Applied Biochemistry and Biotechnology最新文献

Mosquitoes rely heavily on olfactory cues for locating suitable oviposition sites, with microbial communities in aquatic habitats playing a crucial role in producing volatile organic compounds (VOCs) that influence mosquito behaviour. In this study, we isolated Bacillus subtilis DHB13 from the breeding habitat of Culex quinquefasciatus, a major vector of several human diseases. The partial 16S rRNA gene sequence of the isolate has been submitted to NCBI GenBank with the accession number PV698100. The identity and resistance profile of the strain was confirmed through biochemical and antibiotic susceptibility tests. The bacterial suspension demonstrated a notable oviposition activity index (OAI) of 0.77 ± SE, with moderate variation among treatments (F(3, 8) = 3.631, p = 0.0642). Multiple comparison analysis (Tukey's test) showed that OAI values for DHB13-treated media did not differ significantly from natural habitat water but were significantly higher than the sterile control, indicating a biologically relevant attraction of gravid female mosquitoes. LC-MS analysis of the bacterial culture supernatant revealed the presence of three cresol derivatives: diisopropyl-m-cresol, 3-ethyl-p-cresol, and 6-ethyl-o-cresol. These compounds were evaluated through molecular docking against Cx. quinquefasciatus Odorant Binding Protein 1 (CxOBP1), a protein known to mediate olfactory-driven oviposition behaviour. However, mosquito olfaction involves several OBPs, receptors, and enzymes, so interaction with CxOBP1 represents only part of this complex sensory system. Molecular docking revealed strong binding of CxOBP1 with diisopropyl-m-cresol (-6.7 kcal/mol), 3-ethyl-p-cresol (-6.2 kcal/mol), and 6-ethyl-o-cresol (-5.9 kcal/mol), indicating potential oviposition attractant activity. All three ligands were found to bind within a conserved binding pocket of CxOBP1, behavioural assays confirmed the oviposition-stimulant properties of the bacterial suspension, indicating that the detected compounds mimic natural semio-chemicals such as p-cresol, previously recognized as an oviposition cue. These findings reinforce the role of microbiota in shaping mosquito reproductive behaviour through the production of volatile attractants. Moreover, they highlight the potential of using microbial VOCs as environmentally sustainable tools for mosquito surveillance and vector control. This integrative approach linking microbial ecology, chemical analysis, and mosquito behaviour provides novel insights for the development of attractant-based control strategies.

Lysophosphatidic acid acyltransferase (LPAAT) is a pivotal enzyme in the de novo biosynthesis of phosphatidic acid (PA), playing a central role in glycerophospholipid assembly and triacylglycerol (TAG) accumulation. Myrmecia incisa is a green microalga notable for its high content of arachidonic acid (ArA), yet the molecular mechanism underlying ArA enrichment in TAG remains unclear. In this study, a putative LPAAT gene from M. incisa, designated MiLPAAT, was identified and cloned, followed by systematic structural and functional characterization. Sequence analysis revealed that MiLPAAT contains a conserved PlsC domain and the characteristic H(X)₄D and EGTR motifs. Bioinformatic predictions identified at least one transmembrane domain at the N-terminus, supporting its identity as an integral membrane protein. This was further confirmed by membrane fractionation and Western blot analysis, which demonstrated its association with the membrane fraction. Phylogenetic analysis further demonstrated its close evolutionary relationship to LPAAT homologs in other green algae. Heterologous expression in Escherichia coli, coupled with in vitro enzymatic assays, confirmed that the recombinant MiLPAAT protein possesses LPAAT activity, catalyzing the acylation of LPA with various acyl-CoAs. Among the substrates tested, MiLPAAT exhibited the highest catalytic efficiency toward ArA-CoA (104.8 ± 3.2 nmol/mg/min), followed by oleoyl-CoA (81.5 ± 2.7 nmol/mg/min) and palmitoyl-CoA (68.4 ± 2.1 nmol/mg/min), consistent with the ArA-rich TAG composition observed in M. incisa. Immunogold labeling and immunohistochemical localization experiments revealed that MiLPAAT is predominantly localized at the plasma membrane. Findings of the present study suggest that MiLPAAT plays a critical role in PA biosynthesis and assembly of ArA into TAG in M. incisa, providing a novel target for microalgal lipid metabolic engineering.

