Improving in vitro screening compounds anti-Trypanosoma cruzi by GFP-expressing parasites.

BACKGROUND: Conventional microscopic counting is a widely utilised method for evaluating the trypanocidal effects of drugs on intracellular amastigotes. This is a low-cost approach, but it is time-consuming and reliant on the expertise of the microscopist. So, there is a pressing need for developing technologies to enhance the efficiency of low-cost anti-Trypanosoma cruzi drug screening. OBJECTIVES: In our laboratory, we aimed to expedite the screening of anti-T. cruzi drugs by implementing a fluorescent method that correlates emitted fluorescence from green fluorescent protein (GFP)-expressing T. cruzi (Tc-GFP) with cellular viability. METHODS: Epimastigotes (Y strain) were transfected with the pROCKGFPNeo plasmid, resulting in robust and sustained GFP expression across epimastigotes, trypomastigotes, and intracellular amastigotes. Tc-GFP epimastigotes and intracellular amastigotes were exposed to a serial dilution of benznidazole (Bz). Cell viability was assessed through a combination of microscopic counting, MTT, and fluorimetry. FINDINGS: The fluorescence data indicated an underestimation of the activity of Bz against epimastigotes (IC50 75 µM x 14 µM). Conversely, for intracellular GFP-amastigotes, both fluorimetry and microscopy yielded identical IC50 values. Factors influencing the fluorimetry approach are discussed. MAIN CONCLUSIONS: Our proposed fluorometric assessment is effective and can serve as a viable substitute for the time-consuming microscopic counting of intracellular amastigotes.

A vector system for single and tandem expression of cloned genes and multi-colour fluorescent tagging in Haloferax volcanii.

Archaeal cell biology is an emerging field expected to identify fundamental cellular processes, help resolve the deep evolutionary history of cellular life, and contribute new components and functions in biotechnology and synthetic biology. To facilitate these, we have developed plasmid vectors that allow convenient cloning and production of proteins and fusion proteins with flexible, rigid, or semi-rigid linkers in the model archaeon Haloferax volcanii. For protein subcellular localization studies using fluorescent protein (FP) tags, we created vectors incorporating a range of codon-optimized fluorescent proteins for N- or C-terminal tagging, including GFP, mNeonGreen, mCherry, YPet, mTurquoise2 and mScarlet-I. Obtaining functional fusion proteins can be challenging with proteins involved in multiple interactions, mainly due to steric interference. We demonstrated the use of the new vector system to screen for improved function in cytoskeletal protein FP fusions, and identified FtsZ1-FPs that are functional in cell division and CetZ1-FPs that are functional in motility and rod cell development. Both the type of linker and the type of FP influenced the functionality of the resulting fusions. The vector design also facilitates convenient cloning and tandem expression of two genes or fusion genes, controlled by a modified tryptophan-inducible promoter, and we demonstrated its use for dual-colour imaging of tagged proteins in H. volcanii cells. These tools should promote further development and applications of archaeal molecular and cellular biology and biotechnology.

[Establishment of Dual Fluorescent Labeled Human High Bone Metastasis 
Lung Adenocarcinoma Cell Line and Transcriptomic Characterization Analysis].

