Search Results (43)

Petrochemical-based plastics are widely used due to their convenience and low cost, with polyethylene (PE) being the most produced globally. However, the lack of efficient and sustainable treatment methods for conventional plastic wastes has led to severe environmental pollution. A new fungus strain capable of degrading PE was isolated from soil samples collected at a waste disposal site in Henan province and identified as Aspergillus sydowii W144. After 30 days of incubation under solid-state culture conditions, the strain demonstrated significant oxidative depolymerization of low-density polyethylene (LDPE). FTIR results revealed a substantial increase in the carbonyl index of the LDPE film, while differential scanning calorimetry (DSC) analysis detected an enhanced crystallinity in the LDPE film. Notably, distinct pitting and erosion marks were observed on the surface of LDPE film using scanning electron microscopy (SEM). Quantitative analysis showed a weight loss rate of 6.39% and a reduction in Weight-Average Molecular Weight (Mw) by 50.93%. Among currently identified PE-degrading strains polyethylene, A. sydowii W144 exhibits particularly outstanding depolymerization efficiency, especially on untreated PE. Based on the whole-genome data of A. sydowii W144, a preliminary model of the putative polyethylene degradation pathway in A. sydowii W144 was constructed through homology-based sequence analysis and by referencing previously reported polyethylene degradation pathways. Laccase/multicopper oxidase plays a key role in the initial oxidation of PE. Heterologous expression of the candidate gene laccase4 in Pichia pastoris yielded an active enzyme (~56 kDa) with a laccase activity of 460 U/L, confirming its functionality. This study provides a novel microbial resource and potential enzymatic tools for PE biodegradation. The strain exhibits a promising application in complex ecosystems for PE pollution. IMPORTANCE: The polyethylene-degrading strain A. sydowii W144 isolated in this study exhibits highly efficient depolymerization capabilities, particularly under solid-state culture conditions. Genomic sequencing analysis enabled the construction of a potential polyethylene (PE) degradation pathway and facilitated the identification of key laccase and multicopper oxidase genes involved in this process. The isolation of this novel strain enriches the microbial resources available for PE waste treatment and offers new insights into the mechanisms of plastic biodegradation. Full article

Background/Objectives: SARS-CoV-2 continues to generate antigenically divergent variants that reduce the breadth of existing vaccine-induced antibody responses. Fc-fusion subunit vaccines offer advantages in stability, antigen display, and Fc-mediated immune engagement. This study aimed to design and evaluate a tetravalent RBD–Fc fusion construct incorporating RBDs from Wuhan-Hu-1 and Omicron BA.4/BA.5 and to determine whether this configuration can induce broad antibody recognition across SARS-CoV-2 variants. The objective was to assess its feasibility, biochemical properties, and initial immunogenicity. Methods: Immune responses to the construct were first assessed using the C-ImmSim simulation platform. The full-length fusion was synthesized, subcloned into pcDNA3.1(+), expressed in HEK293 cells, and purified by Protein G affinity chromatography. Protein integrity was evaluated by reducing SDS–PAGE. BALB/c mice (female, 8 weeks) were immunized with a prime–boost–boost schedule, and sera were analyzed by ELISA, considering binding to Wuhan-Hu-1, Omicron BA.4/BA.5, and a panel of RBD variants. Results: In silico analysis predicted coordinated antigen clearance, class switching, memory B- and CD4⁺ T-cell formation, and transient cytokine induction. The recombinant protein was expressed efficiently, yielding a major ~56 kDa band and a ~23 kDa RBD fragment. Vaccinated mice generated strong IgG responses to Wuhan-Hu-1 and BA.4/BA.5 RBDs and showed broad binding to major variant RBDs. Conclusions: The tetravalent RBD–Fc fusion vaccine was successfully produced and elicited broad antibody binding across SARS-CoV-2 variants, supporting its potential as a versatile protein-based vaccine platform. Full article

