and mammalian models. Available data from various studies indicate that the accumulation of MNPs in mammalian models and human cells may result in adverse consequences, in terms of epithelial toxicity, immune toxicity, and the disruption of the gut microbiota. The paper also discusses the current research limitations and prospects in this field, aiming to provide a scientific basis and reference for further studies on the toxic mechanisms of micro- and nanoplastics.

Hereditary spastic paraplegias (HSPs) are a heterogeneous group of mono-genetic inherited neurological disorders, whose primary manifestation is the disruption of the pyramidal system, observed as a progressive impaired gait and leg spasticity in patients. Despite the large list of genes linked to this group, which exceeds 80 loci, the number of cellular functions which the gene products engage is relatively limited, among which endoplasmic reticulum (ER) morphogenesis appears central. Mutations in genes encoding ER-shaping proteins are the most common cause of HSP, highlighting the importance of correct ER organisation for long motor neuron survival. However, a major bottleneck in the study of ER morphology is the current lack of quantitative methods, with most studies to date reporting, instead, on qualitative changes. Here, we describe and apply a quantitative image-based screen to identify genetic modifiers of ER organisation using a mammalian cell culture system. An analysis reveals significant quantitative changes in tubular ER and dense sheet ER organisation caused by the siRNA-mediated knockdown of HSP-causing genes ATL1 and RTN2. This screen constitutes the first attempt to examine ER distribution in cells in an automated and high-content manner and to detect genes which impact ER organisation. [ABSTRACT FROM AUTHOR]

DNA replication is a fundamental process ensuring the maintenance of the genome each time cells divide. This is particularly relevant early in development when cells divide profusely, later giving rise to entire organs. Here, we analyze and compare the genome replication progression in human embryonic stem cells, induced pluripotent stem cells, and differentiated cells. Using single-cell microscopic approaches, we map the spatio-temporal genome replication as a function of chromatin marks/compaction level. Furthermore, we mapped the replication timing of subchromosomal tandem repeat regions and interspersed repeat sequence elements. Albeit the majority of these genomic repeats did not change their replication timing from pluripotent to differentiated cells, we found developmental changes in the replication timing of rDNA repeats. Comparing single-cell super-resolution microscopic data with data from genome-wide sequencing approaches showed comparable numbers of replicons and large overlap in origins numbers and genomic location among developmental states with a generally higher origin variability in pluripotent cells. Using ratiometric analysis of incorporated nucleotides normalized per replisome in single cells, we uncovered differences in fork speed throughout the S phase in pluripotent cells but not in somatic cells. Altogether, our data define similarities and differences on the replication program and characteristics in human cells at different developmental states. [ABSTRACT FROM AUTHOR]

Infrared spectroscopy has emerged as a promising technique for studying the composition of biological samples like lipids that play important roles in cellular functions and are involved in various diseases. For this reason, lipids are a target of interest in many biomedical studies. The objective of the present study is to utilize Fourier-Transform Infrared (FT-IR) spectroscopy to examine the main lipid components of human cells (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, C18 ceramide, sphingosine-1-phosphate, ceramide-1-phosphate, sphingomyelin, cholesterol, and triolein). FT-IR analysis on the previously mentioned lipid samples was performed in Attenuated Total Reflection (ATR) mode. The obtained spectra clearly evidence the contributions of the different functional groups that are present in the examined samples. Detailed assignments of spectral features were carried out in agreement with the literature. Similarities and differences among the different types of commercial lipid samples are evidenced and discussed, with particular attention to phospholipid and sphingolipid components. A quantitative analysis of phosphatidylinositol and sphingomyelin spectra using a ratiometric approach is reported. Moreover, a reconstruction procedure of FT-IR spectra of complex lipids useful for chemometrics applications is described. These representative examples of the potential use of the results of the present study can certainly contribute to a larger use of FT-IR spectroscopy in lipidomics.

In this study, we investigated the properties of ascorbic acid (vitamin C), which is a naturally occurring water-soluble vitamin. Our goal is to evaluate its pro-oxidative and/or antioxidant capabilities. To do this, we initially used a confocal laser scanning microscope (CLSM) to visualize the differentiation pattern in U-937 cells under the treatment of variable concentrations of ascorbic acid. Prior to induction, U-937 cells showed a spherical morphology. After treatment, significant morphological changes were observed in the form of prominent pseudopodia and amoeboid structures. Interestingly, pseudopodia incidences increased with an increase in ascorbic acid concentrations. In addition, our analysis of protein modification using anti-malondialdehyde antibodies showed changes in more than one protein. The findings reveal the link between the differentiation of U-937 cells into macrophages and the protein modifications triggered by the production of reactive oxygen species when U-937 cells are exposed to ascorbic acid. Furthermore, the transformation of ascorbic acid from a pro-oxidative to an antioxidant property is also demonstrated.