BACKGROUND: Bone is a common site for metastasis in lung adenocarcinoma, but the mechanism behind lung adenocarcinoma bone metastasis is still unclear. And currently, there is a lack of easily traceable and stable lung adenocarcinoma bone metastasis cell models, which limits the research on the mechanism of lung adenocarcinoma bone metastasis. The establishment of human lung adenocarcinoma cell line that are highly metastatic to bone, labeled with green fluorescent proteins (GFP) and fireflies luciferase (LUC), along with transcriptomic characterization, would be beneficial for research on lung adenocarcinoma bone metastasis and provide new experimental methods. METHODS: The human lung adenocarcinoma cell line A549-GFP-LUC was injected into nude mice via the left ventricle to construct a bone metastasis model, and was domesticated in vivo for three consecutive times to obtain the human high bone metastasis lung adenocarcinoma cell line A549-GFP-LUC-BM3; cell counting kit-8 (CCK-8), colony formation assay, scratch wound assays, Transwell assay and Western blot were used to compare the proliferation and invasion abilities of A549-GFP-LUC-BM3 with the parental cells. A549-GFP-LUC-BM3 cells and parental cells were further analyzed by transcriptomic sequencing. RESULTS: Human high-bone metastatic lung adenocarcinoma cells A549-GFP-LUC-BM3 was successfully established. Compared to parental cells, this cells exhibited a significantly higher incidence of bone metastasis and enhanced in vitro proliferation, migration, and invasion abilities. Transcriptomic sequencing results revealed that the A549-GFP-LUC-BM3 cell line had 2954 differentially expressed genes compared to the parental cells, with 1021 genes up-regulated and 1933 genes down-regulated. Gene Ontology (GO) functional enrichment analysis indicated that the differentially expressed genes were primarily localized in cellular components such as the cell periphery. The molecular functions identified as significantly enriched included signaling receptor activity, calcium ion binding, and extracellular matrix structural constituent. Additionally, the biological processes found to be enriched were cell adhesion and biological adhesion. The enrichment analysis conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that the differentially expressed genes were primarily involved in the metabolism of xenobiotics by cytochrome P450, retinol metabolism, drug metabolism-cytochrome P450, cell adhesion molecules, steroid hormone biosynthesis, and the nuclear factor kappa B (NF-κB) signaling pathway. CONCLUSIONS: The highly bone-metastatic human lung adenocarcinoma cell line with GFP and luciferase double labeling was successfully established. The biological behavior and transcriptome sequencing of the cell line suggest that it has a high bone-metastatic potential.

Ser/Leu-swapped cell-free translation system constructed with natural/in vitro transcribed-hybrid tRNA set.

The Ser/Leu-swapped genetic code can act as a genetic firewall, mitigating biohazard risks arising from horizontal gene transfer in genetically modified organisms. Our prior work demonstrated the orthogonality of this swapped code to the standard genetic code using a cell-free translation system comprised of 21 in vitro transcribed tRNAs. In this study, to advance this system for protein engineering, we introduce a natural/in vitro transcribed-hybrid tRNA set. This set combines natural tRNAs from Escherichia coli (excluding Ser, Leu, and Tyr) and in vitro transcribed tRNAs, encompassing anticodon-swapped tRNASerGAG and tRNALeuGGA. This approach reduces the number of in vitro transcribed tRNAs required from 21 to only 4. In this optimized system, the production of a model protein, superfolder green fluorescent protein, increases to 3.5-fold. With this hybrid tRNA set, the Ser/Leu-swapped cell-free translation system will stand as a potent tool for protein production with reduced biohazard concerns in future biological endeavors.

Cytotoxicity of Quantum Dots in Receptor-Mediated Endocytic and Pinocytic Pathways in Yeast.

Despite the promising applications of the use of quantum dots (QDs) in the biomedical field, the long-lasting effects of QDs on the cell remain poorly understood. To comprehend the mechanisms underlying the toxic effects of QDs in yeast, we characterized defects associated with receptor-mediated endocytosis (RME) as well as pinocytosis using Saccharomyces cerevisiae as a model in the presence of cadmium selenide/zinc sulfide (CdSe/ZnS) QDs. Our findings revealed that QDs led to an inefficient RME at the early, intermediate, and late stages of endocytic patch maturation at the endocytic site, with the prolonged lifespan of GFP fused yeast fimbrin (Sac6-GFP), a late marker of endocytosis. The transit of FM1-43, a lipophilic dye from the plasma membrane to the vacuole, was severely retarded in the presence of QDs. Finally, QDs caused an accumulation of monomeric red fluorescent protein fused carbamoyl phosphate synthetase 1 (mRFP-Cps1), a vacuolar lumen marker in the vacuole. In summary, the present study provides novel insights into the possible impact of CdSe/ZnS QDs on the endocytic machinery, enabling a deeper comprehension of QD toxicity.

Preparation of Chitosan Nanoparticles through a Readily Solvent-Exchange Process for Efficient and Enhanced Gene Delivery.

