Search Results (64)

Excessive exhaust backpressure (EBP) in modern diesel engines disrupts gas exchange, increases residual gas fraction (RGF), and reduces combustion efficiency. Traditional experimental approaches, including simulations and bench testing, are often time-consuming and costly, which has driven growing interest in artificial neural networks (ANNs) for accurately modelling complex engine behavior. This research introduces an ANN model designed to predict the impact of EBP on the performance and emissions of a diesel engine across varying compression ratio (CR) of 12, 14, 16, and 18 and engine load (25%, 50%, 75%, and 100%) conditions. The ANN model was developed and optimised using genetic algorithms (GA) and particle swarm optimisation (PSO). It was then trained using data from an experimentally validated one-dimensional computational fluid dynamics (1D-CFD) model developed through GT-Power GT-ISE v2024, simulating engine responses under variation CR, load, and EBP conditions. The optimised ANN architecture, featuring an optimal (3-14-10) configuration, was trained using the Levenberg–Marquardt back propagation algorithm. The performance of the model was assessed using statistical criteria, including the coefficient of determination (R²), root mean square error (RMSE), and k-fold cross-validation, by comparing its predictions with both experimental and simulated data. Results indicate that the optimised ANN model outperformed the baseline ANN and other machine learning (ML) models, attaining an R² of 0.991 and an RMSE of 0.011. It reliably predicts engine performance and emissions under varying EBP conditions while offering insights for engine control, optimisation, diagnostics, and thermodynamic mechanisms. The overall prediction error ranged from 1.911% to 2.972%, confirming the model’s robustness in capturing performance and emission outcomes. Full article

Daptomycin (DAP) is a therapeutic option for vancomycin-resistant Enterococcus faecium (VRE) infections, but DAP resistance may occur during treatment. Previously, we identified a mutation within the E. faecium lafB gene that induces hypersusceptibility to DAP. The lafB gene encodes a glycosyltransferase involved in lipoteichoic acid anchor synthesis, which makes it a promising target for enhancing DAP efficacy. In this study, we characterized E. faecium LafB protein (EfLafB) biophysical properties, used AlphaFold3 to predict LafB in silico three-dimensional structure, and determined lafB gene mutation’s role in virulence, comparing E. faecium HBSJRP18 (DAP-hypersusceptible) and a lafB revertant, HBSJRP18_2.7, and analyzing bacterial growth kinetics, biofilm formation ability, and virulence in a Galleria mellonella model. After gene cloning and expressing and purifying EfLafB, circular dichroism and SEC-MALS assays revealed its monomeric nature under in vitro conditions, with approximately a 40 kDa molecular mass and a melting temperature of 50 °C. In silico prediction indicated that LafB is an αβ-type protein with two domains conforming to the GT-4 family glycosyltransferases. These results are further supported by the highly conserved amino acids (E257, D91, R184, and K185), likely involved in UDP-Glc binding. The studied lafB gene mutation resulted in a significant decrease in bacterial growth and virulence in the invertebrate model. Full article

Climatic changes threaten many forms of crop production as well as adversely affecting global ecosystems and human activities. There are two principal ways in which the balance of the global carbon cycle can be restored, firstly by decreasing anthropogenic CO₂ emissions and secondly by increasing the rates of carbon sequestration. Even if emissions are successfully reduced to net zero over the coming decades, it will still be essential to reduce atmospheric CO₂ concentrations to preindustrial levels. This can only be achieved by global-scale carbon sequestration of the order of gigatonnes (Gt) of CO₂ annually. Over recent decades, engineering approaches have been proposed to tackle carbon sequestration. However, their technological effectiveness has yet to be demonstrated at a global scale, with even the most optimistic current values at less than 0.1 Gt CO₂/yr, i.e., 50–100-fold less than required to meet IPCC targets for 2050. In contrast, biological carbon sequestration already operates as a proven global mechanism that also has the potential for increased effectiveness by harnessing high-yield tropical vegetation including perennial crops with sequestration values already exceeding 1 Gt CO₂/yr. This review will contrast engineering and biological approaches to carbon sequestration with a particular focus on the potential for perennial crops, especially in the tropics. The major conclusions are that (i) the 2 Gt CO₂/yr capacity of biological carbon sequestration already dwarfs that of all engineering approaches at 0.0013 Gt CO₂/yr, (ii) biological sequestration is proven to operate at global scale, and (iii) compared to engineering approaches, it will be orders of magnitude less expensive to upscale further in the coming decades. Full article