One of the ways to regulate the sensitivity of human cells to the influenza virus is to knock out genes of the innate immune response. Promising targets for the knockout are genes of the interferon-inducible transmembrane protein (IFITM) family, in particular the IFITM3 gene, whose product limits the entry of a virus into the cell by blocking the fusion of the viral and endosomal membranes. In this study, by means of genome-editing system CRISPR/Cas9, monoclonal cell lines with an IFITM3 knockout were obtained based on WI-38 VA13 cells (human origin). It was found that such cell lines are more sensitive to infection by influenza A viruses of various subtypes. Nevertheless, this feature is not accompanied by an increased titer of newly formed viral particles in a culture medium. [ABSTRACT FROM AUTHOR]

Abstract Background Neuronal networks receive and deliver information to regulate bodily functions while the vascular network provides oxygen, nutrients, and signaling molecules to tissues. Neurovascular interactions are vital for both tissue development and maintaining homeostasis in adulthood; these two network systems align and reciprocally communicate with one another. Although communication between network systems has been acknowledged, the lack of relevant in vitro models has hindered research at the mechanistic level. For example, the current used in vitro neurovascular models are typically established to be short-term (≤ 7 days) culture models, and they miss the supporting vascular mural cells. Methods In this study, we utilized human induced pluripotent stem cell (hiPSC) -derived neurons, fluorescence tagged human umbilical vein endothelial cells (HUVECs), and either human bone marrow or adipose stem/stromal cells (BMSCs or ASCs) as the mural cell types to create a novel 3D neurovascular network-on-a-chip model. Collagen 1–fibrin matrix was used to establish long-term (≥ 14 days) 3D cell culture in a perfusable microphysiological environment. Results Aprotinin-supplemented endothelial cell growth medium-2 (EGM-2) supported the simultaneous formation of neuronal networks, vascular structures, mural cell differentiation, and the stability of the 3D matrix. The formed neuronal and vascular networks were morphologically and functionally characterized. Neuronal networks supported vasculature formation based on direct cell contacts and by dramatically increasing the secretion of angiogenesis-related factors in multicultures in contrast to cocultures without neurons. Both utilized mural cell types supported the formation of neurovascular networks; however, the BMSCs seemed to boost neurovascular networks to greater extent. Conclusions Overall, our study provides a novel human neurovascular network model that is applicable for creating in vivo-like tissue models with intrinsic neurovascular interactions. The 3D neurovascular network model on chip forms an initial platform for the development of vascularized and innervated organ-on-chip and further body-on-chip concepts and offers the possibility for mechanistic studies on neurovascular communication both under healthy and in disease conditions. Video Abstract

Additive manufacturing (3D printing) has been deployed across multiple platforms to fabricate bioengineered tissues. We demonstrate the use of a Thermal Inkjet Pipette System (TIPS) for targeted delivery of cells onto manufactured substrates to design bio-bandages. Two cell lines – HEK 293 (kidney) and K7M2 wt (bone) – were applied using TIPS. We demonstrate a novel means for targeted cell delivery to a hydrogel support structure. These cell/support constructs (bio-bandages) had a high viability for survival and growth over extended periods. Combining a flexible biosupport with application of cells via TIPS printing now for the first time allows for custom cell substrate constructs with various densities to be deployed for regenerative medicine applications.

Plants are an inexhaustible source of bioactive compounds beneficial for contrasting oxidative stress, leading to many degenerative pathologies. Brassica rapa L. subsp. rapa is well known for its nutraceutical properties among edible vegetable species. In our work, we aimed to explore an eco-friendly way to enhance the beneficial dietary phytochemicals in this vast world of crop-growing plants at selected light quality conditions. White broad-spectrum (W) and red–blue (RB) light regimes were used for growing brassica microgreens. The organic extracts were tested on keratinocytes upon oxidative stress to explore their capability to act as natural antioxidant cell protectors. Our results show that both W and RB extracts caused a notable reduction in reactive oxygen species (ROS) levels induced by H2O2. Interestingly, according to its higher contents of polyphenols and flavonoids, the RB was more efficient in reducing ROS amount and DNA damage than the W extract, particularly at the lowest concentration tested. However, at higher concentrations (up to 100 μg/mL), the antioxidant effect reached a plateau, and there was little added benefit. These findings confirm that RB light effectively increases the antioxidant compounds in Brassica rapa L. microgreens, thus contributing to their enhanced activity against oxidative-induced genotoxicity compared to microgreens grown under W light. [ABSTRACT FROM AUTHOR]

Infrared spectroscopy has emerged as a promising technique for studying the composition of biological samples like lipids that play important roles in cellular functions and are involved in various diseases. For this reason, lipids are a target of interest in many biomedical studies. The objective of the present study is to utilize Fourier-Transform Infrared (FT-IR) spectroscopy to examine the main lipid components of human cells (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, C18 ceramide, sphingosine-1-phosphate, ceramide-1-phosphate, sphingomyelin, cholesterol, and triolein). FT-IR analysis on the previously mentioned lipid samples was performed in Attenuated Total Reflection (ATR) mode. The obtained spectra clearly evidence the contributions of the different functional groups that are present in the examined samples. Detailed assignments of spectral features were carried out in agreement with the literature. Similarities and differences among the different types of commercial lipid samples are evidenced and discussed, with particular attention to phospholipid and sphingolipid components. A quantitative analysis of phosphatidylinositol and sphingomyelin spectra using a ratiometric approach is reported. Moreover, a reconstruction procedure of FT-IR spectra of complex lipids useful for chemometrics applications is described. These representative examples of the potential use of the results of the present study can certainly contribute to a larger use of FT-IR spectroscopy in lipidomics. [ABSTRACT FROM AUTHOR]