In view of the excellent prospects of gene therapy and the potential safety and immunogenicity issues challenged by viral vectors, it is of great significance to develop a nonviral vector with low toxicity and low cost. In this work, we report a chitosan nanoparticle (CSNP) to be used as a gene vector prepared through a facile solvent-exchange strategy. Chitosan is first dissolved in ionic liquid 1-ethyl-3-methylimidazolium acetate (EMIM Ac), and then, the solvent is exchanged with water/phosphate-buffered saline (PBS) to remove ionic liquid, forming a final CSNP dispersion after ultrasonication. The prepared CSNP shows a positive surface charge and can condense green fluorescent protein-encoding plasmid (pGFP) at weight ratios (CSNP/pGFP) of 5/1 or higher. Dynamic light scattering size and ζ-potential characterization and gel retardation results confirm the formation of CSNP/pGFP complexes. Compared with plain pGFP, efficient cellular internalization and significantly enhanced green fluorescent protein (GFP) expression are observed by using CSNP as a plasmid vector. Benefitting from the intrinsic biocompatibility, low cost, low immunogenicity, and abundant sources of chitosan, as well as the facile preparation and the efficient gene transfection capacity of CSNP, it is believed that this CSNP could be used as a nonviral gene vector with great clinical translational potentials.

Using Dimensionality Reduction to Visualize Phenotypic Changes in High-Throughput Microscopy.

High-throughput microscopy has enabled screening of cell phenotypes at unprecedented scale. Systematic identification of cell phenotype changes (such as cell morphology and protein localization changes) is a major analysis goal. Because cell phenotypes are high-dimensional, unbiased approaches to detect and visualize the changes in phenotypes are still needed. Here, we suggest that changes in cellular phenotype can be visualized in reduced dimensionality representations of the image feature space. We describe a freely available analysis pipeline to visualize changes in protein localization in feature spaces obtained from deep learning. As an example, we use the pipeline to identify changes in subcellular localization after the yeast GFP collection was treated with hydroxyurea.

iATPSnFR2: A high-dynamic-range fluorescent sensor for monitoring intracellular ATP.

We developed a significantly improved genetically encoded quantitative adenosine triphosphate (ATP) sensor to provide real-time dynamics of ATP levels in subcellular compartments. iATPSnFR2 is a variant of iATPSnFR1, a previously developed sensor that has circularly permuted superfolder green fluorescent protein (GFP) inserted between the ATP-binding helices of the ε-subunit of a bacterial F0-F1 ATPase. Optimizing the linkers joining the two domains resulted in a ~fivefold to sixfold improvement in the dynamic range compared to the previous-generation sensor, with excellent discrimination against other analytes, and affinity variants varying from 4 µM to 500 µM. A chimeric version of this sensor fused to either the HaloTag protein or a suitable spectrally separated fluorescent protein provides an optional ratiometric readout allowing comparisons of ATP across cellular regions. Subcellular targeting the sensor to nerve terminals reveals previously uncharacterized single-synapse metabolic signatures, while targeting to the mitochondrial matrix allowed direct quantitative probing of oxidative phosphorylation dynamics.

Screening signal peptidase based on split-GFP assembly technology to promote the secretion of alkaline protease AprE in Bacillus amyloliquefaciens.

Improving the ability of bacteria to secrete protein is essential for large-scale production of food enzymes. However, due to the lack of effective tracking technology for target proteins, the optimization of the secretory system is facing many problems. In this study, we utilized the split-GFP system to achieve self-assembly into mature GFP in Bacillus amyloliquefaciens and successfully tracked the alkaline protease AprE. The split-GFP system was employed to assess the signal peptidases, a crucial component in the secretory system, and signal peptidase sipA was identified as playing a role in the secretion of AprE. Deletion of sipA resulted in a higher accumulation of the precursor protein of AprE compared to other signal peptidase deletion strains. To explore the mechanism of signal peptidase on signal peptide, molecular docking and calculation of free energy were performed. The action strength of the signal peptidase is determined by its binding affinity with the tripeptides at the C-terminal of the signal peptide. The functions of signal peptides YdbK and NucB rely on sipA, and overexpression of sipA by integrating it into genome of B. amyloliquefaciens increased the activity of extracellular AprE by 19.9 %. These findings provide insights into enhancing the secretion efficiency of chassis strains.

