Search Results (1,184)

The rise in early-age temperature concrete structures, driven by the exothermic reactions during cement hydration, significantly increases the risk of thermal cracking. To address this issue, the construction industry employs several strategies, including the partial substitution of cement with ground granulated blast furnace slag (GGBS) due to its lower heat of hydration. Accurately predicting the hydration temperature of concrete is critical for preventing thermal cracking. This task becomes more complex, with fluctuating ambient temperatures influencing hydration kinetics and heat dissipation. Previous studies often assume adiabatic or isothermal conditions, thus overlooking the impact of ambient temperature variations. This paper presents an innovative finite element modelling (FEM) approach to simulate the hydration temperature progression in in situ concrete slabs, incorporating the effects of ambient temperature fluctuations. Isothermal calorimetry curves were adjusted using the Arrhenius-based approach to express the cement hydration rate as a function of ambient temperature. The FEM outcomes, validated with semi-adiabatic calorimetry tests, demonstrate the model’s capability to forecast temperature development in in situ concrete under varying ambient conditions. Additionally, the study examines the influence of partial cement replacement with GGBS on thermal behaviour, revealing that while GGBS effectively reduces thermal reactions at higher contents, its efficacy diminishes with rising ambient temperatures. Full article

The nine-member CLC gene family of Cl⁻ chloride-transporting membrane proteins is divided into plasma membrane-localized Cl⁻ channels and endo-/lysosomal Cl⁻/H⁺ antiporters. Accessory proteins have been identified for ClC-K and ClC-2 channels and for the lysosomal ClC-7, but not the other CLCs. Here, we identified TMEM9 Domain Family Member B (TMEM9B), a single-span type I transmembrane protein of unknown function, to strongly interact with the neuronal endosomal ClC-3 and ClC-4 transporters. Co-expression of TMEM9B with ClC-3 or ClC-4 dramatically reduced transporter activity in Xenopus oocytes and transfected HEK cells. For ClC-3, TMEM9B also induced a slow component in the kinetics of the activation time course, suggesting direct interaction. Currents mediated by ClC-7 were hardly affected by TMEM9B, and ClC-1 currents were only slightly reduced, demonstrating specific interaction with ClC-3 and ClC-4. We obtained strong evidence for direct interaction by detecting significant Förster Resonance Energy Transfer (FRET), exploiting fluorescence lifetime microscopy-based (FLIM-FRET) techniques between TMEM9B and ClC-3 and ClC-4, but hardly any FRET with ClC-1 or ClC-7. The discovery of TMEM9B as a novel interaction partner of ClC-3 and ClC-4 might have important implications for the physiological role of these transporters in neuronal endosomal homeostasis and for a better understanding of the pathological mechanisms in CLCN3- and CLCN4-related pathological conditions. Full article

The presence of the odorant 2-methylisoborneol (2-MIB) in drinking water sources is undesirable. Although 2-MIB production is known to be influenced by temperature, its regulation at the gene level and its relationship with Chlorophyll-a (Chl-a) at different temperatures remain unclear. This study investigates the impact of temperature on 2-MIB production and related gene expression in Pseudanabaena strains PD34 and PD35 isolated from Lake Paldang, South Korea. The strains were cultured at three temperatures (15, 25, and 30 °C) to examine cell growth, 2-MIB production, and mic gene expression levels. 2-MIB production per cell increased with higher temperatures, whereas mic gene expression levels were higher at lower temperatures, indicating a complex regulatory mechanism involving post-transcriptional and enzyme kinetics factors. Additionally, the relationship between Chl-a and 2-MIB involved in metabolic competition was analyzed, suggesting that high temperatures appear to favor 2-MIB synthesis more than Chl-a synthesis. The distinct difference in the total amount of the two products and the proportion of 2-MIB between the two strains partially explains the variations in 2-MIB production. These findings highlight the significant effect of temperature on 2-MIB biosynthesis in Pseudanabaena and provide a valuable background for gene data-based approaches to manage issues regarding 2-MIB in aquatic environments. Full article