Abstract Background Cas12a (formerly known as Cpf1), the class II type V CRISPR nuclease, has been widely used for genome editing in mammalian cells and plants due to its distinct characteristics from Cas9. Despite being one of the most robust Cas12a nucleases, LbCas12a in general is less efficient than SpCas9 for genome editing in human cells, animals, and plants. Results To improve the editing efficiency of LbCas12a, we conduct saturation mutagenesis in E. coli and identify 1977 positive point mutations of LbCas12a. We selectively assess the editing efficiency of 56 LbCas12a variants in human cells, identifying an optimal LbCas12a variant (RVQ: G146R/R182V/E795Q) with the most robust editing activity. We further test LbCas12a-RV, LbCas12a-RRV, and LbCas12a-RVQ in plants and find LbCas12a-RV has robust editing activity in rice and tomato protoplasts. Interestingly, LbCas12a-RRV, resulting from the stacking of RV and D156R, displays improved editing efficiency in stably transformed rice and poplar plants, leading to up to 100% editing efficiency in T 0 plants of both plant species. Moreover, this high-efficiency editing occurs even at the non-canonical TTV PAM sites. Conclusions Our results demonstrate that LbCas12a-RVQ is a powerful tool for genome editing in human cells while LbCas12a-RRV confers robust genome editing in plants. Our study reveals the tremendous potential of these LbCas12a variants for advancing precision genome editing applications across a wide range of organisms.

In recent years, sphingolipids have attracted significant attention due to their pivotal role in cellular functions and physiological diseases. A valuable tool for investigating the characteristics of sphingolipids can be represented via FT-IR spectroscopy, generally recognized as a very powerful technique that provides detailed biochemical information on the examined sample with the unique properties of sensitivity and accuracy. In the present paper, some fundamental aspects of sphingolipid components of human cells are summarized, and the most relevant articles devoted to the FT-IR spectroscopic studies of sphingolipids are revised. A short description of different FT-IR experimental approaches adopted for investigating sphingolipids is also given, with details about the most commonly used data analysis procedures. The present overview of FT-IR investigations, although not exhaustive, attests to the relevant role this vibrational technique has played in giving significant insight into many aspects of this fascinating class of lipids.

Mutations in the small genome present in mitochondria often result in severe pathologies. Different genetic strategies have been explored, aiming to rescue such mutations. A number of these strategies were based on the capacity of human mitochondria to import RNAs from the cytosol and designed to repress the replication of the mutated genomes or to provide the organelles with wild-type versions of mutant transcripts. However, the mutant RNAs present in mitochondria turned out to be an obstacle to therapy and little attention has been devoted so far to their elimination. Here, we present the development of a strategy to knockdown mitochondrial RNAs in human cells using the transfer RNA-like structure of Brome mosaic virus or Tobacco mosaic virus as a shuttle to drive trans -cleaving ribozymes into the organelles in human cell lines. We obtained a specific knockdown of the targeted mitochondrial ATP6 mRNA, followed by a deep drop in ATP6 protein and a functional impairment of the oxidative phosphorylation chain. Our strategy provides a powerful approach to eliminate mutant organellar transcripts and to analyse the control and communication of the human organellar genetic system. [ABSTRACT FROM AUTHOR]

Background: Neuronal networks receive and deliver information to regulate bodily functions while the vascular network provides oxygen, nutrients, and signaling molecules to tissues. Neurovascular interactions are vital for both tissue development and maintaining homeostasis in adulthood; these two network systems align and reciprocally communicate with one another. Although communication between network systems has been acknowledged, the lack of relevant in vitro models has hindered research at the mechanistic level. For example, the current used in vitro neurovascular models are typically established to be short-term (≤ 7 days) culture models, and they miss the supporting vascular mural cells. Methods: In this study, we utilized human induced pluripotent stem cell (hiPSC) -derived neurons, fluorescence tagged human umbilical vein endothelial cells (HUVECs), and either human bone marrow or adipose stem/stromal cells (BMSCs or ASCs) as the mural cell types to create a novel 3D neurovascular network-on-a-chip model. Collagen 1–fibrin matrix was used to establish long-term (≥ 14 days) 3D cell culture in a perfusable microphysiological environment. Results: Aprotinin-supplemented endothelial cell growth medium-2 (EGM-2) supported the simultaneous formation of neuronal networks, vascular structures, mural cell differentiation, and the stability of the 3D matrix. The formed neuronal and vascular networks were morphologically and functionally characterized. Neuronal networks supported vasculature formation based on direct cell contacts and by dramatically increasing the secretion of angiogenesis-related factors in multicultures in contrast to cocultures without neurons. Both utilized mural cell types supported the formation of neurovascular networks; however, the BMSCs seemed to boost neurovascular networks to greater extent. Conclusions: Overall, our study provides a novel human neurovascular network model that is applicable for creating in vivo-like tissue models with intrinsic neurovascular interactions. The 3D neurovascular network model on chip forms an initial platform for the development of vascularized and innervated organ-on-chip and further body-on-chip concepts and offers the possibility for mechanistic studies on neurovascular communication both under healthy and in disease conditions. DtMPMfM_nPvk_pFj8pUo8r Video Abstract [ABSTRACT FROM AUTHOR]

Fourier transform infrared (FTIR) spectroscopy is a powerful tool for analyzing the biochemical properties of biological samples such as proteins, cellular materials, and tissues. It provides objective information on samples and has been adopted in many research areas of biomedical and biotechnological interest. FTIR spectroscopy can be performed using different approaches at the macro and micro levels allowing the examination of an incredibly broad class of materials. However, it has become evident that the choice of proper spectra acquisition geometries and the modalities of sample preparation in FTIR spectroscopy analysis require special consideration, especially for certain classes of materials such as cells and tissues. In the present paper, we described the different procedures used for preparing and analyzing different types of biological and biotechnological samples when the more largely available approaches are employed using a commercial FTIR spectrometer. Some basic aspects of data analysis procedures are presented in an Appendix. A certain number of our previous experimental results are reported for demonstrating once more the versatility and the potentiality of FTIR spectroscopy. [ABSTRACT FROM AUTHOR]