Aegiceras corniculatum (AC), a mangrove species widely recognized for its diverse pharmacological properties, has attracted significant attention recently due to its antioxidant, anti-inflammatory, and antimicrobial activities. Despite this, the neuroprotective potential of AC, particularly in the context of neurodegenerative disorders such as Parkinson's disease (PD), remains largely unexplored. Due to the growing interest in plant-derived compounds for the management of PD, investigating the therapeutic relevance of AC could provide new insights into alternative strategies for neuroprotection. The present study aimed to evaluate the neuroprotective ability of the leaf extract of AC against PD. Preliminary phytochemical analyses were conducted to identify the presence of bioactive compounds in the AC extract (ACE). The antioxidant capacity of ACE was evaluated using three standard assays: 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, ferric reducing antioxidant power (FRAP) assay, and hydrogen peroxide (H₂O₂) scavenging assay. We used the MPTP-induced PD model established in an SH-SY5Y human neuroblastoma cell line as well as in the U87-MG glioblastoma cell line. MTT assay was conducted to measure the cell viability, and further, 2',7'-dichlorofluorescin diacetate (DCFDA) assay was used to measure the reactive oxygen species (ROS). Glutathione peroxidase (GPx) enzyme activity was determined by a chromogenic reaction-based assay. Western blot analysis was used to assess the expression levels of SNCA protein. The ACE was found to contain a diverse range of phytochemicals, including polyphenols, flavonoids, and terpenoids. Among the ethanolic, hydroethanolic, and aqueous extracts evaluated, ethanolic ACE (eACE) exhibited the highest antioxidant activity. The SH-SY5Y cell line demonstrated significantly higher neuroprotective potential to eACE treatment compared to the U87-MG cell line. Moreover, eACE markedly attenuated intracellular ROS levels and enhanced glutathione peroxidase (GP_X) enzymatic activity. In line with these effects, eACE also significantly downregulated the expression of the SNCA gene, suggesting its potential modulatory role in oxidative stress-related neurodegeneration. AC exhibited notable neuroprotective effects in an in vitro model of PD. These effects are likely mediated through the attenuation of oxidative stress and neuroinflammation, inhibition of apoptosis, and preservation of cellular energy metabolism. The results suggest that the extract of AC may provide important insights for developing therapies that modify the progression of PD. Elucidating the underlying mechanisms could inform the creation of innovative treatments designed to slow or alter the trajectory of PD. The present study emphasizes the potential benefits of AC with contemporary scientific investigation to address the unmet needs of patients suffering from PD.

The release of heavy metals in aquatic systems may cause serious health hazards to humans and other living organisms. Adsorption is a cost-effective and efficient method for heavy metal removal. Biochar is an effective adsorbent for removing heavy metals from aquatic systems because of its larger surface area, functional group, and porous structure. In this study, hardwood-based biochar was used to remove Pb²⁺, Ni²⁺, and Cu²⁺ ions from synthetically prepared wastewater. The highest percentage adsorption of Pb²⁺, Ni²⁺, and Cu²⁺ ions was found to be 90.8 ± 1.9, 92.9 ± 2.1, and 87.9 ± 1.8%, respectively. The adsorption capacity of hardwood-based biochar for Pb²⁺, Ni²⁺, and Cu²⁺ ions was found to be 15.89 ± 0.8, 9.24 ± 0.6 and 11.61 ± 0.9 mg/g. The pseudo first-order rate constant for Pb²⁺, Ni²⁺, and Cu²⁺ ions has been evaluated to be 0.023 ± 0.02, 0.030 ± 0.01, and 0.026 ± 0.02 min⁻¹. The heavy metal ion-loaded biochar was also used as an electrode material for supercapacitors after the successful removal of heavy metal ions. The specific capacitance of Pb²⁺, Ni²⁺, and Cu²⁺ loaded hardwood-based biochar was found to be 203 F/g, and maximum power and energy densities were observed as 3600 W/kg and 3.5 Wh/kg, respectively. After 5000 cycles, the material was able to achieve a retention of 84.9%. Thus, biochar derived from hardwoods shows promise as an electrode material for supercapacitors and as an effective adsorbent for the removal of heavy metals. To improve performance, future studies should focus on surface modification, process upgradation, and assessment in real wastewater treatment and energy storage systems.

