Polyethylene terephthalate nanoplastics cause oxidative stress induced cell death in Saccharomyces cerevisiae.

Polyethylene terephthalate (PET) is a common plastic widely used in food and beverage packaging that poses a serious risk to human health and the environment due to the continual rise in its production and usage. After being produced and used, PET accumulates in the environment and breaks down into nanoplastics (NPs), which are then consumed by humans through water and food sources. The threats to human health and the environment posed by PET-NPs are of great concern worldwide, yet little is known about their biological impacts. Herein, the smallest sized PET-NPs so far (56 nm) with an unperturbed PET structure were produced by a modified dilution-precipitation method and their potential cytotoxicity was evaluated in Saccharomyces cerevisiae. Exposure to PET-NPs decreased cell viability due to oxidative stress induction revealed by the increased expression levels of stress response related-genes as well as increased lipid peroxidation. Cell death induced by PET-NP exposure was mainly through apoptosis, while autophagy had a protective role.

Understanding the stability of a plastic-degrading Rieske iron oxidoreductase system.

Rieske oxygenases (ROs) are a diverse metalloenzyme class with growing potential in bioconversion and synthetic applications. We postulated that ROs are nonetheless underutilized because they are unstable. Terephthalate dioxygenase (TPADO PDB ID 7Q05) is a structurally characterized heterohexameric α3ß3 RO that, with its cognate reductase (TPARED), catalyzes the first intracellular step of bacterial polyethylene terephthalate plastic bioconversion. Here, we showed that the heterologously expressed TPADO/TPARED system exhibits only ~300 total turnovers at its optimal pH and temperature. We investigated the thermal stability of the system and the unfolding pathway of TPADO through a combination of biochemical and biophysical approaches. The system's activity is thermally limited by a melting temperature (Tm) of 39.9°C for the monomeric TPARED, while the independent Tm of TPADO is 50.8°C. Differential scanning calorimetry revealed a two-step thermal decomposition pathway for TPADO with Tm values of 47.6 and 58.0°C (ΔH = 210 and 509 kcal mol-1, respectively) for each step. Temperature-dependent small-angle x-ray scattering and dynamic light scattering both detected heat-induced dissociation of TPADO subunits at 53.8°C, followed by higher-temperature loss of tertiary structure that coincided with protein aggregation. The computed enthalpies of dissociation for the monomer interfaces were most congruent with a decomposition pathway initiated by ß-ß interface dissociation, a pattern predicted to be widespread in ROs. As a strategy for enhancing TPADO stability, we propose prioritizing the re-engineering of the ß subunit interfaces, with subsequent targeted improvements of the subunits.

Improving plastic degrading enzymes via directed evolution.

Plastic degrading enzymes have immense potential for use in industrial applications. Protein engineering efforts over the last decade have resulted in considerable enhancement of many properties of these enzymes. Directed evolution, a protein engineering approach that mimics the natural process of evolution in a laboratory, has been particularly useful in overcoming some of the challenges of structure-based protein engineering. For example, directed evolution has been used to improve the catalytic activity and thermostability of polyethylene terephthalate (PET)-degrading enzymes, although its use for the improvement of other desirable properties, such as solvent tolerance, has been less studied. In this review, we aim to identify some of the knowledge gaps and current challenges, and highlight recent studies related to the directed evolution of plastic-degrading enzymes.

Microplastics and non-natural cellulosic particles in Spanish bottled drinking water.

This investigation explored the presence of microplastics (MPs) and artificial cellulosic particles (ACPs) in commercial water marketed in single use 1.5 L poly(ethylene terephthalate) bottles. In this work we determined a mass concentration of 1.61 (1.10-2.88) µg/L and 1.04 (0.43-1.82) µg/L for MPs and ACPs respectively in five top-selling brands from the Spanish bottled water market. Most MPs consisted of white and transparent polyester and polyethylene particles, while most ACPs were cellulosic fibers likely originating from textiles. The median size of MPs and ACPs was 93 µm (interquartile range 76-130 µm) and 77 µm (interquartile range 60-96 µm), respectively. Particle mass size distributions were fitted to a logistic function, enabling comparisons with other studies. The estimated daily intake of MPs due to the consumption of bottled water falls within the 4-18 ng kg-1 day-1 range, meaning that exposure to plastics through bottled water probably represents a negligible risk to human health. However, it's worth noting that the concentration of plastic found was much higher than that recorded for tap water, which supports the argument in favour of municipal drinking water.

