Search Results (60)

Background: Alterations in protein abundance profiles in yeast deletion strains are frequently utilized to gain insights into cellular functions and regulatory networks, most of which are conserved in higher eukaryotes. Methods: This study investigates the impact of protein extraction methodologies on the whole proteome analysis of S. cerevisiae, comparing detergent-based lysis versus mechanical lysis with silica beads. We evaluated the proteomic profiles of wild-type and two yeast deletion strains, siz1Δ and nfi1Δ (siz2Δ), which are SUMO E3 ligases. Combining isobaric TMTpro-labeling with mass spectrometry using real-time search MS3, we profiled over 4700 proteins, covering approximately 80% of the yeast proteome. Results: Hierarchical clustering and principal component analyses revealed that the choice of protein extraction method significantly influenced the proteomic data, overshadowing the genetic variances among these strains. Notably, the detergent-based lysis showed superior performance in extracting proteins compared to mechanical lysis. Despite minimal proteomic alterations among strains, we observed consistent changes regardless of the lysis strategy in proteins such as Ino1, Rep1, Rep2, Snz1, and Fdh1 in both SUMO E3 ligase deletion strains, implying potential redundant mechanisms of control for these proteins. Conclusion: These findings underscore the importance of method selection at each step of sample preparation in proteomic studies and enhance our comprehension of cellular adaptations to genetic perturbations. Full article

We report the synthesis of highly enantiopure perdeuterated poly-L-lactic acid and poly-D-lactic acid polymers with well-defined molecular weight by polymerization of perdeuterated lactides. Enantiopure D- and L-lactic acid-d₄ monomers were synthesized from sodium pyruvate-d₃ using D- and L-lactate dehydrogenase enzymes (D-LDH and L-LDH) as biocatalysts. The reduced form of the co-enzyme nicotinamide adenine dinucleotide-d₁ (NADH-d₁) was generated in situ from the oxidized form nicotinamide adenine dinucleotide (NAD⁺) by formate dehydrogenase (FDH)-catalyzed oxidation of sodium formate-d₁ to carbon dioxide with concerted reduction of NAD⁺ to NADH-d₁. For the conversion of the perdeuterated lactic acid monomers to the corresponding lactide dimers, we developed a process for generating these compounds in the high purity needed for the final anionic ring-opening polymerization step. This method enabled the generation of a range of perdeuterated polylactic acid polymers that are highly suitable for the characterization of polymer structure and dynamics using neutron scattering, infrared and nuclear magnetic resonance spectroscopy methods that are sensitive to deuterium. Furthermore, these deuterium-labeled polymers are well-suited to the study of the biodegradation of PLA-based plastics. Full article

This study examines the acoustic properties of vowels in foreigner-directed speech (FDS) in interactions between female Omani-Arabic-speaking employers and their foreign domestic helpers (FDHs). Particularly, it investigates whether Arabic corner vowels /i:/, /a:/, and /u:/ undergo acoustic adaptations in FDS. The study also explores the influence of foreign interlocutors’ psycholinguistic characteristics, such as degree of foreign accent, religion, and length of residence (LoR), on the extent of these adaptations. Data were collected from 22 Omani-Arabic-speaking women interacting with their 22 FDHs and with a native speaker (NS) confederate using a spot-the-difference task. Acoustic measures including vowel space area, formant frequency measures (F1 and F2), fundamental frequency (f0), intensity, and duration were compared across speech directed at FDHs and the NS. The results revealed that FDS exhibited greater vowel space expansion, higher F1, and increased pitch (f0) and intensity compared to speech directed at the NS confederate. However, FDS did not significantly affect F2 values. Unexpectedly, vowel duration in FDS was shorter than in speech directed at the NS. Furthermore, the psycholinguistic factors of foreign interlocutors had no significant effect on vowel space expansion in FDS. These findings provide evidence that FDS is characterized by heightened prosodic and acoustic features, potentially contributing to clearer speech. Additionally, the study highlights that NSs employ FDS when interacting with foreigners perceived to have a foreign accent. Full article