Despite their ecological importance and unique evolutionary history, reptiles remain underrepresented in immunological research. The innate immunity of the cottonmouth (Agkistrodon piscivorus), a semi-aquatic pit viper native to the southeastern United States, was characterized to provide insight into the molecular and cellular mechanisms underlying its first line of defense against pathogens. Plasma collected from wild A. piscivorus exhibited strong antibacterial activities against both Gram-negative and Gram-positive bacteria. In addition, plasma from A. piscivorus showed potent hemolytic activities in unsensitized sheep red blood cell (SRBC) hemolysis assays. This activity was concentration-, time-, and temperature-dependent. In addition, the hemolytic activity was inhibited by mild heat treatment (56 °C, 30 min) of plasma and proteases and also by EDTA, suggesting that the hemolytic activity was due to the presence of serum complement proteins. SDS-PAGE analysis of plasma proteins isolated from a mannan-agarose affinity column revealed the presence of a protein with a mass of 36 kDa, raising the strong possibility that the lectin pathway of complement activation is active. The EC₅₀ for hemolysis of SRBCs by plasma from A. piscivorus was approximately 10–100× lower than that of any other reptilian species described. This is the first study to characterize innate immunity in A. piscivorus. Full article

Rice seed storage proteins (SSPs) play a critical role in determining the nutritional quality, cooking properties, and digestibility of rice. To enhance seed quality, CRISPR/Cas9 genome editing was applied to modify SSP composition by targeting genes encoding 13 kDa prolamins and type A glutelins. Three CRISPR/Cas9 constructs were designed: one specific to the 13 kDa prolamin subfamily and two targeting conserved GluA glutelin regions. Edited T₀ and T₁ lines were generated and analyzed using InDel analysis, SDS-PAGE, Bradford assay, and RP-HPLC. Insertions were more frequent than deletions, accounting for 56% and 74% of mutations in prolamin and glutelin genes, respectively. Editing efficiency varied between sgRNAs. All lines with altered protein profiles contained InDels in target genes. SDS-PAGE confirmed the absence or reduction in bands corresponding to 13 kDa prolamins or GluA subunits, showing consistent profiles among lines carrying the same construct. Quantification revealed significant shifts in SSP composition, including increased albumin and globulin content. Prolamin-deficient lines showed reduced prolamins, while GluA-deficient lines exhibited increased prolamins. Total protein content was significantly elevated in all edited lines, suggesting enrichment in lysine-rich fractions. These findings demonstrate that CRISPR/Cas9-mediated editing of SSP genes can effectively reconfigure the rice protein profile and enhance its nutritional value. Full article

Membrane technology is primarily used for the separation and purification of biotechnological products, which contain proteins and enzymes. Membrane fouling during crossflow filtration remains a significant challenge. This study aims to initially validate crossflow filtration models, particularly related to pore-blocking mechanisms, through a comparative analysis with dead-end filtration models. One crossflow microfiltration (MF) and six consecutive ultrafiltration (UF) stages were implemented to concentrate laccase extracts from Pleurotus ostreatus 202 fungi. The complete pore-blocking mechanism significantly impacts the MF, UF 1000, UF 100 and UF 10 stages, with the highest related filtration constant (Kb_F) estimated at 12.60 × 10⁻⁴ (m⁻¹). Although the intermediate pore-blocking mechanism appears across all filtration stages, UF 100 is the most affected, with an associated filtration constant (Ki_F) of 16.70 (m⁻¹). This trend is supported by the highest purification factor (6.95) and the presence of 65, 62 and 56 kDa laccases in the retentate. Standard pore blocking occurs at the end of filtration, only in the MF and UF 1000 stages, with filtration constants (Ks_F) of 29.83 (s^−0.5m^−0.5) and 31.17 (s^−0.5m^−0.5), respectively. The absence of cake formation and the volume of permeate recovered indicate that neither membrane was exposed to exhaustive fouling that could not be reversed by backwashing. Full article