Characterization and elimination efficiency of volatile organic compounds in mechanically recycled polyethylene terephthalate at various recycling stages.

The recycling of polyethylene terephthalate (PET) stands as an effective strategy for mitigating plastic pollution and reducing resource waste. The study aimed to investigate the characterization and elimination efficiency of volatile organic compounds (VOCs) present in rPET at various recycling stages using comprehensive two-dimensional gas chromatography-quadrupole-time-of-flight-mass spectrometry coupled with chemometrics. The results revealed that 52, 135, 95, 44, and 33 VOCs, mostly classified into three chemical groups, were tentatively identified in virgin - PET (v-PET), cold water washed - rPET (C-rPET), decontaminated - rPET (D-rPET), melt-extruded - rPET (M-rPET), and solid-state polycondensation - rPET (S-rPET), respectively. Regarding the VOCs with high and median detection frequencies, fatty acyls showed the highest elimination efficiency (100 % and 92 %), followed by organooxygen compounds (81 % and 99 %), others (97 % and 95 %), and benzene and substituted derivatives (82 % and 95 %) in term of HS-SPME. Following the recycling process, there was a general decrease in the concentration of almost all VOCs, as evidenced by the substantial reduction of o-Xylene, hexanoic acid, octanal, and D-limonene from 18.11, 22.43, 30.74, and 7.41 mg/kg to 0, 0, 3.97, and 0 mg/kg, respectively. However, it was noteworthy that the VOCs identified in the samples were not completely extracted, owing to the limitations of HS-SPME. Furthermore, chemometrics analysis indicated significant discrimination among VOCs from vPET, C-rPET, D-rPET, and M-rPET, while indistinct differences were observed between M-rPET and S-rPET. This study contributes to the enhancement of the recycling process and emphasizes the importance of safeguarding consumer health in terms of elimination of VOCs.

Sonicated polyethylene terephthalate nano- and micro-plastic-induced inflammation, oxidative stress, and autophagy in vitro.

The environmental presence of nano- and micro-plastic particles (NMPs) is suspected to have a negative impact on human health. Environmental NMPs are difficult to sample and use in life science research, while commercially available plastic particles are too morphologically uniform. Additionally, this NMPs exposure exhibited biological effects, including cell internalization, oxidative stress, inflammation, cellular adaptation, and genotoxicity. Therefore, developing new methods for producing heterogenous NMPs as observed in the environment is important as reference materials for research. Thus, we aimed to generate and characterize NMPs suspensions using a modified ultrasonic protocol and to investigate their biological effects after exposure to different human cell lines. To this end, we produced polyethylene terephthalate (PET) NMPs suspensions and characterized the particles by dynamic light scattering and scanning electron microscopy. Ultrasound treatment induced polymer degradation into smaller and heterogeneous PET NMPs shape fragments with similar surface chemistry before and after treatment. A polydisperse suspension of PET NMPs with 781 nm in average size and negative surface charge was generated. Then, the PET NMPs were cultured with two human cell lines, A549 (lung) and HaCaT (skin), addressing inhalation and topical exposure routes. Both cell lines interacted with and have taken up PET NMPs as quantified via cellular granularity assay. A549 but not HaCaT cell metabolism, viability, and cell death were affected by PET NMPs. In HaCaT keratinocytes, large PET NMPs provoked genotoxic effects. In both cell lines, PET NMPs exposure affected oxidative stress, cytokine release, and cell morphology, independently of concentration, which we could relate mechanistically to Nrf2 and autophagy activation. Collectively, we present a new PET NMP generation model suitable for studying the environmental and biological consequences of exposure to this polymer.

