Intracellular Polymer Research Articles

Partial least squares regression based rapid quantification of intracellular biopolymers from a Sudan black absorption assay

Rapid quantitation of intracellular polyhydroxyalkanoate (PHA) is essential for monitoring and possibly intervening in ongoing bioprocesses. The traditional multistep protocol involving cell separation, lysis, organic solvent extraction, and purification of intracellular polymers is time intensive. In this study, indirect Sudan black (ISB) adsorption data combined with culture optical density and intracellular protein measurements were used to develop partial least squares (PLS) regression models to predict the intracellular PHA concentration in Cupriavidus necator. ISB analysis supported the hypothesized partitioning of Sudan black into residual biomass, PHA, and abiotic compartments. The PLS regression model developed with autoscaled data offered excellent fit on cross-validation (R2CV of 0.987) and independent prediction (R2P of 0.994) sets. The method limit of detection for the PLS model (0.036 g l−1) was reasonably low to track biopolymer accumulation in microbial culture, even in early growth phase. The conservative estimate suggests a significantly shorter processing time with the machine learning-based approach (190 min) than with the traditional protocol (940 min) while retaining analytical interchangeability.

An Update on Polyphosphate In Vivo Activities.

Polyphosphate (polyP) is an evolutionary ancient inorganic molecule widespread in biology, exerting a broad range of biological activities. The intracellular polymer serves as an energy storage pool and phosphate/calcium ion reservoir with implications for basal cellular functions. Metabolisms of the polymer are well understood in procaryotes and unicellular eukaryotic cells. However, functions, regulation, and association with disease states of the polymer in higher eukaryotic species such as mammalians are just beginning to emerge. The review summarises our current understanding of polyP metabolism, the polymer's functions, and methods for polyP analysis. In-depth knowledge of the pathways that control polyP turnover will open future perspectives for selective targeting of the polymer.

Evaluating the effect of antibiotics on aerobic granular sludge treatment of pharmaceutical wastewater.

Aerobic granular sludge (AGS) has been widely applied in pharmaceutical wastewater treatment due to its advantages such as high biomass and excellent settling performance. However, the influence of commonly found antibiotics in pharmaceutical wastewater on the operational efficiency of AGS has been poorly explored. This study investigated the effects of tetracycline (TE) on AGS treating pharmaceutical wastewater at room temperature and analyzed the related mechanisms. The results demonstrate a dose-dependent relationship between TE's effects on AGS. At concentrations below the threshold of 0.1 mg/L, the effects are considered trivial. In contrast, TE with more than 2.0 mg/L reduces the performance of AGS. In the 6.0 mg/L TE group, COD, TN, and TP removal efficiencies decreased to 72.6-75.5, 54.6-58.9, and 71.6-75.8%, respectively. High concentrations of TE reduced sludge concentration and the proportion of organic matter in AGS, leading to a decline in sludge settling performance. Elevated TE concentrations stimulated extracellular polymeric substance secretion, increasing polymeric nitrogen and polymeric phosphorus content. Intracellular polymer analysis revealed that high TE concentrations reduced polyhydroxyalkanoates but enhanced glycogen metabolism. Enzyme activity analysis disclosed that high TE concentrations decreased the activity of key enzymes associated with nutrient removal.

Biodegradation of poly-3-hydroxybutyrate after soil inoculation with microbial consortium: Soil microbiome and plant responses to the changed environment

Biodegradable plastics play a vital role in addressing global plastics disposal challenges. Poly-3-hydroxybutyrate (P3HB) is a biodegradable bacterial intracellular storage polymer with substantial usage potential in agriculture. Poly-3-hydroxybutyrate and its degradation products are non-toxic; however, previous studies suggest that P3HB biodegradation negatively affects plant growth because the microorganisms compete with plants for nutrients. One possible solution to this issue could be inoculating soil with a consortium of plant growth-promoting and N-fixing microorganisms. To test this hypothesis, we conducted a pot experiment using lettuce (Lactuca sativa L. var. capitata L.) grown in soil amended with two doses (1 % and 5 % w/w) of P3HB and microbial inoculant (MI). We tested five experimental variations: P3HB 1 %, P3HB 1 % + MI, P3HB 5 %, P3HB 5 % + MI, and MI, to assess the impact of added microorganisms on plant growth and P3HB biodegradation. The efficient P3HB degradation, which was directly dependent on the amount of bioplastics added, was coupled with the preferential utilization of P3HB as a carbon (C) source. Due to the increased demand for nutrients in P3HB-amended soil by microbial degraders, respiration and enzyme activities were enhanced. This indicated an increased mineralisation of C as well as nitrogen (N), sulphur (S), and phosphorus (P). Microbial inoculation introduced specific bacterial taxa that further improved degradation efficiency and nutrient turnover (N, S, and P) in P3HB-amended soil. Notably, soil acidification related to P3HB was not the primary factor affecting plant growth inhibition. However, despite plant growth-promoting rhizobacteria and N2-fixing microorganisms originating from MI, plant biomass yield remained limited, suggesting that these microorganisms were not entirely successful in mitigating the growth inhibition caused by P3HB.