The L-gulonolactone oxidase enzyme (GULO) catalyzes the last step of L-ascorbic acid (vitamin C) biosynthesis. This enzymatic activity is lost in primates. The full-length rat GULO has been previously produced in plants and demonstrated to be active. In this study, we compared the activity of two variants of GULO produced in Escheriachia coli cells, full-length rat GULO (fGULO) and its C-terminal catalytic domain (cGULO). The expression and purification of the recombinant proteins were optimized, and their biological activity was confirmed by two methods, the GULO activity assay in the protein extracts and the ‘in-gel’ staining for GULO activity. Both variants of recombinant GULO were biologically active in both assays. However, cGULO is more promising than fGULO for ascorbic acid production because it is more efficiently produced by bacteria. Furthermore, the optimal activities of the fGULO and cGULO recombinant proteins were observed at pH 7 and 6.5, and at temperatures of 40 and 30 °C, respectively. Kinetic studies revealed that at low substrate concentrations, K_m values for fGULO and cGULO were 53.5 ± 5 and 42 ± 6.3 µM, respectively. Full article

Extracellular electron transfer (EET) by sulfate-reducing bacteria (SRB), such as Desulfovibrio ferrophilus IS5, enables bacterial interactions with minerals, which are vital for biogeochemical cycling and environmental chemistry. Here, we explore the direct EET mechanisms through outer-membrane cytochromes (OMCs) using IS5 as a model SRB. We employed nanostructured electrodes arrayed with 0, 50, 200, and 500 nm long nanowires (NWs) coated with indium–tin–doped oxide to examine the impact of electrode morphology on the direct EET efficacy. Compared to flat electrodes, NW electrodes significantly enhanced current production in IS5 with OMCs. However, this enhancement was diminished when OMC expression was reduced. Differential pulse voltammetry revealed that NW electrodes specifically augmented redox peaks associated with OMCs without affecting those related to redox mediators, suggesting that NWs foster direct EET through OMCs. Scanning electron microscopy observations following electrochemical analyses revealed a novel vertical cell attachment and aggregation on NW electrodes, contrasting with the horizontal monolayer cell attachment on flat electrodes. This study presents the first evidence of the critical role of electrode nanoscale topography in modulating SRB cell orientation and aggregation behavior. The findings underscore the significant influence of electrode morphology on the direct EET kinetics, highlighting the potential impact of mineral morphology on mineral reduction and biogeochemical processes. Full article

Bacillus subtilis is a model organism used to study molecular processes in Gram-positive bacteria. Sigma factor B, which associates with RNA polymerase, is one of the transcriptional regulators involved in the cell’s response to environmental stress. Experiments have proven that the amounts of free σ^B (SigB) are controlled by a system of anti- (RsbW) and anti-anti-sigma (RsbV) factors expressed from the same operon as SigB. Moreover, the phosphorylation state of RsbV is controlled by phosphatases RsbP and RsbU, which directly dephosphorylate RsbV. A set of chemical equations describing the network controlling the levels of free SigB was converted to a set of differential equations quantifying the dynamics of the network. The solution of these equations allowed the simulation of the kinetic behavior of the network and its components under real conditions reflected in the time series of protein expression. In this study, the time series of protein expression measured by mass spectrometry were utilized to investigate the role of phosphatases RsbU/RsbP in transmitting the environmental signal. Additionally, the influence of kinetic constants and the amounts of other network components on the functioning of the network was investigated. A comparison with the same simulation performed using a transcriptomic dataset showed that while the time series between the proteomic and transcriptomic datasets are not correlated, the results are the same. This indicates that when modeling is performed within one dataset, it does not matter whether the data come from the mRNA or protein level. In summary, the computational results based on experimental data provide a quantitative insight into the functioning of the SigB-dependent circuit and offer a template for the quantitative study of similar systems. Full article

Bacterial ubiquitous Toxin-Antitoxin (TA) systems are considered to be important survival mechanisms during stress conditions. In regular environmental conditions, the antitoxin blocks the toxin, whereas during imbalanced conditions, the antitoxin concentration decreases, exposing the bacteria cell to a range of toxic events. The most evident consequence of this disequilibrium is cell growth arrest, which is the reason why TAs are generally described as active in the function of bacterial growth kinetics. Virulence-associated proteins B and C (VapBC) are a family of type II TA system, in which VapC is predicted to display the toxic ribonuclease activity while VapB counteracts this activity. Previously, using in silico data, we designated four VapBC TA modules in Leptospira interrogans serovar Copenhageni, the main etiological agent of human leptospirosis in Brazil. The present study aimed to obtain the proteins and functionally characterize the VapBC-1 module. The expression of the toxin gene vapC in E. coli did not decrease the cell growth rate in broth culture, as was expected to happen within active TA modules. However, interestingly, when the expression of the toxin was compared to that of the complexed toxin and antitoxin, cell viability was strongly affected, with a decrease of three orders of magnitude in colony forming unity (CFU). The assumption of the affinity between the toxin and the antitoxin was confirmed in vivo through the observation of their co-purification from cultivation of E. coli co-expressing vapB-vapC genes. RNAse activity assays showed that VapC-1 cleaves MS2 RNA and ribosomal RNA from L. interrogans. Our results indicate that the VapBC-1 module is a potentially functional TA system acting on targets that involve specific functions. It is very important to emphasize that the common attribution of the functionality of TA modules cannot be defined based merely on their ability to inhibit bacterial growth in a liquid medium. Full article