Application of an alchemical free energy method for the prediction of thermostable DuraPETase variants.

Non-equilibrium (NEQ) alchemical free energy calculations are an emerging tool for accurately predicting changes in protein folding free energy resulting from amino acid mutations. In this study, this method in combination with the Rosetta ddg monomer tool was applied to predict more thermostable variants of the polyethylene terephthalate (PET) degrading enzyme DuraPETase. The Rosetta ddg monomer tool efficiently enriched promising mutations prior to more accurate prediction by NEQ alchemical free energy calculations. The relative change in folding free energy of 96 single amino acid mutations was calculated by NEQ alchemical free energy calculation. Experimental validation of ten of the highest scoring variants identified two mutations (DuraPETaseS61M and DuraPETaseS223Y) that increased the melting temperature (Tm) of the enzyme by up to 1 °C. The calculated relative change in folding free energy showed an excellent correlation with experimentally determined Tm resulting in a Pearson's correlation coefficient of r = - 0.84. Limitations in the prediction of strongly stabilizing mutations were, however, encountered and are discussed. Despite these challenges, this study demonstrates the practical applicability of NEQ alchemical free energy calculations in prospective enzyme engineering projects. KEY POINTS: • Rosetta ddg monomer enriches stabilizing mutations in a library of DuraPETase variants • NEQ free energy calculations accurately predict changes in Tm of DuraPETase • The DuraPETase variants S223Y, S42M, and S61M have increased Tm.

Smart Graphene Textiles for Biopotential Monitoring: Laser-Tailored Electrochemical Property Enhancement.

While most of the research in graphene-based materials seeks high electroactive surface area and ion intercalation, here, we show an alternative electrochemical behavior that leverages graphene's potential in biosensing. We report a novel approach to fabricate graphene/polymer nanocomposites with near-record conductivity levels of 45 Ω sq-1 and enhanced biocompatibility. This is realized by laser processing of graphene oxide in a sandwich structure with a thin (100 µm) polyethylene terephthalate film on a textile substrate. Such hybrid materials exhibit high conductivity, low polarization, and stability. In addition, the nanocomposites are highly biocompatible, as evidenced by their low cytotoxicity and good skin adhesion. These results demonstrate the potential of graphene/polymer nanocomposites for smart clothing applications.

Exploring the substrate spectrum of phylogenetically distinct bacterial polyesterases.

The rapid escalation of plastic waste accumulation presents a significant threat of the modern world, demanding an immediate solution. Over the last years, utilization of the enzymatic machinery of various microorganisms has emerged as an environmentally friendly asset in tackling this pressing global challenge. Thus, various hydrolases have been demonstrated to effectively degrade polyesters. Plastic waste streams often consist of a variety of different polyesters, as impurities, mainly due to wrong disposal practices, rendering recycling process challenging. The elucidation of the selective degradation of polyesters by hydrolases could offer a proper solution to this problem, enhancing the recyclability performance. Towards this, our study focused on the investigation of four bacterial polyesterases, including DaPUase, IsPETase, PfPHOase, and Se1JFR, a novel PETase-like lipase. The enzymes, which were biochemically characterized and structurally analyzed, demonstrated degradation ability of synthetic plastics. While a consistent pattern of polyesters' degradation was observed across all enzymes, Se1JFR stood out in the degradation of PBS, PLA, and polyether PU. Additionally, it exhibited comparable results to IsPETase, a benchmark mesophilic PETase, in the degradation of PCL and semi-crystalline PET. Our results point out the wide substrate spectrum of bacterial hydrolases and underscore the significant potential of PETase-like enzymes in polyesters degradation.

A meta-analysis of risk factors for a Dacron-cuffed catheter related infection in hemodialysis.