Light-mediated intracellular polymerization.

The synthesis of synthetic intracellular polymers offers groundbreaking possibilities in cellular biology and medical research, allowing for novel experiments in drug delivery, bioimaging and targeted cancer therapies. These macromolecules, composed of biocompatible monomers, are pivotal in manipulating cellular functions and pathways due to their bioavailability, cytocompatibility and distinct chemical properties. This protocol details two innovative methods for intracellular polymerization. The first one uses 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959) as a photoinitiator for free radical polymerization under UV light (365 nm, 5 mW/cm2). The second method employs photoinduced electron transfer-reversible addition-fragmentation chain-transfer polymerization with visible light (470 nm, 100 mW/cm2). We further elaborate on isolating these intracellular polymers by streptavidin/biotin interaction or immobilized metal ion affinity chromatography for polymers tagged with biotin or histidine. The entire process, from polymerization to isolation, takes ~48 h. Moreover, the intracellular polymers thus generated demonstrate significant potential in enhancing actin polymerization, in bioimaging applications and as a novel avenue in cancer treatment strategies. The protocol extends to animal models, providing a comprehensive approach from cellular to systemic applications. Users are advised to have a basic understanding of organic synthesis and cell biology techniques.

Comparison of batch, fed-batch and continuous operation modes for scalable polyhydroxyalkanoate (PHA) production and carbon sequestration from phenol

Polyhydroxyalkanoates (PHA) are biodegradable intracellular polymers, and a renewable alternative to conventional plastics. To reduce PHA's cost, this study combined an unconventional carbon source, i.e. a toxic aromatics phenol, with an acclimated consortium, to develop a scalable PHA production process. The resultant process performance was systematically compared across three operational modes: batch, fed-batch, and continuous. Phenol toxicity caused inhibition in the batch mode, reducing the PHA synthesis rate but resulting in a high PHA content in cells (PHA% > 50 %). Fed-batch feeding alleviated such inhibition, facilitating a high PHA titer and yield from the substrate. Interestingly, a higher feeding rate in the fed-batch reactor improved its performance, probably by providing a suitable food-to-microbe ratio for the biomass. Using a continuous mode also reduced phenol toxicity, but resulted in more synthesis of non-PHA cellular materials (NPCM). The difference might have been caused by different metabolic states and the presence of intermediates in the reactors. Comparing all 14 conditions under the three modes, feeding phenol more gradually tended to channel substrate away from PHA to NPCM synthesis. The volumetric productivity generally ranged from 3 to 12 mg L−1 min−1, while the biomass productivity was between 2 and 11 mg g initial CDW−1 min−1. A 3-stage, production-harvest and starvation-production process was then designed to simulate real industrialized manufacturing, where the operational modes had a long-term effect on biomass' productivity. Fed-batch mode performed the best, as it fixed 1/3 of phenol's organic carbon in PHA product, suggesting high potential for future application.