In this study, we present the design, implementation, and successful use of digital droplet PCR (ddPCR) for the monitoring of chimeric antigen receptor T-cell (CAR-T) expansion in patients with B-cell malignancies treated with different CAR-T products at our clinical center. Initially, we designed a specific and highly sensitive ddPCR assay targeting the junction between the 4-1BB and CD3ζ domains of tisa-cel, normalized with RPP30, and validated it using blood samples from the first tisa-cel-treated patient in Switzerland. We further compared this assay with a published qPCR (quantitative real-time PCR) design. Both assays showed reliable quantification of CAR-T copies down to 20 copies/µg DNA. The reproducibility and precision were confirmed through extensive testing and inter-laboratory comparisons. With the introduction of other CAR-T products, we also developed a corresponding ddPCR assay targeting axi-cel and brexu-cel, demonstrating high specificity and sensitivity with a limit of detection of 20 copies/µg DNA. These assays are suitable for CAR-T copy number quantification across multiple sample types, including peripheral blood, bone marrow, and lymph node biopsy material, showing robust performance and indicating the presence of CAR-T cells not only in the blood but also in target tissues. Longitudinal monitoring of CAR-T cell kinetics in 141 patients treated with tisa-cel, axi-cel, or brexu-cel revealed significant expansion and long-term persistence. Peak expansion correlated with clinical outcomes and adverse effects, as is now well known. Additionally, we quantified the CAR-T mRNA expression, showing a high correlation with DNA copy numbers and confirming active transgene expression. Our results highlight the quality of ddPCR for CAR-T monitoring, providing a sensitive, precise, and reproducible method suitable for clinical applications. This approach can be adapted for future CAR-T products and will support the monitoring and the management of CAR-T cell therapies. Full article

Recently, prokaryotic laccases from lactic acid bacteria (LAB), which can degrade biogenic amines, were discovered. A laccase enzyme has been cloned from Oenococcus oeni, a very important LAB in winemaking, and it has been expressed in Escherichia coli. This enzyme has similar characteristics to those previously isolated from LAB as the ability to oxidize canonical substrates such as 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,6-dimethoxyphenol (2,6-DMP), and potassium ferrocyanide K₄[Fe(CN₆)], and non-conventional substrates as biogenic amines. However, it presents some distinctiveness, the most characteristic being its psychrophilic behaviour, not seen before among these enzymes. Psychrophilic enzymes capable of efficient catalysis at low temperatures are of great interest due to their potential applications in various biotechnological processes. In this study, we report the discovery and characterization of a new psychrophilic laccase, a multicopper oxidase (MCO), from the bacterium Oenococcus oeni. The psychrophilic laccase gene, designated as LcOe 229, was identified through the genomic analysis of O. oeni, a Gram-positive bacterium commonly found in wine fermentation. The gene was successfully cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. Biochemical characterization of the psychrophilic laccase revealed its optimal activity at low temperatures, with a peak at 10 °C. To our knowledge, this is the lowest optimum temperature described so far for laccases. Furthermore, the psychrophilic laccase demonstrated remarkable stability and activity at low pH (optimum pH 2.5 for ABTS), suggesting its potential for diverse biotechnological applications. The kinetic properties of LcOe 229 were determined, revealing a high catalytic efficiency (kcat/Km) for several substrates at low temperatures. This exceptional cold adaptation of LcOe 229 indicates its potential as a biocatalyst in cold environments or applications requiring low-temperature processes. The crystal structure of the psychrophilic laccase was determined using X-ray crystallography demonstrating structural features similar to other LAB laccases, such as an extended N-terminal and an extended C-terminal end, with the latter containing a disulphide bond. Also, the structure shows two Met residues at the entrance of the T1Cu site, common in LAB laccases, which we suggest could be involved in substrate binding, thus expanding the substrate-binding pocket for laccases. A structural comparison of LcOe 229 with Antarctic laccases has not revealed specific features assigned to cold-active laccases versus mesophilic. Thus, further investigation of this psychrophilic laccase and its engineering could lead to enhanced cold-active enzymes with improved properties for future biotechnological applications. Overall, the discovery of this novel psychrophilic laccase from O. oeni expands our understanding of cold-adapted enzymes and presents new opportunities for their industrial applications in cold environments. Full article