Tetrahydrofolate (THF), the biologically active form of folate, serves as a crucial carrier of one-carbon units essential for synthesizing cellular components such as amino acids and purine nucleotides in vivo. It also acts as an important precursor for the production of pharmaceuticals, including folinate and L-5-methyltetrahydrofolate (L-5-MTHF). In this study, we developed an efficient enzyme cascade system for the production tetrahydrofolate from folate, incorporating NADPH recycling, and explored its application in the synthesis of L-5-MTHF, a derivative of tetrahydrofolate. To achieve this, we first screened dihydrofolate reductases (DHFRs) from various organisms, identifying SmDHFR from Serratia marcescens as the enzyme with the highest catalytic activity. We then conducted a comparative analysis of formate dehydrogenases (FDHs) from different sources, successfully establishing an NADPH recycling system. To further enhance biocatalytic efficiency, we optimized key reaction parameters, including temperature, pH, enzyme ratio, and substrate concentration. To address the challenge of pH mismatch in dual-enzyme reactions, we employed an enzymatic microenvironment regulation strategy. This involved covalently conjugating SmDHFR with a superfolder green fluorescent protein mutant carrying 30 surface negative charges (−30sfGFP), using the SpyCatcher/SpyTag system. This modification resulted in a 2.16-fold increase in tetrahydrofolate production, achieving a final yield of 4223.4 µM. Finally, we extended the application of this tetrahydrofolate synthesis system to establish an enzyme cascade for L-5-MTHF production with NADH recycling. By incorporating methylenetetrahydrofolate reductase (MTHFR), we successfully produced 389.8 μM of L-5-MTHF from folate and formaldehyde. This work provides a novel and efficient pathway for the biosynthesis of L-5-MTHF and highlights the potential of enzyme cascade systems in the production of tetrahydrofolate-derived compounds. Full article

Anaerobic digestion is a critical technology for pollution control, resource capacity enhancement, and sludge management, necessitating improvements in its efficiency. Formate serves as an electron carrier in syntrophic oxidation of volatile fatty acids (VFAs) during anaerobic digestion. The accumulation of formate can exert an inhibitory effect on the anaerobic digestion process. However, the stress concentration and the mechanism of the formate are not as simple as theoretical calculations based on thermodynamics. Thus, we investigated the response to different concentrations of formate in the syntrophic oxidation of propionate. The anaerobic sludge system and syntrophic co-culture system were applied. The propionate showed more stable degradation when formate dosage ranged from 5 to 10 mM. However, when the formate dosage reached 50 mM, the concentration of propionate was significantly higher than that of CK group, and the propionate metabolism was significantly inhibited. The reduction in functional flora and homogeneous metabolic pathways were found to be unfavorable for the stable progression of syntrophic propionate metabolism. Thus, the enhancement of homoacetogenesis can be a strategy adopted by the sludge system to alleviate formate stress. The methylmalonyl-CoA (MMC) pathway was inhibited under formate stress; the downregulation of RNA transcription of formate dehydrogenase (FDH) and hydrogenase (Hyd) related to MMC pathway may be the main reason for the inhibition of syntrophic propionate oxidation. The anaerobic sludge experiment and the co-culture experiment elucidated the mechanism of action of formate from both macroscopic rules and microscopic molecular mechanisms, respectively. Full article

Strengthening primary healthcare (PHC) is vital for enhancing efficiency and improving access, clinical outcomes, and population well-being. The World Health Organization emphasizes the role of effective PHC in reducing healthcare costs and boosting productivity. With growing healthcare demands and limited resources, efficient management is critical. Background/Objectives: Building on this point, this study aimed to evaluate the efficiency of PHC units across Greece, focusing on Health Centers (HCs) and Local Health Units (ToMYs). The objective was to assess their efficiency levels and identify factors contributing to observed inefficiencies. This study explores a novel research area by being the first to assess the efficiency of restructured primary healthcare facilities in Greece, utilizing 2019 data—the first year operational data became available for the newly established ToMY facilities following recent healthcare reforms. Methods: We applied a comprehensive suite of non-parametric methods, including Data Envelopment Analysis (DEA) under variable, constant, increasing, and decreasing returns to scale (VRS, CRS, IRS/NDRS, DRS/NIRS) assumptions, along with the Free Disposal Hull (FDH) model, all oriented toward output maximization. Efficiency scores were refined using bootstrapping to calculate 95% confidence intervals, and efficient units were ranked via the super-efficiency model. Outliers were identified and removed through the data cloud algorithm. For the first time at this scale, the final sample included the vast majority of PHC facilities in Greece—234 Health Centers and 94 Local Health Units—with inputs categorized into three human resource types: medical, nursing/paramedical, and administrative/other staff. Outputs encompassed scheduled visits, emergency visits, and pharmaceutical prescription visits. This diverse and comprehensive application of DEA methods represents a novel approach to evaluating PHC efficiency in Greece, with potential relevance to broader healthcare contexts. Results: The analysis revealed significant inefficiencies and differences in technical efficiency between HCs and ToMYs. HCs could nearly double their outputs (VRS score: 1.92), while ToMYs could increase theirs by 58% (VRS score: 1.58). Scale efficiency scores were closer, with HCs slightly more aligned with their optimal scale (1.17 vs. 1.20 for ToMYs). Conclusions: There is significant potential to improve efficiency in PHC, with variations depending on unit characteristics and regional differences. This evaluation provides a foundation for policymakers to identify areas for improvement and enhance the overall performance of healthcare services in Greece. Full article