Anthocyanins, as natural pigments belonging to the flavonoid group, play a crucial role in plant reproduction, stress resistance and human fitness. Kiwifruit, which is rich in anthocyanins, demonstrates significant potential for promoting health benefits. Although light is widely recognized as an inducer of anthocyanin accumulation, we observed that kiwifruit accumulates more anthocyanin after bagging treatment. This unexpected finding suggests that anthocyanin biosynthesis in kiwifruit may also be regulated by other environmental or physiological factors influenced by bagging, such as humidity, temperature, or gas exchange. This implies that bagging may trigger specific regulatory pathways that promote anthocyanin accumulation through multiple environmental cues beyond light. Therefore, RNA-seq was performed to find the potential pathway. A total of 260 differentially expressed genes were found, including 8 transcription factors and 1 anthocyanin biosynthesis gene F3GT1 (glucosyltransferase). Dual-luciferase reporter assays revealed that bHLH transcription factors could activate the promoter of F3GT1 by 2.45-fold. We infer that bagging treatment increases the kiwifruit anthocyanin content through the bHLH291-F3GT1 pathway. This study not only highlights the potential agricultural applications and commercial value of bagging treatment but also provides new theoretical support for improving fruit coloration and optimizing breeding strategies. Full article

L-asparaginase (L-ASNase) is a key industrial enzyme significant for cancer therapy and the food industry for reducing dietary acrylamide. The hyperthermophilic L-ASNase from Thermococcus sibiricus (TsAI) was previously shown to exhibit high activity and thermostability and is promising for biotechnology. To gain insights into structure-functional relationships of TsAI, determination of the substrate specificity, kinetic parameters, structural characterization, and molecular docking were performed. TsAI characteristics were compared with the TsAI^D54G/T56Q mutant, which exhibited increased activity after a double mutation in the substrate-binding region. TsAI and TsAI^D54G/T56Q were found to display high activity towards D-asparagine—62% and 21% of L-asparaginase activity, respectively—and low L-glutaminase coactivity of ~5%. Restoring the mesophilic-like triad GSQ in the mutant resulted in a two-fold increase in activity towards L-asparagine compared with TsAI. Crystal structures of TsAI forms solved at 1.9 Å resolution revealed that double mesophilic-like mutation increased the flexibility of the loop M51-L57, located in close proximity to the active site. Structural superposition and mutational analysis indicate that mobility of this loop is essential for the activity of thermo-ASNases. Molecular docking, without taking into account the temperature factor, showed that, in contrast to L-asparagine interaction, D-asparagine orientation in the TsAI and TsAI^D54G/T56Q active sites is similar and not optimal for catalysis. Under real conditions, high temperatures can induce structural changes that reduce L-ASNase discrimination towards D-asparagine. Overall, the obtained structural and biochemical data provide a basis for a more detailed understanding of thermo-ASNase functioning and possibilities to engineer improved variants for future biotechnological application. Full article

The demand for L-tryptophan (L-Trp) has been rapidly increasing across various industries, including pharmaceuticals, food, and animal feed. However, traditional production methods have been unable to efficiently meet this growing demand. Hence, this study aimed to develop strategies for enhancing L-Trp production in Escherichia coli. Firstly, an L-Trp-producing strain was selected and subjected to atmospheric and room temperature plasma (ARTP) mutagenesis to generate a mutant library. This was followed by high-throughput screening using an L-Trp-specific riboswitch and a yellow fluorescent protein (YFP)-based biosensor in a flow cytometric cell sorting (FACS) system. Among the screened mutants, GT3938 exhibited a 1.94-fold increase in L-Trp production. Subsequently, rational metabolic engineering was applied to GT3938 by knocking out the L-Trp intracellular transporter gene (tnaB), enhancing the expression of the aromatic amino acid exporter (YddG) and optimizing precursor supply pathways. The resulting strain, zh08, achieved an L-Trp titer of 3.05 g/L in shake-flask fermentation, representing a 7.71-fold improvement over the original strain. This study demonstrated an effective strategy for industrial strain development by integrating biosensor-assisted, high-throughput screening with rational metabolic engineering. Full article