Bovine infectious rhinotracheitis (IBR), caused by bovine alphaherpesvirus 1 (BoAHV1), poses significant challenges to the global cattle industry due to its high contagiousness and economic impact. In our study, we successfully isolated a BoAHV1 strain from suspected infected bovine nasal mucus samples in Yanji city, revealing genetic similarities with strains from Sichuan, Egypt, and the USA, while strains from Xinjiang, Beijing, Hebei, and Inner Mongolia showed more distant associations, indicating potential cross-border transmission. Additionally, our investigation of BoAHV1 infection dynamics within host cells revealed early upregulation of gB, which is critical for sustained infection, while the expression of gC and gD showed variations compared to previous studies. These findings enhance our understanding of BoAHV1 diversity and infection kinetics, underscoring the importance of international collaboration for effective surveillance and control strategies. Furthermore, they lay the groundwork for the development of targeted therapeutics and vaccines to mitigate the impact of IBR on the cattle industry. Full article

Human cytomegalovirus (HCMV) represents a highly medically important pathogen which has constantly been the subject of both molecular and clinical investigations. HCMV infections, especially those in high-risk patients, still raise many unanswered questions, so current investigations are focused on viral pathogenesis, vaccine development, and options for antiviral drug targeting. To this end, the use of suitable viral strains as well as recombinant reporter constructs in cultured cells and model systems has specific significance. We previously reported on the application of various herpesviruses that express green, red, or related fluorescent proteins, especially in the fields of virus–host interaction and antiviral research. Here, we characterized a recombinant version of the clinically relevant and cell type-adaptable HCMV strain TB40, which expresses firefly luciferase as a quantitative reporter of viral replication (TB40-FLuc). The data provide evidence for five main conclusions. First, HCMV TB40-FLuc is employable in multiple settings in primary human cells. Second, viral reporter signals are easily quantifiable, even at early time points within viral replication. Third, the FLuc reporter reflects the kinetics of viral intracellular replication, cascade-like viral IE-E-L protein production, and progeny release. Fourth, as relates to specific applications of the TB40-FLuc system, we demonstrated the reliability of quantitative antiviral compound determination in multi-well formats and its independence from fluorescence-based measurements in the case of autofluorescent inhibitors. Finally, we illustrated increased reporter sensitivity in comparison to other recombinant HCMVs. In essence, recombinant HCMV TB40-FLuc combines several molecular properties that are considered beneficial in studies on viral host tropism, replication efficiency, and antiviral drug assessment. Full article

Oxidative stress in the human lung is caused by both internal (e.g., inflammation) and external stressors (smoking, pollution, and infection) to drive pathology in a number of lung diseases. Cellular damage caused by oxidative damage is reversed by several pathways, one of which is the antioxidant response. This response is regulated by the transcriptional factor NRF2, which has the ability to regulate the transcription of more than 250 genes. In disease, this balance is overwhelmed, and the cells are unable to return to homeostasis. Several pharmacological approaches aim to improve the antioxidant capacity by inhibiting the interaction of NRF2 with its key cytosolic inhibitor, KEAP1. Here, we evaluate an alternative approach by overexpressing NRF2 from chemically modified RNAs (cmRNAs). Our results demonstrate successful expression of functional NRF2 protein in human cell lines and primary cells. We establish a kinetic transcriptomic profile to compare antioxidant response gene expression after treatment of primary human bronchial epithelial cells with either KEAP1 inhibitors or cmRNAs. The key gene signature is then applied to primary human lung fibroblasts and alveolar macrophages to uncover transcriptional preferences in each cell system. This study provides a foundation for the understanding of NRF2 dynamics in the human lung and provides initial evidence of alternative ways for pharmacological interference. Full article