Traditional acrylate grouting materials often suffer from mechanical performance degradation and interfacial bonding failure under long-term water immersion, significantly limiting their application in pressurized water environments. This study proposes a composite crosslinking synergistic strategy to address these challenges. By constructing a dual-network structure through polyethylene glycol diacrylate (PEG500DA) and a monofunctional crosslinker (PEG-MA), and systematically optimizing the material formulation by regulating the triethanolamine content to control gelation time, the mechanical and hydraulic stability of the material was significantly enhanced. Increasing the acrylate concentration to 35% achieved an optimal balance between a slurry viscosity (8.3 mPa·s) and mechanical performance, with tensile strength reaching 76 kPa and the compressive strength of the sand-solidified body measuring 440 kPa. At a PEG500DA/PEG-MA ratio of 2:3, the material exhibited both high tensile strength (78 kPa) and exceptional ductility (elongation at break > 407%), with a compressive strength of 336 kPa for the sand-solidified body. When the total crosslinker content exceeded 5%, the 28-day water absorption and volume expansion rates were effectively reduced to 12% and 11%, respectively. Under simulated pressurized water conditions, the modified material demonstrated a water-pressure resistance of 300 kPa after 1 day, stabilizing at 350 kPa after 56 days—a 75% improvement over commercial products. This study provides an innovative solution for long-term anti-seepage applications in complex hydrogeological environments, offering significant advancements in material design and engineering reliability. Full article

Temporal variations in diurnal internal tides (ITs) and near-inertial waves (NIWs) in the southern South China Sea (SCS) are characterized, based on two 13-month moored current observations. Diurnal ITs, dominated by O₁ and K₁, are found to exhibit spring–neap cycles of about 14 days and significant seasonal variations. The incoherent components explain 54% and 56% of the total energy in the diurnal band, which further complicates its temporal variabilities. As for NIWs, wind energy input serves as the primary energy source and three strong events are observed. Tropical cyclone RAI passed through two moorings during the event 1 period, and triggered a peak near-inertial kinetic energy of 19.55 J m⁻³ (18.82 J m⁻³) at two moorings. After generation, the NIWs propagated downward to around 300 m, becoming the most intense event observed at DA2. In contrast, the NIWs response to tropical cyclone NOCK’s passage during event 3 was relatively weaker. The near-inertial KE generated by NOCK was confined to depths shallower than 150 m, with the average near-inertial KE being only 85% (52%) of that during event 1 for two moorings, despite the near-inertial energy input from NOCK being nearly 400% that of RAI. The modulation of background vorticity is considered the primary factor resulting in the difference in intensity of two NIW events. The penetrating depth of NIWs under the modulation of anticyclonic eddies was more than twice that under the cyclonic eddies. Furthermore, the strongest NIWs during event 2 that were observed below 350 m at mooring 2 (183% stronger than average) were also related to a strong anticyclonic eddy. Full article

Scrub typhus is a mite-borne, re-emerging public health problem in India, particularly in Tamil Nadu, South India. More than 40 serotypes of Orientia tsutsugamushi have been documented worldwide. However, the information on the circulation of its molecular sub-types in India is scanty. A retrospective study was conducted among serologically confirmed cases of scrub typhus. DNA isolated from blood was screened by a nested polymerase chain reaction (nPCR) targeting the GroEL and the 56 kDa type-specific antigen (TSA) genes. Out of 59 samples, 14 partial fragments of GroEL and the twelve 56 kDa genes were PCR-amplified and DNA-sequenced. The neighbor-joining (NJ) analysis indicated three distinct phylogenetic clades, including a novel genotype designated as Ot-Thanjavur-Tamil Nadu (Ot-TJTN, 9 nos. 64.3%); Karp-like (4 nos. 28.6%); and Kuroki-Gilliam type (1 no. 7.1%). Also, phylogenetic analysis of twelve 56 kDa variable domains (VDΙ-ΙΙΙ) of TSA gene sequences revealed a distinctive new genotypic cluster of eight samples (66.6%), and the remaining four (33.4%) were Karp-like genotypes. The Simplot analysis for the similarity and event of recombination testing elucidated the existence of the new genotype of the Ot-TJTN cluster, which was undescribed so far, in the Kato and TA716 lineages. The significant findings recommend further studies to understand the ongoing transmission dynamics of different O. tsutsugamushi strains in vector mites, rodent hosts, and humans in this region. Full article