Since the nanoscale in combination with luminescent properties and prospective applications in different fields of optoelectronics and biomedicine is stimulating a growing interest towards research in the area of cadmium telluride (CdTe) quantum dots (QDs), the method of "green" synthesis of CdTe QDs with the use of the Pleurotus ostreatus mycelium culture as a biological matrix has been developed. The study of their physical and chemical characteristics has shown that the synthesized CdTe QDs are characterized by a crystalline structure and a predominantly spherical morphology and are 3–8 nm in size with the luminescence maximum within the 340–370 nm range. The study of their effects on different types of mammalian cells has shown that CdTe QDs have dose-dependent effects on mouse endothelial cells and human and rat erythrocytes, T- and B-lymphocytes, colorectal cancer cells (Colo 205), and human breast cancer cells (MCF-7). In particular, suppression of proliferative indices of endotheliocytes and an increase in the count of dead cells were observed, which indicates the cytotoxic action of nanocrystalline CdTe and its antiproliferative effect on endothelial cells. At the 5 µM concentration, CdTe QDs exhibited hemolytic activity, due to their action on erythrocytes, and affected adhesive contacts and cancer cell survivability. At the same time, human breast cancer cells (MCF-7) were more sensitive to their action. The data obtained are exclusively important for the understanding of mechanisms underlying the toxicity of CdTe QDs and for their future application in biological and biomedical research. [ABSTRACT FROM AUTHOR]

To ensure proper segregation during mitosis, chromosomes must be efficiently captured by spindle microtubules and subsequently aligned on the mitotic spindle. The efficacy of chromosome interaction with the spindle can be influenced by how widely chromosomes are scattered in space. Here, we quantify chromosome-scattering volume (CSV) and find that it is reduced soon after nuclear envelope breakdown (NEBD) in human cells. The CSV reduction occurs primarily independently of microtubules and is therefore not an outcome of interactions between chromosomes and the spindle. We find that, prior to NEBD, an acto-myosin network is assembled in a LINC complex-dependent manner on the cytoplasmic surface of the nuclear envelope. This acto-myosin network remains on nuclear envelope remnants soon after NEBD, and its myosin-II-mediated contraction reduces CSV and facilitates timely chromosome congression and correct segregation. Thus, we find a novel mechanism that positions chromosomes in early mitosis to ensure efficient and correct chromosome-spindle interactions.

Topoisomerase II (topo II) inhibitors are commonly used as chemotherapy to treat multiple types of cancer, though their use is also associated with the development of therapy related acute leukemias. While the chromosome-damaging effects of etoposide, a topo II poison, have been proposed to act through a threshold mechanism, little is known about the chromosome damaging effects and dose responses for the catalytic inhibitors of the enzyme. The current study was designed to further investigate the potencies and concentration-response relationships of several topoisomerase II inhibitors, including the topoisomerase II poison etoposide, as well as catalytic inhibitors aclarubicin, merbarone, ICRF-154 and ICRF-187 using both a traditional in vitro micronucleus assay as well as a flow-cytometry based version of the assay. Benchmark dose (BMD) analysis was used to identify models that best fit the data and estimate a BMD, in this case the concentration at which a one standard deviation increase above the control frequency would be expected. All of the agents tested were potent in inducing micronuclei in human lymphoblastoid TK6 cells, with significant increases seen at low micromolar, and in the cases of aclarubicin and etoposide, at low nanomolar concentrations. Use of the anti-kinetochore CREST antibody with the microscopy-based assay demonstrated that the vast majority of the micronuclei originated from chromosome breakage. In comparing the two versions of the micronucleus assay, significant increases in micronucleated cells were observed at similar or lower concentrations using the traditional microscopy-based assay. BMD modeling of the data exhibited several advantages and proved to be a valuable alternative for concentration-response analysis, producing points of departure comparable to those derived using traditional no-observed or lowest-observed genotoxic effect level (NOGEL or LOGEL) approaches.

Engineered tissue currently lacks requisite capacity to sustain cell viability and functionality. Here we demonstrate that human RPE cell lines (ARPE-19) can be cultured on ultrathin suspended electrospun nanofibre scaffolds (ENS) composed of hydrophobic polymer polyacrylonitrile (PAN) and a water-soluble aliphatic diamine, without (untreated) or with (treated) fluocinolone acetonide (FA). Cells survived and retained their characteristic morphology for up to 150 days with FA-treated ENS and manifested a morphological epithelial phenotype with expression of biomarkers critical for maintaining retinal physiological characteristics. This novel technique for producing culture substrates provides suitable hydrophilicity and a protective environment for prolonged RPE culture and has immense potential for subretinal transplantation. The findings indicate that FA-treated ENS is an excellent matrix for retaining the differentiated and epithelial phenotype.