Synergistic approach for enhanced production of polyhydroxybutyrate by Bacillus pseudomycoides SAS-B1: Effective utilization of glycerol and acrylic acid through fed-batch fermentation and its environmental impact assessment

The continual rise in the consumption of petroleum-based synthetic polymers raised a significant environmental concern. Bacillus pseudomycoides SAS-B1 is a gram-positive rod-shaped halophilic bacterium capable of accumulating Polyhydroxybutyrate (PHB)-an intracellular biodegradable polymer. In the present study, the optimal conditions for cell cultivation in the seed media were developed. The optimal factors included a preservation age of 14 to 21 days (with 105 to 106 cells/mL), inoculum size of 0.1 % (w/v), 1 % (w/v) glucose, and growth temperature of 30 °C. The cells were then cultivated in a two-stage fermentation process utilizing glycerol and Corn Steep Liquor (CSL) as carbon and nitrogen sources, respectively. PHB yield was effectively increased from 2.01 to 9.21 g/L through intermittent feeding of glycerol and CSL, along with acrylic acid. FTIR, TGA, DSC, and XRD characterization studies were employed to enumerate the recovered PHB and determine its physicochemical properties. Additionally, the study assessed the cradle-to-gate Life Cycle Assessment (LCA) of PHB production, considering net CO2 generation and covering all major environmental impact categories. The production of 1000 kg of PHB resulted in lower stratospheric ozone depletion and comparatively reduced carbon dioxide emissions (2022.7 kg CO2 eq.) and terrestrial ecotoxicity (9.54 kg 1,4-DCB eq.) than typical petrochemical polymers.

Actin Paracrystals Display Double Schottky Diode‐Like Electrical Behavior

AbstractActin filaments are abundant intracellular polymers implicated in cell motility and contraction. Actin also forms paracrystalline structures under various conditions, including exposure to lanthanides and other high ionic strength conditions. Here the electrical properties of purified actin paracrystals induced by either MgCl2 or GdCl3 are tested. Tight electrically seal‐voltage‐clamped crystals display strong rectification with a nonlinear conductance under symmetrical and biionic conditions, including 20 mm GdCl3/140 mm KCl and 10 mm MgCl2/140 mm KCl. Rectification is observed in symmetrical ionic conditions with positive‐to‐negative conductance ratios ranging from 4.18 to 9.00 nS. The current‐to‐voltage relationships can be fitted with theoretical double Schottky equations and resemble the behavior of semiconducting nanotubes. The highly nonlinear electrical properties of paracrystalline actin arrays may help explain the physiology of actin‐rich cellular compartments, such as stereocilia of the cochlea and neuronal dendritic spines also suggest potentially relevant nano‐technological properties of this protein.

Metagenomics, metatranscriptomics, and proteomics reveal the metabolic mechanism of biofilm sequencing batch reactor with higher phosphate enrichment capacity under low phosphorus load

The biofilm sequencing batch reactor (BSBR) process has higher phosphate recovery efficiency and enrichment multiple when the phosphorus load is lower, but the mechanism of phosphate enrichment at low phosphorus load remains unclear. In this study, we operated two BSBR operating under low and high phosphorus load (0.012 and 0.032 kg/(m3·d)) respectively, and used metagenomic, metatranscriptomic, and proteomics methods to analyze the community structure of the phosphorus accumulating organisms (PAOs) in the biofilm, the transcription and protein expression of key functional genes and enzymes, and the metabolism of intracellular polymers. Compared with at high phosphorus load, the BSBR at low phosphorus load have different PAOs and fewer types of PAOs, but in both cases the PAOs must have the PHA, PPX, Pst, and acs genes to become dominant. Some key differences in the metabolism of PAOs from the BSBR with different phosphorus load can be identified as follows. When the phosphorus load is low, the adenosine triphosphoric acid (ATP) and NAD(P)H in the anaerobic stage come from the TCA cycle and the second half of the EMP pathway. The key genes that are upregulated include GAPDH, PGK, ENO, ppdk in the EMP pathway, actP in acetate metabolism, phnB in polyhydroxybutyrate (PHB) synthesis, and aceA, mdh, sdhA, and IDH1 in the TCA cycle. In the meantime, the ccr gene in the PHV pathway is inhibited. As a result, the metabolism of the PAOs features low glycogen with high PHB, Pupt, Prel, and low PHV. That is, more ATP and NAD(P)H flow to phosphorus enrichment metabolism, thus allowing the highly efficient enrichment of phosphorus from low concentration phosphate thanks to the higher abundance of PAOs. The current results provide theoretical support and a new technical option for the enrichment and recovery of low concentrations of phosphate from wastewater by the BSBR process.