OBJECTIVE: To provide theoretical basis for prevention of a Dacron-cuffed catheter related infection (CRI), the risk factors of CRI in hemodialysis patients were systematically evaluated. METHODS: Eight databases, including PubMed, Cochrane library, EMBASE, Web of Science, China National Knowledge Infrastructure (CNKI), Chinese Biomedical Database (CBM), Wanfang Database and Chinese Scientific Journal Database (VIP), were searched to screen out literatures related to the risk factors of long-term indwelling a Dacron-cuffed CRI in hemodialysis. Meta-analysis of risk factors for a Dacron-cuffed CRI in hemodialysis and publication bias test were performed using RevMan 5.4 software. RESULTS: After screening, 13 literatures involving a Dacron-cuffed CRI were included, with a total of 625 patients, and the infection rate was 11.7%. The combined OR value and 95% confidence interval (CI) of all factors were: Combined with Diabetes (1.94, 1.51 ~ 2.50), Hb (1.82, 1.35 ~ 2.44), age (2.38, 1.06 ~ 5.34), catheter indwelling time (1.79, 1.21 ~ 2.66), serum albumin (2.26, 1.25 ~ 4.08), catheter indwelling site (3.29, 1.74 ~ 6.23) and the number of tube placement (5.40, 2.65 ~ 11.02). CONCLUSIONS: The main risk factors for a Dacron-cuffed CRI in hemodialysis were combined with diabetes, hemoglobin level, age, catheter indwelling time, serum albumin level, femoral vein catheter indwelling and catheterization times. In other words, hemodialysis patients are at higher risk of CRI if they have diabetes, or if they have a lower hemoglobin level, or if they are older, or if they have a longer duration of catheterization, or if they have a lower serum albumin level, or if they have a femoral vein catheter, or if they have more catheters.

Manufacturing of PEEK orthodontic baseplate and 3D-printed alloy components from an intraoral scan.

This paper demonstrates a digital manufacturing technique of a removable orthodontic appliance from an intraoral scan. An intraoral scan was made for the maxillary and mandibular arches. 3Shape Orthodontics Appliance Designer produced the virtual Hawley retainer, consisting of alloy components (Adam Clasps and Fitted Labial bow) and a base plate. The base plate design was modified to adapt to inserting the alloy components, which were combined using cold-cured acrylic. The finished Hawley retainer was assessed intraorally. The described technique emphasizes the design specifications of digitally designed and manufactured removable orthodontic appliances. A combination of additive and subtractive techniques was successfully employed to manufacture the alloy components and base plate. This novel method provides an alternative approach to manufacturing removable appliances with computer-aided design (CAD)/computer-aided manufacturing (CAM) technologies. The described process offers a precursor to digital manufacturing of other developed designs of dental appliances.

Effects of microplastics on the structure and function of bacterial communities in sediments of a freshwater lake.

As an emerging pollutant, microplastics (MPs) cause widespread concern around the world owing to the serious threat they pose to ecosystems. In particular, sediments are thought to be the long-term sink for the continual accumulation of MPs in freshwater ecosystems. Polyethylene (PE) and polyethylene terephthalate (PET) have been frequently detected with large concentration variations in freshwater sediments from the lower reaches of the Yangtze River, one of the most economically developed regions in China, characterized by accelerated urbanization and industrialization, high population density and high plastics consumption. However, the impact of PE and PET on the sedimental bacterial community composition and its function has not been well reported for this specific region. Herein, PE and PET particles were added to freshwater sediments to assess the effects of different MP types on the bacterial community and its function, using three concentrations (500, 1500 and 2500 items/kg) per MP and incubations of 35, 105 and 175 days, respectively. This study identified a total of 68 phyla, 211 classes, 518 orders, 853 families and 1745 genera. Specifically, Proteobacteria, Chloroflexi, Acidobacteriota, Actinobacteriota and Firmicutes were the top five phyla. A higher bacterial diversity was obtained in control sediments than in the MP-treated sediments. The presence of MPs, whether PET or PE, had significant impact on the bacterial diversity, community structure and community composition. PICRUSt2 and FAPOTAX predictions demonstrated that MPs could potentially affect the metabolic pathways and ecologically functional groups of bacteria in the sediment. Besides the MP-related factors, such as the type, concentration and incubation time, the physicochemical parameters had an effect on the structure and function of the bacterial community in the freshwater sediment. Taken together, this study provides useful information for further understanding how MPs affect bacterial communities in the freshwater sediment of the lower reaches of the Yangtze River, China.