In recent years, sphingolipids have attracted significant attention due to their pivotal role in cellular functions and physiological diseases. A valuable tool for investigating the characteristics of sphingolipids can be represented via FT-IR spectroscopy, generally recognized as a very powerful technique that provides detailed biochemical information on the examined sample with the unique properties of sensitivity and accuracy. In the present paper, some fundamental aspects of sphingolipid components of human cells are summarized, and the most relevant articles devoted to the FT-IR spectroscopic studies of sphingolipids are revised. A short description of different FT-IR experimental approaches adopted for investigating sphingolipids is also given, with details about the most commonly used data analysis procedures. The present overview of FT-IR investigations, although not exhaustive, attests to the relevant role this vibrational technique has played in giving significant insight into many aspects of this fascinating class of lipids. [ABSTRACT FROM AUTHOR]

Introduction: The synthesis of proteins is a fundamental process in the life-span of all cells. The activation of ribosomes on transcripts is the starting signal for elongation and, in turn, the translation of an mRNA. Thereby, most mRNAs circulate between single (monosomes) and multi ribosomal particles (polysomes), a process that defines their translational activity. The interplay between monosomes and polysomes is thought to crucially impact translation rate. How monosomes and polysomes are balanced during stress remains, however, elusive.Methods: Here, we set out to investigate the monosome and polysome levels as well as their kinetics under different translational stress conditions including mTOR inhibition, downregulation of the eukaryotic elongation factor 2 (eEF2) and amino acid depletion.Results: By using a timed ribosome runoff approach in combination with polysome profiling, we found that the used translational stressors show very distinct effects on translation. However, they all had in common that the activity of monosomes was preferentially affected. This adaptation seems to be needed for sufficient translation elongation. Even under harsh conditions such as amino acid starvation, we detected active polysomes while monosomes were mostly inactive. Hence, it is plausible that cells compensate the reduced availability of essential factors during stress by adapting the levels of active monosomes to favor sufficient elongation.Discussion: These results suggest that monosome and polysome levels are balanced under stress conditions. Together, our data argue for the existence of translational plasticity that ensure sufficient protein synthesis under stress conditions, a process that is necessary for cell survival and recovery.

The toxic effects of four cationic porphyrins on various human cells were studied in vitro. It was found that, under dark conditions, porphyrins are almost nontoxic, while, under the action of light, the toxic effect was observed starting from nanomolar concentrations. At a concentration of 100 nM, porphyrins caused inhibition of metabolism in the MTT test in normal and cancer cells. Furthermore, low concentrations of porphyrins inhibited colony formation. The toxic effect was nonlinear; with increasing concentrations of various porphyrins, up to about 1 μM, the effect reached a plateau. In addition to the MTT test, this was repeated in experiments examining cell permeability to trypan blue, as well as survival after 24 h. The first visible manifestation of the toxic action of porphyrins is blebbing and swelling of cells. Against the background of this process, permeability to porphyrins and trypan blue appears. Subsequently, most cells (even mitotic cells) freeze in this swollen state for a long time (24 and even 48 h), remaining attached. Cellular morphology is mostly preserved. Thus, it is clear that the cells undergo mainly necrotic death. The hypothesis proposed is that the concentration dependence of membrane damage indicates a limited number of porphyrin targets on the membrane. These targets may be any ion channels, which should be considered in photodynamic therapy. [ABSTRACT FROM AUTHOR]

In a recent study, we showed in an in vitro murine cerebellar microvascular endothelial cell (cerebEND) model as well as in vivo in rats that Tumor-Treating Fields (TTFields) reversibly open the blood–brain barrier (BBB). This process is facilitated by delocalizing tight junction proteins such as claudin-5 from the membrane to the cytoplasm. In investigating the possibility that the same effects could be observed in human-derived cells, a 3D co-culture model of the BBB was established consisting of primary microvascular brain endothelial cells (HBMVEC) and immortalized pericytes, both of human origin. The TTFields at a frequency of 100 kHz administered for 72 h increased the permeability of our human-derived BBB model. The integrity of the BBB had already recovered 48 h post-TTFields, which is earlier than that observed in cerebEND. The data presented herein validate the previously observed effects of TTFields in murine models. Moreover, due to the fact that human cell-based in vitro models more closely resemble patient-derived entities, our findings are highly relevant for pre-clinical studies. [ABSTRACT FROM AUTHOR]

Plants are an inexhaustible source of bioactive compounds beneficial for contrasting oxidative stress, leading to many degenerative pathologies. Brassica rapa L. subsp. rapa is well known for its nutraceutical properties among edible vegetable species. In our work, we aimed to explore an eco-friendly way to enhance the beneficial dietary phytochemicals in this vast world of crop-growing plants at selected light quality conditions. White broad-spectrum (W) and red–blue (RB) light regimes were used for growing brassica microgreens. The organic extracts were tested on keratinocytes upon oxidative stress to explore their capability to act as natural antioxidant cell protectors. Our results show that both W and RB extracts caused a notable reduction in reactive oxygen species (ROS) levels induced by H2O2. Interestingly, according to its higher contents of polyphenols and flavonoids, the RB was more efficient in reducing ROS amount and DNA damage than the W extract, particularly at the lowest concentration tested. However, at higher concentrations (up to 100 μg/mL), the antioxidant effect reached a plateau, and there was little added benefit. These findings confirm that RB light effectively increases the antioxidant compounds in Brassica rapa L. microgreens, thus contributing to their enhanced activity against oxidative-induced genotoxicity compared to microgreens grown under W light.