The large-scale production of soybean proteins results in the generation of a significant volume of wastewater, containing a substantial amount of valuable β-amylase. The β-amylase enzyme was purified from the soybean whey wastewater using a three-step process, including alcohol precipitation, ion-exchange chromatography, and gel filtration chromatography. The specific activity of the purified β-amylase was 29,700 U/mg, with an enzyme activity recovery of 17.3% and purification fold of 16.5. The β-amylase had a molecular mass of around 56 kDa and an isoelectric point (pI) value of 4.8. The β-amylase exhibited optimal activity at 55 °C and reasonable stability between 30 °C and 40 °C. The enzyme demonstrated an optimum pH of 6.0 and relative stability in the pH range of 5.0–8.0. Furthermore, the β-amylase activity was stimulated by PMSF, Tween-20, Tween-40, Tween-60, Tween-80, and Triton X-100. In terms of substrate preference, the enzyme hydrolyzed potato starch worked most effectively, followed by amylose, amylopectin, soluble starch, maltose, and pullulan. The purified β-amylase showed K_m and V_max values of 3.62 μM and 1.04 μM/ (g protein min), respectively. The purification process was simple and yielded high purification and recovery. The β-amylase from soybean whey wastewater has potential applications in the food and beverage industries. Full article

Babesia duncani (B. duncani), a protozoan parasite prevalent in North America, is a significant threat for human health. Given the regulatory role of pyruvate kinase I (PyK I) in glycolytic metabolism flux and ATP generation, PyK I has been considered the target for drug intervention for a long time. In this study, B. duncani PyK I (BdPyK I) was successfully cloned, expressed, and purified. Polyclonal antibodies were confirmed to recognize the native BdPyK I protein (56 kDa) using Western blotting. AlphaFold software predicted the three-dimensional structure of BdPyK I, and molecular docking with small molecules was conducted to identify potential binding sites of inhibitor on BdPyK I. Moreover, inhibitory effects of six inhibitors (tannic acid, apigenin, shikonin, PKM2 inhibitor, rosiglitazone, and pioglitazone) on BdPyK I were examined under the optimal enzymatic conditions of 3 mM PEP and 3 mM ADP, and significant activity reduction was found. Enzyme kinetics and growth inhibition assays further confirmed the reliability of these inhibitors, with PKM2 inhibitor, tannic acid, and apigenin exhibiting the highest selectivity index as specific inhibitors for B. duncani. Subsequently, key amino acid residues were mutated in both BdPyK I and Homo sapiens pyruvate kinase I (HPyK I), and two differential amino acid residues (isoleucine and phenylalanine) were identified between HPyK I and BdPyK I through PyK activity detection experiments. These findings lay foundation for understanding the role of PyK I in the growth and development of B. duncani, providing insights for babesiosis prevention and drug development. Full article