Variants in the GBA1 gene, encoding the lysosomal enzyme glucosylceramidase beta 1 (GCase), are linked to Parkinson’s disease (PD) and Gaucher disease (GD). Heterozygous variants increase PD risk, while homozygous variants lead to GD, a lysosomal storage disorder. Some GBA1 variants impair GCase maturation in the endoplasmic reticulum, blocking lysosomal transport and causing glucosylceramide accumulation, which disrupts lysosomal function. This study explores therapeutic strategies to address these dysfunctions. Using Site-directed Enzyme Enhancement Therapy (SEE-Tx^®), two structurally targeted allosteric regulators (STARs), GT-02287 and GT-02329, were developed and tested in GD patient-derived fibroblasts with relevant GCase variants. Treatment with GT-02287 and GT-02329 improved the folding of mutant GCase, protected the GCase_Leu444Pro variant from degradation, and facilitated the delivery of active GCase to lysosomes. This enhanced lysosomal function and reduced cellular stress. These findings validate the STARs’ mechanism of action and highlight their therapeutic potential for GCase-related disorders, including GD, PD, and Dementia with Lewy Bodies. Full article

The dysregulation of fatty acid (FA) metabolism is linked to various brain diseases, including Alzheimer’s disease (AD). Mass spectrometry imaging (MSI) allows for the visualization of FA distribution in brain tissues but is often limited by low detection sensitivity and high background interference. In this work, we introduce a novel on-tissue chemical derivatization method for FAs using Girard’s Reagent T (GT) as a derivatization reagent combined with 2-chloro-1-methylpyridinium iodide (CMPI) as a coupling reagent and triethylamine (TEA) to provide a basic environment for the reaction. This method significantly enhances the detection sensitivity of FAs, achieving a 1000-fold improvement over traditional negative ion mode analysis. Our method enabled us to observe a notable depletion of oleic acid in the corpus callosum of AD mouse model brain tissue sections compared to wild-type control brain tissue sections. The reliability of our method was validated using LC-MS/MS, which confirmed the presence of eight distinct GT-labeled FAs across various tissue locations. This approach not only improves FA detection in brain tissues but also has the potential to provide a deeper understanding of FA dynamics associated with AD pathogenesis. Full article

The penile tubular urethra forms by canalization of the urethral plate without forming an obvious urethral groove in mice, while the urethral epithelium forms a fully open urethral groove before urethra closure through the distal-opening-proximal-closing process in humans and guinea pigs. Our knowledge of the mechanism of penile development is mainly based on studies in mice. To reveal how the fully opened urethral groove forms in humans and guinea pigs, we compared the expression patterns and levels of key developmental genes using in situ hybridization and quantitative PCR during glans and preputial development between guinea pigs and mice. Our results revealed that, compared with mouse preputial development, which started before sexual differentiation, preputial development in guinea pigs was delayed and initiated at the same time that sexual differentiation began. Fgf10 was mainly expressed in the urethral epithelium in developing genital tubercle (GT) of guinea pigs. The relative expression of Shh, Fgf8, Fgf10, Fgfr2, and Hoxd13 was reduced more than 4-fold in the GT of guinea pigs compared to that of mice. Hedgehog and Fgf inhibitors induced urethral groove formation and restrained preputial development in cultured mouse GT, while Shh and Fgf10 proteins induced preputial development in cultured guinea pig GT. Our discovery suggests that the differential expression of Shh and Fgf10/Fgfr2 may be the main reason a fully opened urethral groove forms in guinea pigs, and it may be similar in humans as well. Full article

Background/Objectives: This study aims to investigate whether Signal Transducer and Activator of Transcription 4 (STAT4) influences the anti-tumor immune response and is possibly involved in the initiation or relapse of pituitary adenomas (PAs) by examining STAT4 polymorphisms and serum levels. This research seeks to uncover potential connections that could inform future therapeutic strategies and improve our understanding of PA pathogenesis. Materials and Methods: This study was conducted at the Laboratory of Ophthalmology, Lithuanian University of Health Sciences. DNA was extracted from peripheral venous blood samples, and the genotyping of four STAT4 SNPs (rs7574865, rs10181656, rs7601754, and rs10168266) was performed using real-time PCR with TaqMan^® Genotyping assays. The serum STAT4 levels were measured via ELISA, and the optical density was read at 450 nm. Genotype frequencies, allele distributions, and serum STAT4 levels were statistically analyzed to assess associations with pituitary adenoma occurrence. Results: A binary logistic regression revealed that the STAT4 rs7574865 GT + GG genotypes vs. TT were associated with 1.7-fold increased odds of PA occurrence under the dominant genetic model (p = 0.012). The stratification by gender showed no significant associations in females; however, in males, the STAT4 rs10168266 CC + CT genotypes compared to TT were linked to 2.5-fold increased odds of PA under the dominant genetic model (p = 0.005). STAT4 rs10181656, rs7574865, rs7601754, and rs10168266 were analyzed to evaluate the associations with the pituitary adenoma size. We found that the STAT4 rs7574865 GG genotype was statistically significantly less frequent in the macro PA group compared to in the reference group (p = 0.012). For PA relapse, the rs7574865 G allele was less frequent in the PA group without relapse (p = 0.012), and the GT + GG genotypes were associated with a 1.8-fold increase in the PA group without relapse occurrence (p = 0.008). The serum STAT4 levels were higher in the PA patients compared to those of the reference group (p < 0.001). Elevated STAT4 serum levels were observed in PA patients with the STAT4 rs10181656 CC or CG genotypes (CC: p = 0.004; CG: p = 0.023), and with the rs7574865 GG or GT genotypes (GG: p = 0.003; GT: p = 0.021). The PA patients with the STAT4 rs7601754 AA genotype exhibited higher serum levels compared to those of the reference group (p < 0.001). Similarly, higher serum levels were found in the PA patients with the STAT4 rs10168266 CC or CT genotypes (CC: p = 0.004; CT: p = 0.027). A haplotype frequency analysis revealed no statistically significant results. Conclusions: The STAT4 genotypes were significantly associated with the PA occurrence, size, and relapse. Elevated serum STAT4 levels were observed in the PA patients, highlighting its potential role in PA pathogenesis. Full article