Small nucleolar RNAs (snoRNAs) are a highly expressed class of non-coding RNAs known for their role in guiding post-transcriptional modifications of ribosomal RNAs and small nuclear RNAs. Emerging studies suggest that snoRNAs are also implicated in regulating other vital cellular processes, such as pre-mRNA splicing and 3′-processing of mRNAs, and in the development of cancer and viral infections. There is an emerging body of evidence for specific snoRNA's involvement in the optimal replication of RNA viruses. In order to investigate the expression pattern of snoRNAs during influenza A viral infection, we performed RNA sequencing analysis of the A549 human cell line infected by influenza virus A/Puerto Rico/8/1934 (H1N1). We identified 66 that were upregulated and 55 that were downregulated in response to influenza A virus infection. The increased expression of most C/D-box snoRNAs was associated with elevated levels of 5'- and 3'-short RNAs derived from this snoRNA. Analysis of the poly(A)+ RNA sequencing data indicated that most of the differentially expressed snoRNAs synthesis was not correlated with the corresponding host genes expression. Furthermore, influenza A viral infection led to an imbalance in the expression of genes responsible for C/D small nucleolar ribonucleoprotein particles' biogenesis. In summary, our results indicate that the expression pattern of snoRNAs in A549 cells is significantly altered during influenza A viral infection. [ABSTRACT FROM AUTHOR]

Over the course of evolution, the centrosome function has been conserved in most eukaryotes, but its core architecture has evolved differently in some clades, with the presence of centrioles in humans and a spindle pole body (SPB) in yeast. Similarly, the composition of these two core elements has diverged, with the exception of Centrin and SFI1, which form a complex in yeast to initiate SPB duplication. However, it remains unclear whether this complex exists at centrioles and whether its function has been conserved. Here, using expansion microscopy, we demonstrate that human SFI1 is a centriolar protein that associates with a pool of Centrin at the distal end of the centriole. We also find that both proteins are recruited early during procentriole assembly and that depletion of SFI1 results in the loss of the distal pool of Centrin, without altering centriole duplication. Instead, we show that SFI1/Centrin complex is essential for centriolar architecture, CEP164 distribution, and CP110 removal during ciliogenesis. Together, our work reveals a conserved SFI1/Centrin module displaying divergent functions between mammals and yeast. Synopsis: The SFI1/Centrin complex is crucial for centrosome duplication in yeast. This study demonstrates the evolutionary conservation of this complex at human centrioles, which has a crucial role in maintaining centriole architecture and ciliogenesis. Human SFI1 is a bona fide centriole protein.SFI1 forms an interdependent complex with centrin at the centriolar distal end.The role of the SFI1/Centrin complex in yeast centrosome duplication is not conserved in human.SFI1 depletion impacts centriole architecture integrity.SFI1 depletion impairs CEP164 organization, CP110 removal and ciliogenesis. [ABSTRACT FROM AUTHOR]

In this study, we propose a method for the separate determination of both types of adrenergic receptors based on radioligand binding analysis using 125I-iodocyanopindolol, comprising three measurements: (1) without competing ligands, (2) in the presence of selective ligand ICI 118,551 (0.25 μM), and (3) in the presence of two selective ligands, ICI 118,551 and CGP 20712 (0.25 μM each). The technique was tested on a model system of two transgenic cell lines ADL-7A and A2R9 with the expression of recombinant β1- and β2-adrenergic receptors. If the ratio of the number of β1-adrenergic receptors to β2-adrenergic receptors is 1 : 10, the measurement error is about 15%. Analysis of nine cell lines representing different types of blood cells showed the presence of β2-adrenergic receptors in Daudi, Raji, Dami, K-562, HL-60, U-937, and THP-1 cells and their absence in Jurkat and MOLT-4 cells. β1-Adrenergic receptors are reliably recorded only in THP-1 cells of monocytic origin. In other cell lines, with the exception of Dami, the number of β1-adrenergic receptors was found below the detection limit, estimated as 250 molecules per cell. Measurements performed on the peripheral blood mononuclear cells of seven healthy donors showed the presence of β2-adrenergic receptors in the range from 1000 to 2500 molecules per cell, while the content of β1-adrenergic receptors in all cases appeared to be on the border or beyond the detection limit. [ABSTRACT FROM AUTHOR]

Circular RNAs (circRNAs) are involved in diverse biological processes. Avian influenza virus (AIV) can cross the species barrier to infect humans. Here, we employed RNA sequencing technology to profile circRNA, microRNA, and mRNA expression in human lung carcinoma cells in response to AIV or human influenza A virus (IAV) infection at viral replication. The analysis revealed that the expression of 475 common circRNAs were significantly regulated. The 381 and 1163 up-regulated circRNAs were induced by AIV at 8 and 16 h, respectively. Subsequently, gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses were also conducted for the AIV-specific up-regulated circRNAs. Moreover, the circRNAs were characterized, of which six were verified by quantitative real-time PCR. We further confirmed that expression of the selected circRNAs only increased following AIV infection. Knocking down the selected circRNAs promoted AIV proliferation, and overexpression of three of the candidate circRNAs restricted AIV replication and proliferation. We further analyzed that AIV-specific up-regulated circRNA mechanisms might function through the ceRNA network to affect the "Endocytosis" pathway and the "Cell cycle process". These data provide the first expression profile of AIV-specific up-regulated circRNAs and shed new light on the pathogenesis of AIV infection. Our findings also suggest that these circRNAs serve an important role in AIV infection. [ABSTRACT FROM AUTHOR]