Gene Transfection Efficiency Improvement with Lipid Conjugated Cationic Carbon Dots.

An ideal vehicle with a high transfection efficiency is crucial for gene delivery. In this study, a type of cationic carbon dot (CCD) known as APCDs were first prepared with arginine (Arg) and pentaethylenehexamine (PEHA) as precursors and conjugated with oleic acid (OA) for gene delivery. By tuning the mass ratio of APCDs to OA, APCDs-OA conjugates, namely, APCDs-0.5OA, APCDs-1.0OA, and APCDs-1.5OA were synthesized. All three amphiphilic APCDs-OA conjugates show high affinity to DNA through electrostatic interactions. APCDs-0.5OA exhibit strong binding with small interfering RNA (siRNA). After being internalized by Human Embryonic Kidney (HEK 293) and osteosarcoma (U2OS) cells, they could distribute in both the cytoplasm and the nucleus. With APCDs-OA conjugates as gene delivery vehicles, plasmid DNA (pDNA) that encodes the gene for the green fluorescence protein (GFP) can be successfully delivered in both HEK 293 and U2OS cells. The GFP expression levels mediated by APCDs-0.5OA and APCDs-1.0OA are ten times greater than that of PEI in HEK 293 cells. Furthermore, APCDs-0.5OA show prominent siRNA transfection efficiency, which is proven by the significantly downregulated expression of FANCA and FANCD2 proteins upon delivery of FANCA siRNA and FANCD2 siRNA into U2OS cells. In conclusion, our work demonstrates that conjugation of CCDs with a lipid structure such as OA significantly improves the gene transfection efficiency, providing a new idea about the designation of nonviral carriers in gene delivery systems.

A Whole-Process Visible Strategy for the Preparation of Rhizomucor miehei Lipase with Escherichia coli Secretion Expression System and the Immobilization.

BACKGROUND: Rhizomucor miehei (RM) lipase is a regioselective lipase widely used in food, pharmaceutical and biofuel industries. However, the high cost and low purity of the commercial RM lipase limit its industrial applications. Therefore, it is necessary to develop cost-effective strategies for large-scale preparation of this lipase. The present study explored the high-level expression of RM lipase using superfolder green fluorescent protein (sfGFP)-mediated Escherichia coli secretion system. RESULTS: The sfGFP(-15) mutant was fused to the C-terminus of RM lipase to mediate its secretion expression. The yield of the fusion protein reached approximately 5.1 g/L with high-density fermentation in 5-L fermentors. Unlike conventional secretion expression methods, only a small portion of the target protein was secreted into the cell culture while majority of the fusion protein was still remained in the cytoplasm. However, in contrast to intracellular expression, the target protein in the cytoplasm could be transported efficiently to the supernatant through a simple washing step with equal volume of phosphate saline (PBS), without causing cell disruption. Hence, the approach facilitated the downstream purification step of the recombinant RM lipase. Moreover, contamination or decline of the engineered strain and degradation or deactivation of the target enzyme can be detected efficiently because they exhibited bright green fluorescence. Next, the target protein was immobilized with anion-exchange and macropore resins. Diethylaminoethyl sepharose (DEAE), a weak-basic anion-exchange resin, exhibited the highest bind capacity but inhibited the activity of RM lipase dramatically. On the contrary, RM lipase fixed with macropore resin D101 demonstrated the highest specific activity. Although immobilization with D101 didn't improve the activity of the enzyme, the thermostability of the immobilized enzyme elevated significantly. The immobilized RM lipase retained approximately 90% of its activity after 3-h incubation at 80 °C. Therefore, D101 was chosen as the supporting material of the target protein. CONCLUSION: The present study established a highly efficient strategy for large-scale preparation of RM lipase. This innovative technique not only provides high-purity RM lipase at a low cost but also has great potential as a platform for the preparation of lipases in the future.

Comparing chemical transfection, electroporation, and lentiviral vector transduction to achieve optimal transfection conditions in the Vero cell line.