Background: The presence of the rs35705950 variant in the MUC5B gene promoter is a critical genetic risk factor in idiopathic pulmonary fibrosis (IPF). It has been associated with usual interstitial pneumonia (UIP) in several interstitial lung diseases (ILDs). In antisynthetase syndrome (ASSD), most high-resolution computed tomography (HRCT) patterns are inflammatory, but up to 13% have UIP, leading to a worse prognosis. Methods: This single-center study included 60 patients with ASSD-ILD. We investigated whether carrying the MUC5B rs35705950 promoter variant was associated with UIP. To estimate the strength of the association between the genotype of the MUC5B rs35705950 promoter variant and the fibrotic pattern we used the odds ratio (cOR), and to assess the effect of confounding variables (age, evolution time, and sex), we performed a logistic regression to obtained the adjusted odds ratio (aOR). Results: The GT genotype of the MUC5B rs35705950 promoter variant is associated with up to a 4-fold increased risk of UIP (cOR 5.0, 95% CI 1.13–22.10), and the effect was even maintained after adjusting for potentially confounding variables such as sex, age, and time to progression (aOR 5.2, 95% CI 1.04–25.89). Conclusions: our study supports the role of MUC5B rs35705950 in ASSD-ILD with UIP. It reinforces that this polymorphism in our population could have a similar genetic basis to that already described in other ILDs that present predominantly fibrotic patterns. Full article

Age-related macular degeneration (AMD) is a major global health problem as it is the leading cause of irreversible loss of central vision in the aging population. Anti-vascular endothelial growth factor (anti-VEGF) therapies are effective but do not respond optimally in all patients. This study investigates the genetic factors associated with susceptibility to AMD and response to treatment, focusing on key polymorphisms in the ARMS2 (rs10490924), IL1B1 (rs1143623), TNFRSF1B (rs1061622), TNFRSF1A (rs4149576), VEGFA (rs3024997), ARMS2, IL1B1, TNFRSF1B, TNFRSF1A, and VEGFA serum levels in AMD development and treatment efficacy. This study examined the associations of specific genetic polymorphisms and serum protein levels with exudative and early AMD and the response to anti-VEGF treatment. The AA genotype of VEGFA (rs3024997) was significantly associated with a 20-fold reduction in the odds of exudative AMD compared to the GG + GA genotypes. Conversely, the TT genotype of ARMS2 (rs10490924) was linked to a 4.2-fold increase in the odds of exudative AMD compared to GG + GT genotypes. In females, each T allele of ARMS2 increased the odds by 2.3-fold, while in males, the TT genotype was associated with a 5-fold increase. Lower serum IL1B levels were observed in the exudative AMD group compared to the controls. Early AMD patients had higher serum TNFRSF1B levels than controls, particularly those with the GG genotype of TNFRSF1B rs1061622. Exudative AMD patients with the CC genotype of TNFRSF1A rs4149576 had lower serum TNFRSF1A levels compared to the controls. Visual acuity (VA) analysis showed that non-responders had better baseline VA than responders but experienced decreased VA after treatment, whereas responders showed improvement. Central retinal thickness (CRT) reduced significantly in responders after treatment and was lower in responders compared to non-responders after treatment. The T allele of TNFRSF1B rs1061622 was associated with a better response to anti-VEGF treatment under both dominant and additive genetic models. These findings highlight significant genetic and biochemical markers associated with AMD and treatment response. This study found that the VEGFA rs3024997 AA genotype reduces the odds of exudative AMD, while the ARMS2 rs10490924 TT genotype increases it. Lower serum IL1B levels and variations in TNFRSF1B and TNFRSF1A levels were linked to AMD. The TNFRSF1B rs1061622 T allele was associated with better anti-VEGF treatment response. These markers could potentially guide risk assessment and personalized treatment for AMD. Full article