Antifouling (AF) coatings containing booster biocides are used worldwide as one of the most cost-effective ways to prevent the attachment of marine organisms to submerged structures. Nevertheless, many of the commercial biocides, such as Econea® (tralopyril), are toxic in marine environments. For that reason, it is of extreme importance that new efficient AF compounds that do not cause any harm to non-target organisms and humans are designed. In this study, we measured the half-maximal inhibitory concentration (IC50) of a promising nature-inspired AF compound, a triazolyl glycosylated chalcone (compound 1), in an immortalized human retinal pigment epithelial cell line (hTERT-RPE-1) and compared the results with the commercial biocide Econea®. We also investigated the effects of these biocides on the cellular lipidome following an acute (24 h) exposure using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS). Our results showed that compound 1 did not affect viability in hTERT-RPE-1 cells at low concentrations (1 μM), in contrast to Econea®, which caused a 40% reduction in cell viability. In total, 71 lipids were found to be regulated upon exposure to 10 µM of both compounds. Interestingly, both compounds induced changes in lipids involved in cell death, membrane modeling, lipid storage, and oxidative stress, but often in opposing directions. In general, Econea® exposure was associated with an increase in lipid concentrations, while compound 1 exposure resulted in lipid depletion. Our study showed that exposure to human cells at sublethal Econea® concentrations results in the modulation of several lipids that are linked to cell death and survival. [ABSTRACT FROM AUTHOR]

In human cells, approximately 30% of all polypeptides enter the secretory pathway at the level of the endoplasmic reticulum (ER). This process involves cleavable amino-terminal signal peptides (SPs) or more or less amino-terminal transmembrane helices (TMHs), which serve as targeting determinants, at the level of the precursor polypeptides and a multitude of cytosolic and ER proteins, which facilitate their ER import. Alone or in combination SPs and TMHs guarantee the initial ER targeting as well as the subsequent membrane integration or translocation. Cytosolic SRP and SR, its receptor in the ER membrane, mediate cotranslational targeting of most nascent precursor polypeptide chains to the polypeptide-conducting Sec61 complex in the ER membrane. Alternatively, fully-synthesized precursor polypeptides and certain nascent precursor polypeptides are targeted to the ER membrane by either the PEX-, SND-, or TRC-pathway. Although these targeting pathways may have overlapping functions, the question arises how relevant this is under cellular conditions and which features of SPs and precursor polypeptides determine preference for a certain pathway. Irrespective of their targeting pathway(s), most precursor polypeptides are integrated into or translocated across the ER membrane via the Sec61 channel. For some precursor polypeptides specific Sec61 interaction partners have to support the gating of the channel to the open state, again raising the question why and when this is the case. Recent progress shed light on the client spectrum and specificities of some auxiliary components, including Sec62/Sec63, TRAM1 protein, and TRAP. To address the question which precursors use a certain pathway or component in intact human cells, i.e., under conditions of fast translation rates and molecular crowding, in the presence of competing precursors, different targeting organelles, and relevant stoichiometries of the involved components, siRNA-mediated depletion of single targeting or transport components in HeLa cells was combined with label-free quantitative proteomics and differential protein abundance analysis. Here, we present a summary of the experimental approach as well as the resulting differential protein abundance analyses and discuss their mechanistic implications in light of the available structural data. [ABSTRACT FROM AUTHOR]

Escherichia coli is one of the most common Gram-negative pathogens and is responsible for infection leading to neonatal meningitis and sepsis. The FtsZ protein is a bacterial tubulin homolog required for cell division in most species, including E. coli. Several agents that block cell division have been shown to mislocalise FtsZ, including the bacteriophage λ-encoded Kil peptide, resulting in defective cell division and a filamentous phenotype, making FtsZ an attractive target for antimicrobials. In this study, we have used an in vitro meningitis model system for studying the effect of bacteriophages on FtsZ using fluorescent E. coli EV36/FtsZ-mCherry and K12/FtsZ-mNeon strains. We show localisation of FtsZ to the bacterial cell midbody as a single ring during normal growth conditions, and mislocalisation of FtsZ producing filamentous multi-ringed bacterial cells upon addition of the known inhibitor Kil peptide. We also show that when bacteriophages K1F-GFP and T7-mCherry were applied to their respective host strains, these phages can inhibit FtsZ and block bacterial cell division leading to a filamentous multi-ringed phenotype, potentially delaying lysis and increasing progeny number. This occurs in the exponential growth phase, as actively dividing hosts are needed. We present that ZapA protein is needed for phage inhibition by showing a phenotype recovery with a ZapA mutant strain, and we show that FtsI protein is also mislocalised upon phage infection. Finally, we show that the T7 peptide gp0.4 is responsible for the inhibition of FtsZ in K12 strains by observing a phenotype recovery with a T7Δ0.4 mutant.

In human cells, approximately 30% of all polypeptides enter the secretory pathway at the level of the endoplasmic reticulum (ER). This process involves cleavable amino-terminal signal peptides (SPs) or more or less amino-terminal transmembrane helices (TMHs), which serve as targeting determinants, at the level of the precursor polypeptides and a multitude of cytosolic and ER proteins, which facilitate their ER import. Alone or in combination SPs and TMHs guarantee the initial ER targeting as well as the subsequent membrane integration or translocation. Cytosolic SRP and SR, its receptor in the ER membrane, mediate cotranslational targeting of most nascent precursor polypeptide chains to the polypeptide-conducting Sec61 complex in the ER membrane. Alternatively, fully-synthesized precursor polypeptides and certain nascent precursor polypeptides are targeted to the ER membrane by either the PEX-, SND-, or TRC-pathway. Although these targeting pathways may have overlapping functions, the question arises how relevant this is under cellular conditions and which features of SPs and precursor polypeptides determine preference for a certain pathway. Irrespective of their targeting pathway(s), most precursor polypeptides are integrated into or translocated across the ER membrane via the Sec61 channel. For some precursor polypeptides specific Sec61 interaction partners have to support the gating of the channel to the open state, again raising the question why and when this is the case. Recent progress shed light on the client spectrum and specificities of some auxiliary components, including Sec62/Sec63, TRAM1 protein, and TRAP. To address the question which precursors use a certain pathway or component in intact human cells, i.e., under conditions of fast translation rates and molecular crowding, in the presence of competing precursors, different targeting organelles, and relevant stoichiometries of the involved components, siRNA-mediated depletion of single targeting or transport components in HeLa cells was combined with label-free quantitative proteomics and differential protein abundance analysis. Here, we present a summary of the experimental approach as well as the resulting differential protein abundance analyses and discuss their mechanistic implications in light of the available structural data.