Orientia tsutsugamushi is responsible for causing scrub typhus (ST) and is the leading cause of acute encephalitis syndrome (AES) in AES patients. A rapid and sensitive method to detect scrub typhus on-site is essential for the timely deployment of control measures. In the current study, we developed a rapid, sensitive, and instrument-free lateral flow assay (LFA) detection method based on CRISPR/Cas12a technology for diagnosing ST (named LoCIST). The method is completed in three steps: first, harnessing the ability of recombinase polymerase for isothermal amplification of the target gene; second, CRISPR/Cas12a-based recognition of the target; and third, end-point detection by LFA. The detection limit of LoCIST was found to be one gene copy of ST genomic DNA per reaction, and the process was complete within an hour. In 81 clinical samples, the assay showed no cross-reactivity with other rickettsial DNA and was 100% consistent with PCR detection of ST. LoCIST demonstrated 97.6% sensitivity and 100% specificity. Overall, the LoCIST offers a novel alternative for the portable, simple, sensitive, and specific detection of ST, and it may help prevent and control AES outbreaks due to ST. In conclusion, LoCIST does not require specialized equipment and poses a potential for future applications as a point-of-care diagnostic. Full article

FSHr antibodies have been shown to inhibit the differentiation of spermatogonia to primary spermatocytes, resulting in infertility without a pathological effect on reproductive organs. The aim of this study was to develop single-chain variable fragments (scFvs) against the follicular-stimulating hormone receptor (anti-FSHr) using phage-display technology and to evaluate the effects of intratesticular administration of the anti-FSHr scFv on testicular function and testosterone production. A phage clone against the extracellular domain of FSHr selected from a scFv phagemid library was analyzed for binding kinetics by surface plasmon resonance. Using ultrasound guidance, three adult macaques (M. fascicularis) were administered with 1 mL of 0.4 mg/mL anti-FSHr scFv (treatment) and 1 mL sterile phosphate buffer solution (control) into the left and right rete testis, respectively. Testicular appearance and volume, ejaculate quality, and serum testosterone levels were recorded on day 0 (before injection) and on days 7, 28, and 56 (after injection). Testicular tissue biopsies were performed on day 7 and day 56 to quantify the mRNA expressions of androgen binding protein (ABP), inhibin subunit beta B (IHBB), and vascular endothelial growth factor A (VEGFA). The results demonstrated that the anti-FSHr scFv molecule was calculated as 27 kDa with a dissociation constant (K_D) of 1.03 µM. The volume of the anti-FSHr scFv-injected testicle was reduced on days 28 and 56 compared with day 0 (p < 0.05). Total sperm number was reduced from day 0 (36.4 × 10⁶ cells) to day 56 (1.6 × 10⁶ cells) (p < 0.05). The percentage of sperm motility decreased from day 0 (81.7 ± 1.0%) to day 7 (23.3 ± 1.9%), day 28 (41.7 ± 53.4%), and day 56 (8.3 ± 1.9%) (p < 0.05). Sperm viability on day 0 was 86.8 ± 0.5%, which reduced to 64.2 ± 1.5%, 67.1 ± 2.2%, and 9.3 ± 1.1% on days 7, 28, and 56, respectively (p < 0.05). The expression of ABP and VEGFA on days 7 (14.2- and 3.2-fold) and 56 (5.6- and 5.5-fold) was less in the scFv-treated testicle compared with the controls (p < 0.05). On day 56, the expression of IHBB was less (p < 0.05) in the treated testis (1.3-fold) compared with the controls. Serum testosterone levels were unchanged throughout the study period (p > 0.05). This study characterized the anti-FSHr scFv and demonstrated that treatment with anti-FSHr ameliorates testicular function without altering testosterone levels, offering a potential alternative contraceptive for the long-tailed macaques. Full article

