Bacterial surface display of PETase mutants and MHETase for an efficient dual-enzyme cascade catalysis.

Biological degradation of PET plastic holds great potential for plastic recycling. However, the high costs associated with preparing free enzymes for degrading PET make it unfeasible for industrial applications. Hence, we developed various cell catalysts by surface-displaying PETase mutants and MHETase using autotransporters in E. coli and P. putida. The efficiency of surface display was enhanced through modifying the host, co-expressing molecular chaperones, and evoluting the autotransporter. In strain EC9F, PET degradation rate was boosted to 3.85 mM/d, 51-fold and 23-fold increase compared to free enzyme and initial strain ED1, respectively. The reusability of cell catalyst EC9F was demonstrated with over 38 % and 30 % of its initial activity retained after 22 cycles of BHET degradation and 3 cycles of PET degradation. The highest reported PET degradation rate of 4.95 mM/d was achieved by the dual-enzyme cascade catalytic system EC9F+EM2 + R, a mixture of cell catalyst EC9F and EM2 with surfactant rhamnolipid.

Development of a synthetic library of humanized nanobodies for targeted IL-6 inhibition.

Interleukin-6 (IL-6) is a cytokine that can bind to IL-6 receptor and induce pleiotropic effects. It serves as a critical biomarker, involved in inflammation amplification, tumor progression, and many other disease developments. Nanobodies, featuring small structure and high affinity, are a powerful and versatile tool in medical diagnostics and therapeutics. Here, based on a scaffold optimized for humanization and stability, we developed a synthetic phage display library that rapidly generated high-affinity and humanized nanobodies, negating the need for animal immunization. Using enhanced green fluorescent protein (eGFP) as a benchmark, we demonstrated that the library produced humanized nanobodies with high function and great intracellular stability. The library was then subjected to screening against IL-6. We identified a standout nanobody, NbL3, which exhibited high affinity (22.16 nM) and stability and significantly inhibited IL-6-enhanced migration on the human breast cancer cell MCF-7 at a relatively low concentration. NbL3's strong blocking activity provides a promising therapeutic alternative for the IL-6-targeted intervention strategy, underscoring the broader potential of our synthetic library as a versatile platform for the development of humanized nanobodies against multiple antigens.

Influence of Visual Stimulus Changes in a Virtual Environment on Postural Control: Focusing on a Hallway Walking Simulation.

The purpose of this study was to clarify the effects of the standing center of gravity sway by providing visual stimulus information as if the subjects were walking in virtual reality (VR) and by monitoring conditions with different corridor widths. We included 25 healthy young individuals in our study. The center of gravity sway was measured during open- and closed-eye static standing using images of walking in corridors of different widths (780 and 1600 mm) presented on a VR and personal computer monitor (Monitor). The parameters measured for the center of gravity sway were swing path length (SPL), height of excursion (HoE), and width of excursion (WoE). The results showed that the SPL and HoE values were significantly greater in the VR group than those in the Monitor group. The greater center of gravity sway in the VR compared with the Monitor group can be attributed to the ability of the head-mounted VR display to cover the entire field of vision and its head-tracking function. There was no change in the center of gravity sway with respect to the corridor width, which may be because the width of the corridor alone did not provide sufficient visual stimulation to affect physical function. This research could lead to further studies which could impact the motivation of patients for rehabilitation therapies.

Real-time display of intracranial subdural electrodes and the brain surface during an electrode implantation procedure using permeable film.

Background: Subdural electrode (SDE) implantation is an important method of diagnosing epileptogenic lesions and mapping brain function, even with the current preference for stereoelectroencephalography. We developed a novel method to assess SDEs and the brain surface during the electrode implantation procedure using brain images printed onto permeable films and intraoperative fluoroscopy. This method can help verify the location of the electrode during surgery and improve the accuracy of SDE implantation. Methods: We performed preoperative imaging by magnetic resonance imaging and computed tomography. Subsequently, the images were edited and fused to visualize the gyrus and sulcus better. We printed the images on permeable films and superimposed them on the intraoperative fluoroscopy display. The intraoperative and postoperative coordinates of the electrodes were obtained after the implantation surgery, and the differences in the locations were calculated. Results: Permeable films were created for a total of eight patients with intractable epilepsy. The median difference of the electrodes between the intraoperative and postoperative images was 4.6 mm (Interquartile range 2.9-7.1). The locations of electrodes implanted outside the operation field were not significantly different from those implanted inside. Conclusion: Our new method may guide the implantation of SDEs into their planned location.