Abstract The rise of nanotechnology and widespread use of engineered nanomaterials in everyday human life has led to concerns regarding their potential effect on human health. Adverse effects of nanomaterials and nanoparticles on various molecular and cellular alterations have been well-studied. In contrast, the role of epigenetic alterations in their toxicity remains relatively unexplored. This review summarizes current evidence of alterations in cytosine DNA methylation and histone modifications in response to nanomaterials and nanoparticles exposures in vivo and in vitro. This review also highlights existing knowledge gaps regarding the role of epigenetic alterations in nanomaterials and nanoparticles toxicity. Additionally, the role of epigenetic changes as potential translational biomarkers for detecting adverse effects of nanomaterials and nanoparticles is discussed.

Here, we report on the nanopatterning of different aromatic polymer substrates achieved by KrF excimer laser treatment. The conditions for the construction of the laser-induced periodic surface structures, the so-called LIPSS pattern, were established by optimized laser fluence and a number of pulses. The polymer substrates were polyethylene naphthalate (PEN), polyethersulfone (PES), and polystyrene (PS), which were chosen since they are thermally, chemically, and mechanically resistant polymers with high absorption coefficients at the excimer laser wavelength. The surface morphology of the treated substrates was investigated by atomic force microscopy and scanning electron microscopy, and the roughness and effective surface area on the modified samples were determined. Elemental concentration was characterized by energy-dispersive (EDX) analysis, surface chemistry was determined with X-ray photoelectron spectroscopy (XPS). The samples with the formation of LIPSS induced by 10 mJ·cm−2 with 1000, 3000, and 6000 pulses were used for subsequent in vitro cytocompatibility tests using human cells from osteosarcoma (U-2 OS). The LIPSS pattern and its ability of significant cell guidance were confirmed for some of the studied samples. Cell morphology, adhesion, and proliferation were evaluated. The results strongly contribute to the development of novel applications using nanopatterned polymers, e.g., in tissue engineering, cell analysis or in combination with metallization for sensor construction. [ABSTRACT FROM AUTHOR]

Titanium dioxide (TiO2) nanoparticles are widely manufactured, with a range of applications in consumer products. Significant toxicity of TiO2 nanoparticles has, however, been recognized, suggesting considerable risk to human health. To evaluate fully their toxicity, assessment of the epigenetic action of these nanoparticles is critical. However, only few studies are available examining the capability of nanoparticles to alter epigenetic integrity. In the present study, the effect of TiO2 nanoparticles exposure on histone modifications, a major epigenetic mechanism, was investigated in human colorectal (Caco-2) and lung (NL20) epithelial cell lines. Histone H3 and H4 modifications were assessed by array analysis using the EpiQuickTM Histone H3 or H4 Modification Multiplex Assay. Seventeen histone modifications were identified with altered levels after exposure to TiO2 nanoparticles. Changes in several selected histone modifications (Caco-2 cells: H3cit, H3K9me3, H3K27me3, H3K36me3, H3K9ac, and H4K8ac; NL20 cells: H3K4me3, H3K9me3, H3K27me3, H3K9ac, and H3K18ac) were verified by Western blot analysis. The results also revealed aberrant expression of histone modifying enzymes in TiO2 exposed cells. Expression levels were determined by array analysis using the Human Epigenetic Chromatin Modification Enzymes RT2 Profiler™ PCR Array, with 12 genes identified in both Caco-2 cells and NL20 cells. qRT-PCR analysis confirmed the array results for several selected histone modifying enzyme genes (ASH1L, CARM1, EHMT2, HAT1, HDAC9, KMT2E, NCOA1, SETDB2, and USP16). The findings from this study clearly demonstrate the impact of TiO2 nanoparticles exposure on histone modification in two human cell lines, supporting potential involvement of this epigenetic mechanism in the toxicity of TiO2 nanoparticles. Hence, for complete assessment of potential risk from nanoparticle exposure, epigenetic studies are critical. [ABSTRACT FROM AUTHOR]

In the context of inefficient antibiotics, antibacterial alternatives are urgently needed to stop the increasing resistance rates in pathogens. This study reports the fabrication and characterization of four promising magnetite-based antibiotic delivery systems for ENT (ear, nose and throat) applications. Magnetite nanoparticles were functionalized with streptomycin and neomycin and some were entrapped in polymeric spheres. The obtained nanomaterials are stable, with spherical morphology, their size ranging from ~2.8 to ~4.7 nm for antibiotic-coated magnetite nanoparticles, and from submicron sizes up to several microns for polymer-coated magnetite–antibiotic composites. Cell viability and antimicrobial tests demonstrated their biocompatibility on human diploid cells and their antibacterial effect against Gram-negative (Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) opportunistic bacteria. The presence of the polymeric coat proved an enhancement in biocompatibility and a slight reduction in the antimicrobial efficiency of the spheres. Our results support the idea that functional NPs and polymeric microsystems containing functional NPs could be tailored to achieve more biocompatibility or more antimicrobial effect, depending on the bioactive compounds they incorporate and their intended application. [ABSTRACT FROM AUTHOR]