Polyhydroxyalkanoates: Biosynthesis Optimization and Design of Antimicrobial Composites

Background. The accumulation of plastic waste negatively affects the environment and human health. Currently, one of the strategies to address this global ecological problem involves the utilization of biodegradable plastics instead in place of synthetic ones. Among them, polyhydroxyalkanoates (PHA) – microbial intracellular polymers – hold a significant position. Their advantages are biodegradability, biocompatibility, and favorable thermomechanical properties. Given these attributes, PHA has significant prospects for use in medicine, agriculture, and the food industry, in particular for packaging food products. Objective. Enchance the efficiency of bacterial synthesis of polyhydroxyalkanoates through nutrient media modification, obtain antimicrobial composites based on PHA, and determine their antimicrobial properties. Methods. The optimization of PHA biosynthesis involved selecting appropriate cultivation conditions, including carbon and nitrogen sources, cultivation time, and working volume. The isolation of PHA from bacterial biomass was achieved through chloroform extraction (mixing for 10 h at 35 °C, with a biomass-to-chloroform ratio of 1:50); followed by precipitation with double the volume of isopropanol. The resulting polymer was then dried to a constant weight at 60 °C. The hydrophobicity of the biopolymer was assessed using the water contact angle measurement. Composites of biopolymers with antimicrobials in the form of films were obtained using two methods: 1) solution casting method; and 2) layering the biocides onto the polymer film. The antimicrobial activity of the resulting composites was determined using the agar diffusion method. Results. Through the optimization of the mineral media and the change of cultivation conditions, it was possible to obtain 0.26–1.45 g/l of polyhydroxyalkanoates (5.1–34.0% PHA from biomass). The R. ruber UCM Ac-288 strain synthesized the maximum amount of biopolymer (34.0% PHA). This study established the ability of Gordonia bacteria to synthesize PHA for the first time. PHA compositions of optimal content were obtained, with hydrophobicity comparable to that of polyethylene packaging films. Antimicrobial pro­perties of biopolymers composites with biocides have been substantiated. Conclusions. The bacterial synthesis of PHA was increased by modifying nutrient media. Composites based on PHA with biocides were developed. It was determined that these composites exhibit antimicrobial properties and high hydrophobicity. Consequently, they hold promise for use as biofilms for packaging and preser­ving food products.

Biotransformation of Lignocellulosic Biomass Hydrolysate into Polyhydroxybutyrate Biopolymer via Ralstonia Eutropha

Currently, a rampant cultural shift is occurring in the modern world to progressively substitute fossil-derived plastics and shift to novel biomaterials that are benign to the environment owing to increased awareness of environmental sustainability along with the implementation of strict regulations worldwide. Polyhydroxyalkanoates (PHAs) are promising intracellular biodegradable polymers that have attracted considerable focus owing to their biocompatibility, biodegradability, non-toxicity and environment-friendly nature to function in diverse applications notably in the pharmaceutical, medical, textile, materials, fuel, agricultural industries. Nonetheless, despite its huge market potential, the commercial growth of PHA is achieved on a small extent only, since the cost-effectiveness of this product is highly debatable owing to the high production cost of processing the carbon substrate. The goal behind this research study is to explore the possibility of exploiting low-cost carbon substrates from low-value lignocellulosic materials that would have otherwise been discarded as waste and add stress to the landfill to manufacture biopolymer compounds that are used in everyday lives as well as to enhance the functionality and yields of glucose from PHA substrates that can undergo industrial upscaling. One of the major challenges of transforming lignocellulosic biomass into fermentable sugars is the recalcitrant nature of the fibre which renders it very resistant to the release of sugars for fermentation. Since lignocellulosic biomass has a specific attribute such as an extremely coordinated matrix which renders it very resistant to the release of sugars for fermentation owing to biological degradation, a pre-treatment phase is necessary prior to the hydrolysis stage for the transformation of the fermentable sugars. This study focuses on the biosynthesis of biopolymers from lignocellulosic biomass through sustainable approaches such as enzyme and microbial activities in order to examine its viability as a replacement for traditional polymers. Cupriavidus Necator H16 (Ralstonia Eutropha) having 8×108 CFU/ml viable colonies were cultured at 30 oC and was inoculated in submerged fermentation of M9 minimal salt medium using 1% reducing sugar from Furcraea Foetida as carbon source. Batch fermentation of PHB in submerged cultivation conducted for a residence time from 0 to 48h resulted in a dry cell weight from 0.32±0.05% to 1.62±0.05%. The nitrogen limiting phase was achieved after 48h and 17.05±0.35% of PHB was extracted from 3ml of the fermentation broth. The PHB yield was dramatically lower than reported optimal yields of 37.55 to 97.80% from works of literature. Nonetheless, Fourier-transform infrared spectroscopy (FTIR) spectroscopy revealed characteristics bands for carbonyl, methine and ester groups along with intermolecular hydrogen bonds in the biopolymer. Sudan Black B and FTIR spectrum demonstrate that PHB biosynthesis successfully bioaccumulates inside the cells of Ralstonia Eutropha using cellulose from Lignocellulosic biomass (LCB) as carbon source. Hence, the process needs to be optimized in terms of variables such as inoculum size, inoculum concentration, incubation time and salt medium conditions in order to maximise the production of PHB from Furcraea Foetida in Ralstonia Eutropha cultivation.