Maritime accidents in the estuary of the River Seine from 2009-2019.

BACKGROUND: In confined waters, ships run a high risk of groundings, contact, sinkings and near misses. In such waters the maritime traffic is dense, the waterway is narrow, the depth is limited, and tides and currents are constantly changing. MATERIALS AND METHODS: From 2009-2019, 75 accidents were investigated in the estuary of the Seine. Weather conditions and perceived fatigue were studied. From May to June 2020, 114 seafarers, 34 pilots and 80 captains, responded to a questionnaire focusing on the use of Pilot Portable Units (PPU) and Electronic Chart Display Information Systems (ECDIS). RESULTS: The 75 accidents corresponded to an average of 6.8 ± 3.2 accidents per year. Groundings were the most frequent accidents (35%, n = 26) followed by contact accidents with the quayside (25%, n = 19), between ships or tugs while manoeuvring (8%, n = 6) or while sailing (1%, n = 1). There was no loss of vessels nor fatalities of crew members. In poor weather conditions, there were 76% more accidents than in normal conditions (4.4 ± 2.5 accidents/10,000 movements versus 2.5 ± 1.9 accidents/10,000 movements, p < 0.03). Almost all the accidents (96%) were related to human errors of judgment (81%), or negligence (53%), or both (39). Perceived fatigue was probably in cause in 6 accidents. Only 3 accidents were related to mechanical causes. Through the questionnaires, 69% of the pilots complained of difficulties in mastering the devices and software. They felt distracted by alarms which affected their attention while navigating. They requested training on a simulator. Concerning ship captains, 83% felt comfortable with ECDIS devices yet only 20% were able to configure the ECDIS correctly. CONCLUSIONS: In the Seine estuary, 75 accidents occurred within the 11 year-study. Risk factors were poor weather conditions and human error. PPU and ECDIS were considered as useful tools in the prevention of accidents. However, pilots and captains requested more thorough training in their use.

Quantitative Performance Evaluation of Commonly Used Colormaps for Image Display in Myocardial Perfusion Imaging: Analysis based on Perceptual Metrics.

Objectives: To quantitatively evaluate the performance of the most used colormaps in image display using perceptual metrics and to what extent these measures are congruent with the true intensity or uptake of pixels at different levels of defect severity in simulated cardiac images. Methods: Six colormaps, labeled "Gray", "Thermal", "Cool", "CEqual", "Siemens" and "S Pet" extracted from FIJI ImageJ software are included. Colormap data are converted from the red, green, blue color space to CIELAB. Perceptual metrics for measuring "color difference" were calculated, including difference (ΔE76) and "speed". The pairwise color difference in every two levels or entries is visualized in a 2-dimensional "heatmap distance matrix" for each colormap. Curves are plotted for each colormap and compared. In addition, to apply this technique to clinical images, simulated short-axis cardiac slices with incremental defect severity (10% grading) were employed. The circumferential profile curves of true pixel intensity, lightness or luminance, and color difference are plotted simultaneously for each defect severity to visualize the concordance of the three curves in various colormaps. Results: In 0% defect, all the curves are at the highest level, except for "s pet", in that the lightness is not at its maximum value. In the phantom with 10% defect (or 90% of maximum value), discrepancies among curves appear. In "Siemens", the ΔE76 drops sharply. In "Siemens" colormap, the ΔE76 drops sharply. In 80% defect, ΔE76 curve, in "gray" colormap drops more slowly than other curves of other colormaps. In "s pet", lightness curve rises paradoxically, although the count intensity and ΔE76 curve match. In 70% defect, again, the curves are in good agreement in "thermal", "Siemens" and "cequal". However, a consistent lag exists in "gray". Up to 50% defect, curves maintain their expected pattern, but in defects more severe than 40%, lightness and ΔE76 curves in "cool" and "cequal" rise paradoxically, and in "thermal", they start to slow down in descent. In "Siemens", falling pattern of the three curves continues. For "s pet" colormap, an erratic pattern of lightness and ΔE76 curves exists. Conclusion: Of 6 colormaps investigated for estimating defect severity, "grayscale" is less favorable than others and "thermal" performs slightly better. "S pet" or rainbow, which is used traditionally by many practitioners, is strongly discouraged. The "Siemens" colormap suffers from decreased discriminating power in the range of mild to moderate/severe. In contrast, the "cool" and "cequal" colormaps outperform the other colormaps employed in this study to some extent, although they have some shortcomings.