Exchangeable aluminum (Al) ions released from acidic soils with pH < 5.5 inhibit root elongation of crops, ultimately leading to yield reduced. It is necessary to identify the quantitative trait locus (QTLs) and candidate genes that confer toxicity resistance to understand the mechanism and improve tolerance of rapeseed. In this study, an F₂ segregating population was derived from a cross between Al-tolerance inbred line FDH188 (R178) and -sensitive inbred line FDH152 (S169), and the F_2:3 were used as materials to map QTLs associated with the relative elongation of taproot (RET) under Al toxicity stress. Based on bulked segregant analysis sequencing (BSA-seq), three QTLs (qAT-A07-1, qAT-A07-2, and qAT-A09-1) were detected as significantly associated with RET, and 656 candidate genes were screened. By combined BSA and RNA-seq analysis, 55 candidate genes showed differentially expressed, including genes encoding ABC transporter G (ABCG), zinc finger protein, NAC, ethylene-responsive transcription factor (ERF), etc. These genes were probably positive factors in coping with Al toxicity stress in rapeseed. This study provides new insight into exploring the QTLs and candidate genes’ response to Al toxicity stress by combined BSA-seq and RNA-seq and is helpful to further research on the mechanism of Al resistance in rapeseed. Full article

Cerebrospinal fluid (CSF) is a fluid critical to brain development, function, and health. It is actively secreted by the choroid plexus, and it emanates from brain tissue due to osmolar exchange and the constant contribution of brain metabolism and astroglial fluid output to interstitial fluid into the ventricles of the brain. CSF acts as a growth medium for the developing cerebral cortex and a source of nutrients and signalling throughout life. Together with perivascular glymphatic and interstitial fluid movement through the brain and into CSF, it also acts to remove toxins and maintain metabolic balance. In this study, we focused on cerebral folate status, measuring CSF concentrations of folate receptor alpha (FOLR1); aldehyde dehydrogenase 1L1, also known as 10-formyl tetrahydrofolate dehydrogenase (ALDH1L1 and FDH); and total folate. These demonstrate the transport of folate from blood across the blood–CSF barrier and into CSF (FOLR1 + folate), and the transport of folate through the primary FDH pathway from CSF into brain FDH + ve astrocytes. Based on our hypothesis that CSF flow, drainage issues, or osmotic forces, resulting in fluid accumulation, would have an associated cerebral folate imbalance, we investigated folate status in CSF from neurological conditions that have a severity association with enlarged ventricles. We found that all the conditions we examined had a folate imbalance, but these folate imbalances were not all the same. Given that folate is essential for key cellular processes, including DNA/RNA synthesis, methylation, nitric oxide, and neurotransmitter synthesis, we conclude that ageing or some form of trauma in life can lead to CSF accumulation and ventricular enlargement and result in a specific folate imbalance/deficiency associated with the specific neurological condition. We believe that addressing cerebral folate imbalance may therefore alleviate many of the underlying deficits and symptoms in these conditions. Full article

Foliar zinc (Zn) application can affect the accumulation and bioaccessibility of cadmium (Cd) and Zn in crops. However, the mechanisms by which foliar Zn application influences Cd and Zn bioaccessibility remain elusive. This study examined the effects of spraying ZnSO₄ and ZnNa₂EDTA on bioaccessibility and chemical forms of Cd and Zn in pakchoi (Brassica chinensis L.) shoots and evaluated human health risks via pakchoi consumption. Spraying ZnSO₄ reduced the concentrations of ethanol-extractable (F_ethanol) and deionized water-extractable (F_d-H2O) Cd, as well as the corresponding bioaccessible Cd concentrations (20.3–66.4%) and attendant health risks of Cd, whereas spraying high-dose ZnNa₂EDTA significantly increased the concentrations of both Cd forms and bioaccessible Cd. Spraying ZnSO₄ and high-dose ZnNa₂EDTA significantly increased the concentrations of Zn in F_ethanol and F_d-H2O and the corresponding bioaccessible Zn concentrations (0.8–8.3-fold). F_ethanol and F_d-H2O were the primary sources of bioaccessible Cd and Zn, contributing more than 59% of the bioaccessible Cd and Zn. These results indicate that foliar Zn application can affect Cd and Zn bioaccessibility in pakchoi mainly by modulating Cd and Zn in F_ethanol and F_d-H2O. These findings provide scientific support for the development of more efficient measures to produce safe and high-quality leafy vegetables from Cd-polluted soils. Full article