Modeling versatile and dynamic anaerobic metabolism for PAOs/GAOs competition using agent-based model and verification via single cell Raman Micro-spectroscopy

Side-stream enhanced biological phosphorus removal process (S2EBPR) has been demonstrated to improve performance stability and offers a suite of advantages compared to conventional EBPR design. Design and optimization of S2EBPR require modification of the current EBPR models that were not able to fully reflect the metabolic functions of and competition between the polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs) under extended anaerobic conditions as in the S2EBPR conditions. In this study, we proposed and validated an improved model (iEBPR) for simulating PAO and GAO competition that incorporated heterogeneity and versatility in PAO sequential polymer usage, staged maintenance-decay, and glycolysis-TCA pathway shifts. The iEBPR model was first calibrated against bulk batch testing experiment data and proved to perform better than the previous EBPR model for predicting the soluble orthoP, ammonia, biomass glycogen, and PHA temporal profiles in a starvation batch testing under prolonged anaerobic conditions. We further validated the model with another independent set of anaerobic testing data that included high-resolution single-cell and specific population level intracellular polymer measurements acquired with single-cell Raman micro-spectroscopy technique. The model accurately predicted the temporal changes in the intracellular polymers at cellular and population levels within PAOs and GAOs, and further confirmed the proposed mechanism of sequential polymer utilization, and polymer availability-dependent and staged maintenance-decay in PAOs. These results indicate that under extended anaerobic phases as in S2EBPR, the PAOs may gain competitive advantages over GAOs due to the possession of multiple intracellular polymers and the adaptive switching of the anaerobic metabolic pathways that consequently lead to the later and slower decay in PAOs than GAOs. The iEBPR model can be applied to facilitate and optimize the design and operations of S2EBPR for more reliable nutrient removal and recovery from wastewater.

Impact of Naproxen on Wastewater Biological Treatment: Focus on Reactor Performance and Mechanisms

Pharmaceutical contamination has emerged as a significant environmental concern; yet, the impact and underlying mechanisms of widely detected naproxen (NPX) on wastewater biological treatment remain poorly understood. To address this knowledge gap, this study investigates the influence of NPX on biological nutrient removal in wastewater treatment under mesophilic conditions, and elucidates the associated mechanisms. The results demonstrate that NPX concentrations below 0.05 mg/L have no significant effect on the removal of pollutants and nutrients in wastewater, whereas the concentrations exceeding 0.5 mg/L hinder the removal of both chemical oxygen demand (COD) and nutrients, with a more pronounced inhibitory effect observed at higher NPX doses. When the concentration of NPX was 2.0 mg/L, the removal efficiency of COD, NH4+-N and phosphate decreased to 81.2~83.6%, 82.5~84.2% and 80.2~82.6%, respectively, which was much lower than that of the blank. Furthermore, NPX leads to a decrease in activated sludge concentration and organic matter content within the sludge. Additionally, NPX promotes the secretion of extracellular polymeric substances (EPS). Periodic investigations reveal that NPX inhibits the biosynthesis of intracellular polymer polyhydroxyalkanoate (PHA), thereby reducing energy production during later stages of degradation. Enzyme activity analysis indicates that high NPX concentrations suppress the activity of key enzymes associated with biological nitrogen and phosphorus removal. These findings provide theoretical insights for the treatment of NPX-containing wastewater using activated sludge processes.