Ovalbumin interaction with polystyrene and polyethylene terephthalate microplastics alters its structural properties.

Contaminating microplastics can interact with food proteins in the food matrix and during digestion. This study investigated adsorption of chicken egg protein ovalbumin to polystyrene (PS, 110 and 260 µm) and polyethylene terephthalate (PET, 140 µm) MPs in acidic and neutral conditions and alterations in ovalbumin structure. Ovalbumin adsorption affinity depended on MPs size (smaller > larger), type (PS > PET) and pH (pH 3 > pH 7). In bulk solution, MPs does not change ovalbumin secondary structure significantly, but induces loosening (at pH 3) and tightening (at pH 7) of tertiary structure. Formed soft corona exclusively consists of full length non-native ovalbumin, while in hard corona also shorter ovalbumin fragments were found. At pH 7 soft corona ovalbumin has rearranged but still preserved level of ordered secondary structure, resulting in preserved thermostability and proteolytic stability, but decreased ability to form fibrils upon heating. Secondary structure changes in soft corona resemble changes in native ovalbumin induced by heat treatment (80 °C). Ovalbumin is abundantly present in corona around microplastics also in the presence of other egg white proteins. These results imply that microplastics contaminating food may bind and change structure and functional properties of the main egg white protein.

Persistence of A-234 nerve agent on indoor surfaces.

Understanding the fundamental physical characteristics of extremely toxic compounds and their behavior across different environments plays a crucial role in assessing their danger. Additionally, this knowledge informs the development of protocols for gathering forensic evidence related to harmful chemicals misuse. In 2018, former Russian spy Sergei Skripal and his daughter were poisoned in Salisbury, England, with a substance later identified as the unconventional nerve agent A-234. Contamination with the compound was found on items inside Skripal's home. The aim of this paper was to determine the persistence of A-234 on selected indoor surfaces. Ceramics, aluminum can, laminated chipboard, polyvinyl chloride (PVC) floor tile, polyethylene terephthalate (PET) bottle, acrylic paint and computer keyboard were used as matrices. The decrease in surface contamination and further fate of the compound was monitored for 12 weeks. Persistence determination involved optimizing the wipe sampling method. Simultaneously, evaporation from the surface and permeation of the contaminant into the matrix were closely monitored. The experimental findings indicate that the nerve agent exhibits remarkable persistence, particularly on impermeable surfaces. Notably, the process of A-234 evaporation plays a minor role in determining its fate, with detectable concentrations observed solely above solid, non-porous surfaces such as ceramics and aluminum can. The surface persistence half-life varied significantly, ranging from 12 min to 478 days, depending on the material. The article has implications for emergency response protocols, decontamination strategies, public health and crime scene investigations.

Enhancing PET Degrading Enzymes: A Combinatory Approach.

Plastic waste has become a substantial environmental issue. A potential strategy to mitigate this problem is to use enzymatic hydrolysis of plastics to depolymerize post-consumer waste and allow it to be reused. Over the last few decades, the use of enzymatic PET-degrading enzymes has shown promise as a great solution for creating a circular plastic waste economy. PsPETase from Piscinibacter sakaiensis has been identified as an enzyme with tremendous potential for such applications. But to improve its efficiency, enzyme engineering has been applied aiming at enhancing its thermal stability, enzymatic activity, and ease of production. Here, we combine different strategies such as structure-based rational design, ancestral sequence reconstruction and machine learning to engineer a more highly active Combi-PETase variant with a melting temperature of 70 °C and optimal performance at 60 °C. Furthermore, this study demonstrates that these approaches, commonly used in other works of enzyme engineering, are most effective when utilized in combination, enabling the improvement of enzymes for industrial applications.