The modern global trend toward sustainable processes that meet the requirements of “green chemistry” provides new opportunities for the broad application of highly active, selective, and specific enzymatic reactions. However, the effective application of enzymes in industrial processes requires the development of systems for the remote regulation of their activity triggered by external physical stimuli, one of which is a low-frequency magnetic field (LFMF). Magnetic nanoparticles (MNPs) transform the energy of an LFMF into mechanical forces and deformations applied to enzyme molecules on the surfaces of MNPs. Here, we demonstrate the up- and down-regulation of two biotechnologically important enzymes, yeast alcohol dehydrogenase (YADH) and soybean formate dehydrogenase (FDH), in aggregates with gold-covered magnetic nanoparticles (GCMNPs) triggered by an LFMF. Two types of aggregates, “dimeric” (with the enzyme attached to several GCMNPs simultaneously), with YADH or FDH, and “monomeric” (the enzyme attached to only one GCMNP), with FDH, were synthesized. Depending on the aggregate type (“dimeric” or “monomeric”), LFMF treatment led to a decrease (down-regulation) or an increase (up-regulation) in enzyme activity. For “dimeric” aggregates, we observed 67 ± 9% and 47 ± 7% decreases in enzyme activity under LFMF exposure for YADH and FDH, respectively. Moreover, in the case of YADH, varying the enzyme or the cross-linking agent concentration led to different magnitudes of the LFMF effect, which was more significant at lower enzyme and higher cross-linking agent concentrations. Different responses to LFMF exposure depending on cofactor presence were also demonstrated. This effect might result from a varying cofactor binding efficiency to enzymes. For the “monomeric” aggregates with FDH, the LFMF treatment caused a significant increase in enzyme activity; the magnitude of this effect depended on the cofactor type: we observed up to 40% enzyme up-regulation in the case of NADP⁺, while almost no effect was observed in the case of NAD⁺. Full article

Multiple cis-acting elements are present in promoter sequences that play critical regulatory roles in gene transcription and expression. In this study, we isolated the cotton FDH (Fiddlehead) gene promoter (pGhFDH) using a real-time reverse transcription-PCR (qRT-PCR) expression analysis and performed a cis-acting elements prediction analysis. The plant expression vector pGhFDH::GUS was constructed using the Gateway approach and was used for the genetic transformation of Arabidopsis and upland cotton plants to obtain transgenic lines. Histochemical staining and a β-glucuronidase (GUS) activity assay showed that the GUS protein was detected in the roots, stems, leaves, inflorescences, and pods of transgenic Arabidopsis thaliana lines. Notably, high GUS activity was observed in different tissues. In the transgenic lines, high GUS activity was detected in different tissues such as leaves, stalks, buds, petals, androecium, endosperm, and fibers, where the pGhFDH-driven GUS expression levels were 3–10-fold higher compared to those under the CaMV 35S promoter at 10–30 days post-anthesis (DPA) during fiber development. The results indicate that pGhFDH can be used as an endogenous constitutive promoter to drive the expression of target genes in various cotton tissues to facilitate functional genomic studies and accelerate cotton molecular breeding. Full article

Unsymmetrical dimethylhydrazine (UDMH) is a common liquid propellant widely used in rocket engines and other applications. The safety of UDMH in service is affected by its slow oxidation during long-term storage to form impurities such as dimethylamine (DMA) and formaldehyde dimethylhydrazone (FDH). How these impurities affect combustion performance is not known, and in order to assess these effects, the present experiments investigated the combustion characteristics of self-igniting fuels and carried out ignition delay time measurements and flame propagation velocity measurements of pure UDMH and its denatured mixtures in a nitrogen tetroxide (NTO) atmosphere. This experiment was carried out to measure the delay time of hedge ignition of pure UDMH and qualitative analysis of its flame propagation properties under vacuum conditions at room temperature (T = 293 K). Ignition delay time measurements and flame propagation characterization were performed under the same experimental conditions for UDMH mixed with 1%, 5% and 10% FDH, UDMH mixed with 1%, 5% and 10% H₂O, UDMH mixed with 1%, 5% and 10% DMA, as well as for UDMH mixed with the same proportions of the three substances (1%, 5% and 10%). The flame propagation characteristics were analyzed. The results showed that the incorporation of DMA, H₂O and FDH in different proportions could inhibit the combustion of UDMH to varying degrees and prolong its ignition delay time. It is worth noting that the introduction of FDH had the least effect on it, and the least effect was observed at a concentration of 1%. In contrast, the effect of DMA on UDMH is more obvious, and the addition of H₂O has the largest increase in the ignition delay time of UDMH. In the flame propagation experiment, the flame of the experimental group adding H₂O can no longer fill the whole experimental window, while the other experimental groups can still make the window full of flame. Combined with the measurements of the ignition delay time, it can be seen that the moisture content has the greatest effect on the combustion characteristics. Full article