BACKGROUND: Transfection is an important analytical method for studying gene expression in the cellular environment. There are some barriers to efficient DNA transfection in host cells, including circumventing the plasma membrane, escaping endosomal compartmentalization, autophagy, immune sensing pathways, and translocating the nuclear envelope. Therefore, it would be very useful to introduce an optimum transfection approach to achieve a high transfection efficiency in the Vero cell line. The aim of this study was to compare various transfection techniques and introduce a highly efficient method for gene delivery in Vero cells. METHODS: In the current study, three transfection methods were used, including chemical transfection, electroporation, and lentiviral vector transduction, to obtain the optimum transfection conditions in the Vero cell line. Vero cells were cultured and transfected with chemical transfection reagents, electroporation, or HIV-1-based lentivectors under different experimental conditions. Transfection efficiency was assessed using flow cytometry and fluorescence microscopy to detect GFP-positive cells. RESULTS: Among the tested methods, TurboFect™ chemical transfection exhibited the highest efficiency. Optimal transfection conditions were achieved using 1 µg DNA and 4 µL TurboFect™ in 6 × 104 Vero cells. CONCLUSION: TurboFect™, a cationic polymer transfection reagent, demonstrated superior transfection efficiency in Vero cells compared with electroporation and lentivirus particles, and is the optimal choice for chemical transfection in the Vero cell line.

Neither injury induced macrophages within the nerve, nor the environment created by Wallerian degeneration is necessary for enhanced in vivo axon regeneration after peripheral nerve injury.

BACKGROUND: Since the 1990s, evidence has accumulated that macrophages promote peripheral nerve regeneration and are required for enhancing regeneration in the conditioning lesion (CL) response. After a sciatic nerve injury, macrophages accumulate in the injury site, the nerve distal to that site, and the axotomized dorsal root ganglia (DRGs). In the peripheral nervous system, as in other tissues, the macrophage response is derived from both resident macrophages and recruited monocyte-derived macrophages (MDMs). Unresolved questions are: at which sites do macrophages enhance nerve regeneration, and is a particular population needed. METHODS: Ccr2 knock-out (KO) and Ccr2gfp/gfp knock-in/KO mice were used to prevent MDM recruitment. Using these strains in a sciatic CL paradigm, we examined the necessity of MDMs and residents for CL-enhanced regeneration in vivo and characterized injury-induced nerve inflammation. CL paradigm variants, including the addition of pharmacological macrophage depletion methods, tested the role of various macrophage populations in initiating or sustaining the CL response. In vivo regeneration, measured from bilateral proximal test lesions (TLs) after 2 d, and macrophages were quantified by immunofluorescent staining. RESULTS: Peripheral CL-enhanced regeneration was equivalent between crush and transection CLs and was sustained for 28 days in both Ccr2 KO and WT mice despite MDM depletion. Similarly, the central CL response measured in dorsal roots was unchanged in Ccr2 KO mice. Macrophages at both the TL and CL, but not between them, stained for the pro-regenerative marker, arginase 1. TL macrophages were primarily CCR2-dependent MDMs and nearly absent in Ccr2 KO and Ccr2gfp/gfp KO mice. However, there were only slightly fewer Arg1+ macrophages in CCR2 null CLs than controls due to resident macrophage compensation. Zymosan injection into an intact WT sciatic nerve recruited Arg1+ macrophages but did not enhance regeneration. Finally, clodronate injection into Ccr2gfp KO CLs dramatically reduced CL macrophages. Combined with the Ccr2gfp KO background, depleting MDMs and TL macrophages, and a transection CL, physically removing the distal nerve environment, nearly all macrophages in the nerve were removed, yet CL-enhanced regeneration was not impaired. CONCLUSIONS: Macrophages in the sciatic nerve are neither necessary nor sufficient to produce a CL response.

A Quantitative Fusion Assay to Study Measles Virus Entry.