The energy sector is the sector that generates the highest amount of environmental contamination, especially in water sources, mostly in the case of coal-based energy production. The aim of this study was to examine a significant contamination source, heavy metal contamination, in coal mining effluents. The current investigation introduces an MOF platform based on zirconium clusters and isophthalic acid with NH₂-MIP-SO₃H mixed amine and sulfonic acid functional groups in order to remove the most common heavy metal ions in coal mining effluents, including Hg, Cd, Pb, and Cu ions. The water matrix and the operational conditions were identified to be very influential in the removal process, such as the pH of water, the initial metal concentration and operating time. NH₂-MIP-SO₃H offers a great removal efficiency of metals starting from 745.83 mg/g for Cd, 673.67 mg/g for Cu, 589.85 mg/g for Hg, and 481.66 mg/g for Pb ions, with the Langmuir equation for equilibrium and pseudo-second-order equation for kinetics being the ideal models to express the equilibrium and kinetic data, respectively. A significant impact of water pH was found to occur, with the NH₂-MIP-SO₃H platform performing best at pH 6. Reuse of NH₂-MIP-SO₃H demonstrates excellent reusability, sustaining 90% of initial performance over eight regeneration cycles. The interaction of functional group-functional metal was the dominant mechanism in the removal process. The NH₂-MIP-SO₃H unique approach to heavy metal removal provides a very hopeful outlook for additional investigations in larger-scale studies. Full article

Background: Sepsis is an uncontrolled systemic inflammatory response to an infection that can result in acute failure of the function of the lung called acute respiratory distress syndrome. Leukocyte recruitment is an important hallmark of acute lung failure in patients with sepsis. Endothelial cells (EC) participate in this process by facilitating tethering, rolling, adhesion, and transmigration of leukocytes via adhesion molecules on their cell surface. In in vivo studies, endothelial nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 and mitogen-activated protein kinase (MAPK) c-Jun intracellular signal transduction pathways were reported to regulate the expression of adhesion molecules. Methods: Mice underwent cecal ligation and puncture (CLP) to induce polymicrobial sepsis and were sacrificed at different time points up to 72 h after sepsis onset. Immunohistochemistry and reverse transcription–quantitative polymerase chain reaction (RT-qPCR) analyses were used to determine the kinetics of nuclear localization of p65 and c-Jun in EC, expression and location of adhesion molecules E-selectin and vascular cell adhesion molecule 1 (VCAM-1). Furthermore, the extent and location of leukocyte recruitment were assessed based on Ly6G staining of neutrophils, cluster determinant (CD) 3 staining of T lymphocytes, and CD68 staining of macrophages. Results: In all pulmonary microvascular beds, we identified p65 and c-Jun nuclear accumulation in a subset of endothelial cells within the first 24 h after CLP-sepsis initiation. E-selectin protein was expressed in a subset of microvessels at 4 and 7 h after sepsis initiation, while VCAM-1 was expressed in a scattered pattern in alveolar tissue and microvessels, without discernible changes during sepsis development. CLP-induced sepsis predominantly promoted the accumulation of neutrophils and T lymphocytes 4 and 7 h after disease onset. Neutrophil accumulation occurred in all pulmonary microvascular beds, while T lymphocytes were present in alveolar tissue and postcapillary venules. Taken together, nuclear localization of p65 and c-Jun in EC and neutrophil recruitment could be associated with induced E-selectin expression in the pulmonary microvessels in CLP-septic mice at the early stage of the disease. In alveolar capillaries, on the other hand, activation of these molecular pathways and leukocyte accumulation occurred in the absence of E-selectin or VCAM-1. Conclusions: Endothelial activation and leukocyte recruitment in sepsis-induced lung injury are regulated by multiple, heterogeneously controlled mechanisms, which vary depending on the type of microvascular bed involved. Full article

Orthoflaviviruses cause a major threat to global public health, and no antiviral treatment is available yet. Zika virus (ZIKV) entry, together with many other viruses, is known to be enhanced by phosphatidylserine (PS) receptors such as T-cell immunoglobulin mucin domain protein 1 (TIM-1). In this study, we demonstrate for the first time, using cell-based electrical impedance (CEI) biosensing, that ZIKV entry is also enhanced by expression of CD300a, another PS receptor. Furthermore, inhibiting CD300a in immature monocyte-derived dendritic cells partially but significantly inhibits ZIKV replication. As we have previously demonstrated that CEI is a useful tool to study Orthoflavivirus infection in real time, we now use this technology to determine how these PS receptors influence the kinetics of in vitro ZIKV infection. Results show that ZIKV entry is highly sensitive to minor changes in TIM-1 expression, both after overexpression of TIM-1 in infection-resistant HEK293T cells, as well as after partial knockout of TIM-1 in susceptible A549 cells. These results are confirmed by quantification of viral copy number and viral infectivity, demonstrating that CEI is highly suited to study and compare virus-host interactions. Overall, the results presented here demonstrate the potential of targeting this universal viral entry pathway. Full article