Succinic acid (SA), a C4 tricarboxylic acid cycle intermediate, is used as raw material for bulk chemicals and specialty chemicals, such as tetrahydrofuran and 1,4-butanediol, as well as also being used to synthesize the biodegradable biopolymers PBS (polymer poly (butylene succinate)). Actinobacillus succinogenes, which is facultative anaerobic and gram-negative, is one of the most promising natural SA-producing organisms, but genetic engineering of A. succinogenes is rare so far. In this study, a series of engineered strains was constructed using the pLGZ922 expression vector and a cytosine base editor (CBE) based on CRIPSR/Cas9; we found that phosphoenolpyruvate carboxylase (PEPC) was more important for the CO₂ fixation pathway than pyruvate carboxylase (PYC) in A. succinogenes, and the annotated oxaloacetic acid decarboxylase (Asuc_0301 and Asuc_0302) had little correlation with the SA synthesis pathway. The by-product pathway was closely related to cell growth, and overexpression of FDH was beneficial to growth, while the knockout of the ackA gene reduced the growth. For the first time, the hypothetic sugars and SA transporters were mined and identified in A. succinogenes, of which Asuc_0914 was responsible for glucose uptake, and Asuc_0715 and Asuc_0716 constituted SA exporters. This deepens the understanding of SA biosynthesis in A. succinogenes and is also valuable for SA production by fermentation. Full article

The study of 3D cell culture has increased in recent years as a model that mimics the tumor microenvironment (TME), which is characterized by exhibiting cellular heterogeneity, allowing the modulation of different signaling pathways that enrich this microenvironment. The TME exhibits two main cell populations: cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAM). The aim of this study was to investigate 3D cell cultures of non-small cell lung cancer (NSCLC) alone and in combination with short-term cultured dermal fibroblasts (FDH) and with differentiated macrophages of the THP-1 cell line. Homotypic and heterotypic spheroids were morphologically characterized using light microscopy, immunofluorescence and transmission electron microscopy. Cell viability, cycle profiling and migration assay were performed, followed by the evaluation of the effects of some chemotherapeutic and potential compounds on homotypic and heterotypic spheroids. Both homotypic and heterotypic spheroids of NSCLC were generated with fibroblasts or macrophages. Heterotypic spheroids with fibroblast formed faster, while homotypic ones reached larger sizes. Different cell populations were identified based on spheroid zoning, and drug effects varied between spheroid types. Interestingly, heterotypic spheroids with fibroblasts showed similar responses to the treatment with different compounds, despite being smaller. Cellular viability analysis required multiple methods, since the responses varied depending on the spheroid type. Because of this, the complexity of the spheroid should be considered when analyzing compound effects. Overall, this study contributes to our understanding of the behavior and response of NSCLC cells in 3D microenvironments, providing valuable insights for future research and therapeutic development. Full article

Formate dehydrogenases catalyze the reversible oxidation of formate to carbon dioxide. These enzymes play an important role in CO₂ reduction and serve as nicotinamide cofactor recycling enzymes. More recently, the CO₂-reducing activity of formate dehydrogenases, especially metal-containing formate dehydrogenases, has been further explored for efficient atmospheric CO₂ capture. Here, we investigate the nicotinamide binding site of formate dehydrogenase from Rhodobacter capsulatus for its specificity toward NAD⁺ vs. NADP⁺ reduction. Starting from the NAD⁺-specific wild-type RcFDH, key residues were exchanged to enable NADP⁺ binding on the basis of the NAD⁺-bound cryo-EM structure (PDB-ID: 6TG9). It has been observed that the lysine at position 157 (Lys¹⁵⁷) in the β-subunit of the enzyme is essential for the binding of NAD⁺. RcFDH variants that had Glu²⁵⁹ exchanged for either a positively charged or uncharged amino acid had additional activity with NADP⁺. The FdsB^L279R and FdsB^K276A variants also showed activity with NADP⁺. Kinetic parameters for all the variants were determined and tested for activity in CO₂ reduction. The variants were able to reduce CO₂ using NADPH as an electron donor in a coupled assay with phosphite dehydrogenase (PTDH), which regenerates NADPH. This makes the enzyme suitable for applications where it can be coupled with other enzymes that use NADPH. Full article