Synthesis and Properties of Polyhydroxyalkanoates on Waste Fish Oil from the Production of Canned Sprats

The waste fish oil obtained from Baltic sprat waste in the production of canned sprats was studied as a sole carbon substrate for PHA synthesis by the wild-type strain Cupriavidus necator B-10646. Sprat oil contained a set of fatty acids with a chain length from C14 to C24, saturation factor 0.63, and provided bacterial growth and PHA synthesis. Bacteria metabolized fatty acids unevenly utilizing polyenoic acids and not using monoenoic and saturated acids. The bacterial biomass yield and the intracellular polymer concentration were 6.5 ± 0.5 g/L and 65 ± 5% by fed-batch culture in flasks. The synthesized PHAs were three-component copolymers with a predominance (97–98 mol.%) of 3-hydroxybutyrate monomers and small inclusions of 3-hydroxyvalerate and 3-hydroxyhexanoate; the ratio of monomers changed slightly depending on the sprat oil concentration. The series of samples had a temperature (Tmelt) of 158–165 °C, a molecular weight (Mw) of 540–760 kDa, and a degree of crystallinity (Cx) of 66–72%. For the first time, the waste fish oil from the production of sprats studied as a carbon substrate is a promising, affordable, and renewable substrate for PHA biosynthesis.

A robust reprogramming strategy for generating hepatocyte-like cells usable in pharmaco-toxicological studies

BackgroundHigh-throughput pharmaco-toxicological testing frequently relies on the use of established liver-derived cell lines, such as HepG2 cells. However, these cells often display limited hepatic phenotype and features of neoplastic transformation that may bias the interpretation of the results. Alternate models based on primary cultures or differentiated pluripotent stem cells are costly to handle and difficult to implement in high-throughput screening platforms. Thus, cells without malignant traits, optimal differentiation pattern, producible in large and homogeneous amounts and with patient-specific phenotypes would be desirable.MethodsWe have designed and implemented a novel and robust approach to obtain hepatocytes from individuals by direct reprogramming, which is based on a combination of a single doxycycline-inducible polycistronic vector system expressing HNF4A, HNF1A and FOXA3, introduced in human fibroblasts previously transduced with human telomerase reverse transcriptase (hTERT). These cells can be maintained in fibroblast culture media, under standard cell culture conditions.ResultsClonal hTERT-transduced human fibroblast cell lines can be expanded at least to 110 population doublings without signs of transformation or senescence. They can be easily differentiated at any cell passage number to hepatocyte-like cells with the simple addition of doxycycline to culture media. Acquisition of a hepatocyte phenotype is achieved in just 10 days and requires a simple and non-expensive cell culture media and standard 2D culture conditions. Hepatocytes reprogrammed from low and high passage hTERT-transduced fibroblasts display very similar transcriptomic profiles, biotransformation activities and show analogous pattern behavior in toxicometabolomic studies. Results indicate that this cell model outperforms HepG2 in toxicological screening. The procedure also allows generation of hepatocyte-like cells from patients with given pathological phenotypes. In fact, we succeeded in generating hepatocyte-like cells from a patient with alpha-1 antitrypsin deficiency, which recapitulated accumulation of intracellular alpha-1 antitrypsin polymers and deregulation of unfolded protein response and inflammatory networks.ConclusionOur strategy allows the generation of an unlimited source of clonal, homogeneous, non-transformed induced hepatocyte-like cells, capable of performing typical hepatic functions and suitable for pharmaco-toxicological high-throughput testing. Moreover, as far as hepatocyte-like cells derived from fibroblasts isolated from patients suffering hepatic dysfunctions, retain the disease traits, as demonstrated for alpha-1-antitrypsin deficiency, this strategy can be applied to the study of other cases of anomalous hepatocyte functionality.