[Structure motif guided mining of MHET hydrolase and development of a two-enzyme cascade for plastics depolymerization at mild temperature].

The utilization of polyethylene terephthalate (PET) has caused significant and prolonged ecological repercussions. Enzymatic degradation is an environmentally friendly approach to addressing PET contamination. Hydrolysis of mono(2-hydroxyethyl) terephthalate (MHET), a competitively inhibited intermediate in PET degradation, is catalyzed by MHET degrading enzymes. Herein, we employed bioinformatic methods that combined with sequence and structural information to discover an MHET hydrolase, BurkMHETase. Enzymatic characterization showed that the enzyme was relatively stable at pH 7.5-10.0 and 30-45 ℃. The kinetic parameters kcat and Km on MHET were (24.2±0.5)/s and (1.8±0.2) µmol/L, respectively, which were similar to that of the well-known IsMHETase with higher substrate affinity. BurkMHETase coupled with PET degradation enzymes improved the degradation of PET films. Structural analysis and mutation experiments indicated that BurkMHETase may have evolved specific structural features to hydrolyze MHET. For MHET degrading enzymes, aromatic amino acids at position 495 and the synergistic interactions between active sites or distal amino acids appear to be required for MHET hydrolytic activity. Therefore, BurkMHETase may have substantial potential in a dual-enzyme PET degradation system while the bioinformatic methods can be used to broaden the scope of applicable MHETase enzymes.

Molecular Insights into the Enhanced Activity and/or Thermostability of PET Hydrolase by D186 Mutations.

PETase exhibits a high degradation activity for polyethylene terephthalate (PET) plastic under moderate temperatures. However, the effect of non-active site residues in the second shell of PETase on the catalytic performance remains unclear. Herein, we proposed a crystal structure- and sequence-based strategy to identify the key non-active site residue. D186 in the second shell of PETase was found to be capable of modulating the enzyme activity and stability. The most active PETaseD186N improved both the activity and thermostability with an increase in Tm by 8.89 °C. The PET degradation product concentrations were 1.86 and 3.69 times higher than those obtained with PETaseWT at 30 and 40 °C, respectively. The most stable PETaseD186V showed an increase in Tm of 12.91 °C over PETaseWT. Molecular dynamics (MD) simulations revealed that the D186 mutations could elevate the substrate binding free energy and change substrate binding mode, and/or rigidify the flexible Loop 10, and lock Loop 10 and Helix 6 by hydrogen bonding, leading to the enhanced activity and/or thermostability of PETase variants. This work unraveled the contribution of the key second-shell residue in PETase in influencing the enzyme activity and stability, which would benefit in the rational design of efficient and thermostable PETase.

Microbial degradation of low-density polyethylene, polyethylene terephthalate, and polystyrene by novel isolates from plastic-polluted environment.

Biodegradation is an eco-friendly measure to address plastic pollution. This study screened four bacterial isolates that were capable of degrading recalcitrant polymers, i.e., low-density polyethylene, polyethylene terephthalate, and polystyrene. The unique bacterial isolates were obtained from plastic polluted environment. Dermacoccus sp. MR5 (accession no. OP592184) and Corynebacterium sp. MR10 (accession no. OP536169) from Malaysian mangroves and Bacillus sp. BS5 (accession no. OP536168) and Priestia sp. TL1 (accession no. OP536170) from a sanitary landfill. The four isolates showed a gradual increase in the microbial count and the production of laccase and esterase enzymes after 4 weeks of incubation with the polymers (independent experiment set). Bacillus sp. BS5 produced the highest laccase 15.35 ± 0.19 U/mL and showed the highest weight loss i.e., 4.84 ± 0.6% for PS. Fourier transform infrared spectroscopy analysis confirmed the formation of carbonyl and hydroxyl groups as a result of oxidation reactions by enzymes. Liquid chromatography-mass spectrometry analysis showed the oxidation of the polymers to small molecules (alcohol, ethers, and acids) assimilated by the microbes during the degradation. Field emission scanning electron microscopy showed bacterial colonization, biofilm formation, and surface erosion on the polymer surface. The result provided significant insight into enzyme activities and the potential of isolates to target more than one type of polymer for degradation.