Terminal deoxynucleotidyl transferase (TdT) is a template-independent polymerase that catalyzes the addition of deoxynucleoside triphosphates to the 3'-terminus of a DNA strand. While TdT is a key enzyme for developing enzymatic DNA synthesis technologies, its inherent low thermal stability presents a significant limitation. This study aims to improve the thermostability of TdT from Mus musculus through a combination of site-saturation mutagenesis and rational design. Residues for saturation mutagenesis were identified using B-factor analysis and the B-FITTER program, while promising substitutions for rational design were selected using Foldit, ProteinMPNN and CARBonAra. Through several iterations of mutagenesis, we obtained two highly promising variants. The first one, dubbed A4, obtained solely through saturation mutagenesis, showed a 26-fold higher expression level than the WT protein. The second one, dubbed mutant 275, demonstrated exceptional stability, showing no significant loss of activity after 180 min of incubation at 45 °C - conditions under which the wild-type enzyme's half-life was less than 2 min. This corresponds to a > 120-fold increase in stability. Additionally, its melting temperature (Tm) was increased by 6.5 °C. Moreover, mutant 275 demonstrated a 4- to 6-fold increase in catalytic activity at 37 °C. This significant enhancement in thermostability was achieved after four rounds of iterative mutagenesis and is attributed to the formation of a stabilizing salt bridge network on the protein surface, distant from the active site. The obtained thermostable TdT variants serve as robust scaffolds for further engineering to improve activity towards the 3'-reversibly blocked nucleotides required for next-generation enzymatic DNA synthesis.

Objective: To explore the mechanism of S100 Calcium Binding Protein A8 (S100A8)-mediated ferroptosis in vascular dementia (VaD) via the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway.

Methods: To establish models of VaD and study microglial responses, we employed bilateral carotid artery stenosis (BCAS) in mice and oxygen-glucose deprivation (OGD) in BV2 cells, respectively. The cognitive function of mice was assessed by Morris water maze experiments. The pathological changes of brain tissue were observed by Nissl staining and luxol fast blue staining. Inflammatory cytokines were determined by enzyme-linked immunosorbent assay. The co-localization of S100A8 was identified by immunofluorescence. Ferroptosis was assessed by related indices such as intracellular Fe²⁺ content, reactive oxygen species, mitochondrial membrane potential and lipid peroxidation. JAK2/STAT3 pathway activation was evaluated by Western blot.

Results: In BCAS mice, cerebral hypoperfusion triggered the upregulation of S100A8 in hippocampal microglia. This upregulation showed a progressive increase following surgery, peaking at 28 days post-BCAS. Inhibition of S100A8 improved cognitive deficits, attenuated brain damage, reduced neuroinflammation, and suppressed ferroptosis in BCAS mice. In BV2 cells, S100A8 silencing alleviated inflammation and ferroptosis induced by OGD. The anti-inflammatory effects of S100A8 silencing were counteracted by Erastin but amplified by Ferrostatin-1‌. Furthermore, S100A8 inhibition blocked JAK2/STAT3 activation; however, the JAK2/STAT3 agonist Colivelin TFA reversed the protective effects of S100A8 silencing in OGD-treated BV2 cells.

Conclusion: S100A8 mediates ferroptosis in VaD via JAK2/STAT3 pathway.

Considered one of the most prevalent monounsaturated fatty acids (MUFA) from marine and plant sources, omega-9 fatty acids (n-9) have gained worldwide recognition since due to its strong anti-obesogenic properties. These findings highlight the anti-obesity efficacy and mechanism of n-9, gondoic acid (GA) for anti-obesity interventions, offering a novel research perspective at recent research field. The animals (BALB/c male mice) were segregated into six study groups: the control group (C) received normal chow diet; the vehicle control group (VC) received normal chow diet + olive oil; high fat diet (HFD) received by the obese control group (OC); and the treatment-I (T-I), treatment-II (T-II), treatment-III (T-III) group co-administered with GA as follows: 1 mg/kg GA + HFD; 2 mg/kg GA + HFD; 4 mg/kg GA + HFD. In this study, GA was delivered orally once daily for six weeks to diet-induced obese mice. According to the findings, administering GA decreased weight gain, blood lipid profile levels, improved inflammation and adipogenesis. The mRNA expression studies and western blot assay found that GA administration may boost glucose metabolism, enhance the process of thermogenesis and β-oxidation in mitochondria via the AMPK mechanism, reducing HFD-induced adiposity. In brief, our findings can point in a broader direction for the use of GA in obesity based on genetic indicators.