We have adopted a real-time assay based on a dual-split reporter to assess cell-cell fusion mediated by the measles virus (MeV) membrane fusion machinery. This reporter system is comprised of two expression vectors, each encoding a segment of Renilla luciferase fused to a segment of GFP. To regain function, the two segments need to associate, which is dependent on cell-cell fusion between effector cells expressing the MeV fusion machinery and target cells expressing the corresponding MeV receptor. By measuring reconstituted luciferase activity, we can follow the kinetics of cell-cell fusion and quantify the extent of fusion. This assay lends itself to the study of the MeV fusion machinery comprised of the attachment and fusion glycoproteins, the matrix protein, and the MeV receptors. Moreover, entry inhibitors targeting attachment or fusion can be readily screened using this assay. Finally, this assay can be easily adopted to study the entry of other members of the Paramyxoviridae, as we have demonstrated for the henipaviruses.

The role of the correlated motion(s) of the chromophore in photoswitching of green and red forms of the photoconvertible fluorescent protein mSAASoti.

Wild-type SAASoti and its monomeric variant mSAASoti can undergo phototransformations, including reversible photoswitching of the green form to a nonfluorescent state and irreversible green-to-red photoconversion. In this study, we extend the photochemistry of mSAASoti variants to enable reversible photoswitching of the red form. This result is achieved by rational and site-saturated mutagenesis of the M163 and F177 residues. In the case of mSAASoti it is M163T substitution that leads to the fastest switching and the most photostable variant, and reversible photoswitching can be observed for both green and red forms when expressed in eukaryotic cells. We obtained a 13-fold increase in the switching efficiency with the maximum switching contrast of the green form and the appearance of comparable switching of the red form for the C21N/M163T mSAASoti variant. The crystal structure of the C21N mSAASoti in its green on-state was obtained for the first time at 3.0 Å resolution, and it is in good agreement with previously calculated 3D-model. Dynamic network analysis reveals that efficient photoswitching occurs if motions of the 66H residue and phenyl fragment of chromophore are correlated and these moieties belong to the same community.

Motor, somatosensory, and executive cortical areas elicit monosynaptic and polysynaptic neuronal activity in the auditory midbrain.

We recently reported that the central nucleus of the inferior colliculus (the auditory midbrain) is innervated by glutamatergic pyramidal cells originating not only in auditory cortex (AC), but also in multiple 'non-auditory' regions of the cerebral cortex. Here, in anaesthetised rats, we used optogenetics and electrical stimulation, combined with recording in the inferior colliculus to determine the functional influence of these descending connections. Specifically, we determined the extent of monosynaptic excitation and the influence of these descending connections on spontaneous activity in the inferior colliculus. A retrograde virus encoding both green fluorescent protein (GFP) and channelrhodopsin (ChR2) injected into the central nucleus of the inferior colliculus (ICc) resulted in GFP expression in discrete groups of cells in multiple areas of the cerebral cortex. Light stimulation of AC and primary motor cortex (M1) caused local activation of cortical neurones and increased the firing rate of neurones in ICc indicating a direct excitatory input from AC and M1 to ICc with a restricted distribution. In naïve animals, electrical stimulation at multiple different sites within M1, secondary motor, somatosensory, and prefrontal cortices increased firing rate in ICc. However, it was notable that stimulation at some adjacent sites failed to influence firing at the recording site in ICc. Responses in ICc comprised singular spikes of constant shape and size which occurred with a short, and fixed latency (∼ 5 ms) consistent with monosynaptic excitation of individual ICc units. Increasing the stimulus current decreased the latency of these spikes, suggesting more rapid depolarization of cortical neurones, and increased the number of (usually adjacent) channels on which a monosynaptic spike was seen, suggesting recruitment of increasing numbers of cortical neurons. Electrical stimulation of cortical regions also evoked longer latency, longer duration increases in firing activity, comprising multiple units with spikes occurring with significant temporal jitter, consistent with polysynaptic excitation. Increasing the stimulus current increased the number of spikes in these polysynaptic responses and increased the number of channels on which the responses were observed, although the magnitude of the responses always diminished away from the most activated channels. Together our findings indicate descending connections from motor, somatosensory and executive cortical regions directly activate small numbers of ICc neurones and that this in turn leads to extensive polysynaptic activation of local circuits within the ICc.

Electric pulse exposure reduces AAV8 dosage required to transduce HepG2 cells.