Survival and hormone production of isolated mouse follicles in three-dimensional artificial scaffolds after stimulation with bpV(HOpic).

PURPOSE: To preserve fertility before gonadotoxic therapy, ovarian tissue can be removed, cryopreserved, and transplanted back again after treatment. An alternative is the artificial ovary, in which the ovarian follicles are extracted from the tissue, which reduces the risk of reimplantation of potentially remaining malignant cells. The PTEN inhibitor bpV(HOpic) has been shown to activate human, bovine and alpacas ovarian follicles, and it is therefore considered a promising substance for developing the artificial ovary. The purpose of this study was to examine the impact of different scaffolds and the vanadate derivative bpV(HOpic) on mice follicle survival and hormone secretion over 10 days. METHODS: A comparative analysis was performed, studying the survival rates (SR) of isolated mice follicle in four different groups that differed either in the scaffold (polycaprolactone scaffold versus polyethylene terephthalate membrane) or in the medium-bpV(HOpic) versus control medium. The observation period of the follicles was 10 days. On days 2, 6, and 10, the viability and morphology of the follicles were checked using fluorescence or confocal microscopy. Furthermore, hormone levels of estrogen (pmol/L) and progesterone (nmol/L) were determined. RESULTS: When comparing the SR of follicles among the four groups, it was observed that on day 6, the study groups utilizing the polycaprolactone scaffold with bpV(HOpic) in the medium (SR: 0.48 ± 0.18; p = 0.004) or functionalized in the scaffold (SR: 0.50 ± 0.20; p = 0.003) exhibited significantly higher survival rates compared to the group using only the polyethylene terephthalate membrane (SR: 0). On day 10, a significantly higher survival rate was only noted when comparing the polycaprolactone scaffold with bpV(HOpic) in the medium to the polyethylene terephthalate membrane group (SR: 0.38 ± 0.20 versus 0; p = 0.007). Higher levels of progesterone were only significantly associated with better survival rates in the group with the polycaprolactone scaffold functionalized with bpV(HOpic) (p = 0.017). CONCLUSION: This study demonstrates that three-dimensional polycaprolactone scaffolds improve the survival rates of isolated mice follicles in comparison with a conventional polyethylene terephthalate membrane. The survival rates slightly improve with added bpV(HOpic). Furthermore, higher rates of progesterone were also partly associated with improved survival.

A model-driven approach to upcycling recalcitrant feedstocks in Pseudomonas putida by decoupling PHA production from nutrient limitation.

Bacterial polyhydroxyalkanoates (PHAs) have emerged as promising eco-friendly alternatives to petroleum-based plastics since they are synthesized from renewable resources and offer exceptional properties. However, their production is limited to the stationary growth phase under nutrient-limited conditions, requiring customized strategies and costly two-phase bioprocesses. In this study, we tackle these challenges by employing a model-driven approach to reroute carbon flux and remove regulatory constraints using synthetic biology. We construct a collection of Pseudomonas putida-overproducing strains at the expense of plastics and lignin-related compounds using growth-coupling approaches. PHA production was successfully achieved during growth phase, resulting in the production of up to 46% PHA/cell dry weight while maintaining a balanced carbon-to-nitrogen ratio. Our strains are additionally validated under an upcycling scenario using enzymatically hydrolyzed polyethylene terephthalate as a feedstock. These findings have the potential to revolutionize PHA production and address the global plastic crisis by overcoming the complexities of traditional PHA production bioprocesses.