Prime editor, an editing tool based on the CRISPR/Cas9 system, allows for all 12 types of nucleotide exchanges and arbitrary indels in genomic sequences without the need for inducing DNA double-strand breaks. Despite its flexibility and precision, prime editing efficiency is still low and hindered by various factors such as target sites, editing types, and the length of the primer binding site. In this study, we developed a prime editing system by incorporating an RNA motif at the 3′ terminal of the pegRNA and integrating all twin prime editor factors into a single plasmid. These two strategies enhanced prime editing efficiency at target sites by up to 3.58-fold and 2.19-fold, respectively. Subsequently, enhanced prime editor was employed in goat cells and embryos to efficiently insert a 38 bp attB sequence into the Gt(ROSA)26Sor (Rosa26) and C-C motif chemokine receptor 5 (CCR5) loci. The enhanced prime editor can mediate 11.9% and 6.8% editing efficiency in parthenogenetic activation of embryos through embryo microinjection. In summary, our study introduces a modified prime editing system with improved editing and transfection efficiency, making it more suitable for inserting foreign sequences into primary cells and embryos. These results broaden the potential applications of prime editing technologies in the production of transgenic animals. Full article

Background: This study evaluated titers and amplitudes of anti-E2 antibodies (anti-E2-Abs) and neutralizing antibodies against hepatitis C virus (HCV; anti-HCV-nAbs) in HIV/HCV-coinfected individuals over five years after successful HCV treatment completion. Methods: We retrospectively analyzed 76 HIV/HCV-coinfected patients achieving sustained virologic response post-HCV treatment. Plasma levels of anti-E2-Abs and anti-HCV-nAbs against five HCV genotypes (Gt1a, Gt1b, Gt2a, Gt3a, and Gt4a) were determined using ELISA and microneutralization assays, respectively. Statistical analyses comparing the three follow-up time points (baseline, one year, and five years post-HCV treatment) were performed using generalized linear mixed models, adjusting p-values with the false discovery rate (q-value). Results: Compared to baseline, anti-E2-Abs titers decreased at one year (1.9- to 2.3-fold, q-value < 0.001) and five years (3.4- to 9.1-fold, q-value < 0.001) post-HCV treatment. Anti-HCV-nAbs decreased 2.9- to 8.4-fold (q-value < 0.002) at one year and 17.8- to 90.4-fold (q-value < 0.001) at five years post-HCV treatment. Anti-HCV-nAbs titers against Gt3a were consistently the lowest. Nonresponse rates for anti-E2-Abs remained low throughout the follow-up, while anti-HCV-nAbs nonresponse rates increased 1.8- to 13.5-fold (q-value < 0.05) at five years post-HCV treatment, with Gt3a showing the highest nonresponse rate. Conclusions: Humoral immune responses against HCV decreased consistently one and five years post-HCV treatment, regardless of HCV genotype and previous HCV therapy or type of treatment (IFN- or DAA-based therapy). This decline was more pronounced for anti-HCV-nAbs, particularly against Gt3. Full article

The benzofuran core inhibitors HCV-796, BMS-929075, MK-8876, compound 2, and compound 9B exhibit good pan-genotypic activity against various genotypes of NS5B polymerase. To elucidate their mechanism of action, multiple molecular simulation methods were used to investigate the complex systems of these inhibitors binding to GT1a, 1b, 2a, and 2b NS5B polymerases. The calculation results indicated that these five inhibitors can not only interact with the residues in the palm II subdomain of NS5B polymerase, but also with the residues in the palm I subdomain or the palm I/III overlap region. Interestingly, the binding of inhibitors with longer substituents at the C5 position (BMS-929075, MK-8876, compound 2, and compound 9B) to the GT1a and 2b NS5B polymerases exhibits different binding patterns compared to the binding to the GT1b and 2a NS5B polymerases. The interactions between the para-fluorophenyl groups at the C2 positions of the inhibitors and the residues at the binding pockets, together with the interactions between the substituents at the C5 positions and the residues at the reverse β-fold (residues 441–456), play a key role in recognition and the induction of the binding. The relevant studies could provide valuable information for further research and development of novel anti-HCV benzofuran core pan-genotypic inhibitors. Full article