Cutting fluids are the main source of oily wastewater in the metalworking industry. This study deals with the development of antifouling composite hydrophobic membranes for treatment of oily wastewater. The novelty of this study is that a low energy electron-beam deposition technique was applied for a polysulfone (PSf) membrane with a molecular-weight cut-off of 300 kDa, which is promising for use in the treatment of oil-contaminated wastewater, by using polytetrafluoroethylene (PTFE) as target materials. The effect of the thickness of the PTFE layer (45, 660, and 1350 nm) on the structure, composition, and hydrophilicity of membranes was investigated using scanning electron microscopy, water contact angle (WCA) measurements, atomic force microscopy, and FTIR-spectroscopy. The separation and antifouling performance of the reference and modified membranes were evaluated during ultrafiltration of cutting fluid emulsions. It was found that the increase in the PTFE layer thickness results in the significant increase in WCA (from 56° up to 110–123° for the reference and modified membranes respectively) and decrease in surface roughness. It was found that cutting fluid emulsion flux of modified membranes was similar to the flux of the reference PSf-membrane (7.5–12.4 L·m⁻²·h⁻¹ at 6 bar) while cutting fluid rejection (R_CF) of modified membranes increased compared to the reference membrane (R_CF = 58.4–93.3% for modified and R_CF = 13% for the reference PSf membrane). It was established that despite the similar flux of cutting fluid emulsion, modified membranes demonstrate 5–6.5 times higher flux recovery ratio (FRR) compared to the reference membrane. The developed hydrophobic membranes were found to be highly efficient in oily wastewater treatment. Full article

GRTH/DDX25 is a testis-specific DEAD-box family of RNA helicase, which plays an essential role in spermatogenesis and male fertility. There are two forms of GRTH, a 56 kDa non-phosphorylated form and a 61 kDa phosphorylated form (pGRTH). GRTH-KO and GRTH Knock-In (KI) mice with R242H mutation (lack pGRTH) are sterile with a spermatogenic arrest at step 8 of spermiogenesis due to failure of round spermatids (RS) to elongate. We performed mRNA-seq and miRNA-seq analysis on RS of WT, KI, and KO to identify crucial microRNAs (miRNAs) and mRNAs during RS development by establishing a miRNA–mRNA network. We identified increased levels of miRNAs such as miR146, miR122a, miR26a, miR27a, miR150, miR196a, and miR328 that are relevant to spermatogenesis. mRNA–miRNA target analysis on these DE-miRNAs and DE-mRNAs revealed miRNA target genes involved in ubiquitination process (Ube2k, Rnf138, Spata3), RS differentiation, and chromatin remodeling/compaction (Tnp1/2, Prm1/2/3, Tssk3/6), reversible protein phosphorylation (Pim1, Hipk1, Csnk1g2, Prkcq, Ppp2r5a), and acrosome stability (Pdzd8). Post-transcriptional and translational regulation of some of these germ-cell-specific mRNAs by miRNA-regulated translation arrest and/or decay may lead to spermatogenic arrest in KO and KI mice. Our studies demonstrate the importance of pGRTH in the chromatin compaction and remodeling process, which mediates the differentiation of RS into elongated spermatids through miRNA–mRNA interactions. Full article

Improvement of the efficiency of drug penetration into the eye tissues is still an actual problem in ophthalmology. One of the most promising solutions is drug encapsulation in carriers capable of overcoming the cornea/sclera tissue barrier. Formulations on the base of antioxidant enzyme, superoxide dismutase 1 (SOD1), and an inhibitor of angiotensin-converting enzyme, enalaprilat, were prepared by simultaneous inclusion of both drugs into calcium phosphate (CaP) particles in situ with subsequent covering of the particles with 5 kDa chitosan. The formulations obtained were characterized by dynamic light scattering and scanning electron microscopy. Hybrid CaP-chitosan particles co-loaded with SOD1 and enalaprilat had a mean hydrodynamic diameter of 120–160 nm and ζ-potential +20 ± 1 mV. The percentage of the inclusion of SOD1 and enalaprilat in hybrid particles was 30% and 56%, respectively. The ability of SOD1 and enalaprilat to reduce intraocular pressure (IOP) was examined in vivo in normotensive Chinchilla rabbits. It was shown that topical instillations of SOD1/enalaprilat co-loaded hybrid particles were much more effective in decreasing IOP compared to free enzyme or free enalaprilat and even to the same particles that contained a single drug. Thus, the proposed formulations demonstrate potential as prospective therapeutic agents for the treatment of glaucoma. Full article