We demonstrate that applying electric field pulses to hepatocytes, in vitro, in the presence of enhanced green fluorescent protein (EGFP)-expressing adeno-associated virus (AAV8) vectors reduces the viral dosage required for a given transduction level by more than 50-fold, compared to hepatocytes exposed to AAV8-EGFP vectors without electric field pulse exposure. We conducted 48 experimental observations across 8 exposure conditions in standard well plates. The electric pulse exposures involved single 80-ms pulses with 375 V/cm field intensity. Our study suggests that electric pulse exposure results in enhanced EGFP expression in cells, indicative of increased transduction efficiency. The enhanced transduction observed in our study, if translated successfully to an in vivo setting, would be a promising indication of potential reduction in the required dose of AAV vectors. Understanding the effects of electric field pulses on AAV transduction in vitro is an important preliminary step.

Biocomposite thermoplastic polyurethanes containing evolved bacterial spores as living fillers to facilitate polymer disintegration.

The field of hybrid engineered living materials seeks to pair living organisms with synthetic materials to generate biocomposite materials with augmented function since living systems can provide highly-programmable and complex behavior. Engineered living materials have typically been fabricated using techniques in benign aqueous environments, limiting their application. In this work, biocomposite fabrication is demonstrated in which spores from polymer-degrading bacteria are incorporated into a thermoplastic polyurethane using high-temperature melt extrusion. Bacteria are engineered using adaptive laboratory evolution to improve their heat tolerance to ensure nearly complete cell survivability during manufacturing at 135 °C. Furthermore, the overall tensile properties of spore-filled thermoplastic polyurethanes are substantially improved, resulting in a significant improvement in toughness. The biocomposites facilitate disintegration in compost in the absence of a microbe-rich environment. Finally, embedded spores demonstrate a rationally programmed function, expressing green fluorescent protein. This research provides a scalable method to fabricate advanced biocomposite materials in industrially-compatible processes.

Identification and characterization of myeloid cells localized in the tadpole liver cortex in Xenopus laevis.

In the present study, using transgenic frogs that express GFP specifically in myeloid cells under the myeloperoxidase enhancer sequence, we found that myeloperoxidase-positive cells are localized in the liver cortex at the late tadpole stages. Immunohistochemical analysis revealed that myelopoiesis in the liver cortex became evident after st. 50 and reached its peak by st. 56. Transplantation experiments indicated that cells with a high density at the liver cortex were derived from the dorso-lateral plate tissue in the neurula embryo. Analysis of smear samples of the cells isolated from collagenase-treated liver tissues of the transgenic tadpoles indicated that myeloid cells were the major population of blood cells in the larval liver and that, in addition to myeloid colonies, erythroid colonies expanded in entire liver after metamorphosis. Cells that were purified from the livers of transgenic tadpoles according to the GFP expression exhibited the multi-lobed nuclei. The results of present study provide evidence that the liver cortex of the Xenopus tadpole is a major site of granulopoiesis.

Whole-Cell Display of Phospholipase D in Escherichia coli for High-Efficiency Extracellular Phosphatidylserine Production.

Phospholipids are widely utilized in various industries, including food, medicine, and cosmetics, due to their unique chemical properties and healthcare benefits. Phospholipase D (PLD) plays a crucial role in the biotransformation of phospholipids. Here, we have constructed a super-folder green fluorescent protein (sfGFP)-based phospholipase D (PLD) expression and surface-display system in Escherichia coli, enabling the surface display of sfGFP-PLDr34 on the bacteria. The displayed sfGFP-PLDr34 showed maximum enzymatic activity at pH 5.0 and 45 °C. The optimum Ca2+ concentrations for the transphosphatidylation activity and hydrolysis activity are 100 mM and 10 mM, respectively. The use of displayed sfGFP-PLDr34 for the conversion of phosphatidylcholine (PC) and L-serine to phosphatidylserine (PS) showed that nearly all the PC was converted into PS at the optimum conditions. The displayed enzyme can be reused for up to three rounds while still producing detectable levels of PS. Thus, Escherichia coli/sfGFP-PLD shows potential for the feasible industrial-scale production of PS. Moreover, this system is particularly valuable for quickly screening higher-activity PLDs. The fluorescence of sfGFP can indicate the expression level of the fused PLD and changes that occur during reuse.