Sensitive and Accurate Differentiation of Extracellular Polymeric Substance Hydrolysis Using Flow Cytometry during Biofilm Extracellular Polymeric Substance Extraction

Extracellular polymeric substances (EPS) affect the formation, properties, and function of biofilms. Understanding the roles of EPS in biofilm-related processes depends on reliable extraction and evaluation methods. However, differentiating true EPS from intracellular organic polymers released by cell lysis has proven to be difficult during EPS extraction. In this study, thermal and cation exchange resin (CER) treatments were used to extract biofilm EPS. SYTOX Green (SG) dye was used to stain nucleic acid (NA), and the enhanced fluorescence signal (FS) from the NA-SG complex was sensitively recorded by flow cytometry (FC). Results showed that thermal extraction could achieve effective EPS release, but a higher temperature and longer time caused a significant cell activity decline, protein denaturation, and cell lysis. In contrast, CER extraction had no effects on cell activity and organics denaturation, and it only caused minor cell lysis at a high CER dose. Combining all the results indicates that CER extraction using 70 g-CER/g-VSS for 2 h provided reliable and efficient extraction of biofilm EPS: high EPS extraction efficiency with minimal EPS denaturation, cell lysis, and loss of cell activity. FC combined with SG staining should be applicable for evaluating the effectiveness of EPS extraction from any microbial aggregates.

Engineering Cyborg Bacteria Through Intracellular Hydrogelation.

Natural and artificial cells are two common chassis in synthetic biology. Natural cells can perform complex tasks through synthetic genetic constructs, but their autonomous replication often causes safety concerns for biomedical applications. In contrast, artificial cells based on nonreplicating materials, albeit possessing reduced biochemical complexity, provide more defined and controllable functions. Here, for the first time, the authors create hybrid material-cell entities termed Cyborg Cells. To create Cyborg Cells, a synthetic polymer network is assembled inside each bacterium, rendering them incapable of dividing. Cyborg Cells preserve essential functions, including cellular metabolism, motility, protein synthesis, and compatibility with genetic circuits. Cyborg Cells also acquire new abilities to resist stressors that otherwise kill natural cells. Finally, the authorsdemonstrate thetherapeutic potential by showing invasion into cancer cells. This work establishes a new paradigm in cellular bioengineering by exploiting a combination of intracellular man-made polymers and their interaction with the protein networks of living cells.

Cerebral Metabolic Stress Improves after Voxelotor Treatment in Pediatric Sickle Cell Disease

Introduction In sickle cell disease (SCD), hemoglobin S rapidly forms intracellular polymers in the deoxygenated state, resulting in red blood cell sickling, hemolysis, and vaso-occlusion. These changes alter cerebral perfusion and dramatically increase risk for neurocognitive deficits and stroke (Bush et al., 2014; Wang et al., 2021). Voxelotor (Oxbryta®) is an inhibitor of HbS polymerization that has been shown to significantly improve anemia and reduce hemolysis in sickle cell disease. However, it is unknown whether these changes are accompanied by improvements in perfusion and reductions in cerebral metabolic stress. Here we employ two novel optical techniques known as frequency-domain near-infrared spectroscopy (FDNIRS) and diffuse correlation spectroscopy (DCS) to non-invasively monitor regional changes in oxygen extraction fraction (OEF), cerebral blood flow (CBF), and cerebral metabolic rate of oxygen (CMRO2) as markers of cerebral metabolic stress in a pilot cohort of children with SCD beginning voxelotor. Our primary objective was to test the hypothesis that voxelotor decreases CBF, OEF, and CMRO2 from pre-treatment levels. We also explored the relationship between CBF, OEF, and CMRO2 and hemoglobin. Methods Children ages 4 to 17 years old with SCD (HbSS or HbS-thalassemia) were enrolled at Children's Healthcare of Atlanta. Participants who met study criteria started open label voxelotor at recommended dose. Participants were monitored prior to treatment initiation, as well as at weeks 4, 8, and 12 weeks. At each study visit, laboratory measurements of anemia (CBC, Retic) and FDNIRS/DCS measurements of regional cerebral blood flow, oxygen extraction fraction, and oxygen metabolism were made (Lee et al., 2022). Transcranial Doppler ultrasound (TCD, middle cerebral artery, internal carotid artery, and carotid bifurcations) along with markers of hemolysis (LDH, T.Bili) were performed at weeks 0 and 12. Paired Wilcoxon signed-rank tests were used to test whether all measures were significantly different from pre-treatment levels. Linear mixed-effects regression models with random-intercept at the patient level were used to examine the association of the relative change in each outcome measure (OEF, CBF, and CMRO2) from pre-treatment levels and delta changes in laboratory data. These models were two-level and accounted for repeated measures nested within each patient. Results Ten of the 12 recruited patients met study criteria. All patients were HbSS on stable dose of hydroxyurea (Table 1). Following initiation of voxelotor, the median (interquartile range, IQR) blood hemoglobin levels increased by 1.2 (0.4, 1.5) g/dL at week 4 (p=0.023); these increases persisted at weeks 8 and 12. Bilirubin was significantly decreased at week 12 (p=0.008), and LDH increased, although the trend was not statistically significant (p=0.24). While TCD velocities were unchanged at week 12, significant decreases in OEF, CBF, and CMRO2 were observed by week 4 (all p < 0.05) that persisted at weeks 8 and 12 (Table 2). Across all time points, relative changes in OEF, CBF, and CMRO2 from pre-treatment levels were inversely associated with changes in hemoglobin (p = 0.0015, 0.071, and 0.020, respectively). Discussion Non-invasive FDNIRS/DCS was used to successfully measure changes in regional cerebral blood flow and oxygen utilization in response to treatment with voxelotor. Results suggest that voxelotor treatment in children with SCD reduces cerebral metabolic stress by improving oxygen delivery to the tissue. Work is ongoing to validate these measurements with MRI. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Zona Pellucida like Domain Protein 1 (ZPLD1) Polymerization Is Regulated by Two Distinguished Hydrophobic Motifs.

Zona Pellucida Like Domain 1 Protein (ZPLD1) is a main component of the cupula, a gelatinous structure located in the labyrinth organ of the inner ear and involved in vestibular function. The N-glycosylated protein is likely able to organize high-molecular-weight polymers via its zona pellucida (ZP) module, which is common for many extracellular proteins that self-assemble into matrices. In this work, we confirmed that ZPLD1 can form multimers while setting up a cellular model leveraging Madin-Darby canine kidney (MDCK) cells to study protein polymerization. We identified two motifs within ZPLD1 which regulate its polymerization and follow previously published conserved regions, identified across ZP proteins. Mutational depletion of either one of these modules led to diminished or abnormal polymer formation outside of the cells, likely due to altered processing at the plasma membrane. Further, intracellular polymer formation was observed. Proteolytic cleavage during secretion, separating the regulatory motif located distinct of the ZP module from the mature monomer, seems to be necessary to enable polymerization. While the molecular interactions of the identified motifs remain to be proven, our findings suggest that ZPLD1 is a polymer forming ZP protein following an orchestrated mechanism of protein polymerization to finally build up a gelatinous hydrogel.

Effect of biodegradable poly-3-hydroxybutyrate amendment on the soil biochemical properties and fertility under varying sand loads

BackgroundPoly-3-hydroxybutyrate (P3HB) is a bacterial intracellular carbon and energy storage polymer, used as a thermoplastic polyester in a wide array of industrial and agricultural applications. However, how the soil microbiome and fertility are altered by exogenously applied P3HB has been relatively unexplored. This study aimed to assess the effects of P3HB addition to nutrient restricted soil: its biological properties and lettuce (Lactuca sativa L. var. capitata L.) biomass production. The experiment was designed to evaluate impacts of spatial arrangement of the relatively organic-rich (soil organic matter, P3HB particles) versus poor fractions of the matrix with confounding factors such as variable microbial biomass, inherent nutrient/energy status, different water relations (due to variable hydrophysical properties of soil augmented by sand at different ratios).ResultsThe results revealed that P3HB in soils induced inconsistent to contradictory changes in the microbial abundance as well as in most enzymatic activities. The differences were conditioned by the sand content both under P3HB presence or absence. On the other hand, dehydrogenase, urease activities, basal and substrate-induced soil respirations were mostly enhanced by P3HB addition, directly with increasing sand content (several respiration types). Nevertheless, P3HB significantly inhibited lettuce biomass production.ConclusionsP3HB introduction to soil boosts the microbial activity owing to the preferential utilization of P3HB as C source, which depletes soil N and strongly inhibits the plant growth. Enhanced microbial activity in P3HB-amended soils with high sand content (60–80%) suggested that in nutrient-impoverished soil P3HB can temporarily replace SOM as a C source for microbial communities due to the shift of their structure to preferentially P3HB-degrading microbiome.Graphical