Phage-displayed heptapeptide sequence conjugation significantly improves the specific targeting ability of antimicrobial peptides against Staphylococcus aureus.

Broad-spectrum antibacterial drugs often lack specificity, leading to indiscriminate bactericidal activity, which can disrupt the normal microbial balance of the host flora and cause unnecessary cytotoxicity during systemic administration. In this study, we constructed a specifically targeted antimicrobial peptide against Staphylococcus aureus by introducing a phage-displayed peptide onto a broad-spectrum antimicrobial peptide and explored its structure-function relationship through one-factor modification. SFK2 obtained by screening based on the selectivity index and the targeting index showed specific killing ability against S. aureus. Moreover, SFK2 showed excellent biocompatibility in mice and piglet, and demonstrated significant therapeutic efficacy against S. aureus infection. In conclusion, our screening of phage-derived heptapeptides effectively enhances the specific bactericidal ability of the antimicrobial peptides against S. aureus, providing a theoretical basis for developing targeted antimicrobial peptides.

Extraction of Modified Adeno-Associated Virus-Like Particles Displaying Peptides on Their Surface.

Virus-like particles (VLPs) of the adeno-associated virus (AAV) can be produced using the baculovirus expression vector system. Insertion of small peptides on the surface of the AAV or AAV VLPs has been used to redirect the AAV to different target tissues and for vaccine development. Usually, the VLPs self-assemble intracellularly, and an extraction step must be performed before purification. Here, we describe the method we have used to extract AAV VLPs from insect cells successfully with peptide insertions on their surface.

Variations of floral temperature in changing weather conditions.

Floral temperature is a flower characteristic that has the potential to impact the fitness of flowering plants and their pollinators. Likewise, the presence of floral temperature patterns, areas of contrasting temperature across the flower, can have similar impacts on the fitness of both mutualists. It is currently poorly understood how floral temperature changes under the influence of different weather conditions, and how floral traits may moderate these changes. The way that floral temperature changes with weather conditions will impact how stable floral temperatures are over time and their utility to plants and pollinators. The stability of floral temperature cues is likely to facilitate effective plant-pollinator interactions and play a role in the plant's reproductive success. We use thermal imaging to monitor how floral temperatures and temperature patterns of four plant species (Cistus 'snow fire' and 'snow white', Coreopsis verticillata and Geranium psilostemon) change with several weather variables (illumination, temperature; windspeed; cloud cover; humidity and pressure) during times that pollinators are active. All weather variables influenced floral temperature in one or more species. The directionality of these relationships was similar across species. In all species, light conditions (illumination) had the greatest influence on floral temperatures overall. Floral temperature and the extent to which flowers showed contrasting temperature patterns were influenced predominantly by light conditions. However, several weather variables had additional, lesser, influences. Furthermore, differences in floral traits, pigmentation and structure, likely resulted in differences in temperature responses to given conditions between species and different parts of the same flower. However, floral temperatures and contrasting temperature patterns that are sufficiently elevated for detection by pollinators were maintained across most conditions if flowers received moderate illumination. This suggests the presence of elevated floral temperature and contrasting temperature patterns are fairly constant and may have potential to influence plant-pollinator interactions across weather conditions.

Chemical Stability of mRNA/cDNA Complexes: Defining the Limits of mRNA display.

Messenger RNA (mRNA) display is being increasingly adopted for peptide drug candidate discovery. While many conditions have been reported for the affinity enrichment step and in some cases for peptide modification, there is still limited understanding about the versatility of peptide-puromycin-mRNA/cDNA (complementary DNA) complexes. This work explores the chemical stability of mRNA/cDNA hybrid complexes under a range of different fundamental chemical conditions as well as with peptide modification conditions reported in an mRNA display setting. We further compare the stability of full complexes originating from two different mRNA display systems (RaPID and cDNA-TRAP). Overall, these complexes were found to be stable under a broad range of conditions, with some edge conditions benefitting from encoding directly in cDNA rather than mRNA. This should allow for more and broader exploitation of late-stage peptide modification chemistry in mRNA display, with confidence regarding the stability of encoding, and potentially better hit-finding campaigns as a result.

Immersion immunization with recombinant Saccharomyces cerevisiae displaying ORF25 induced protective immunity against cyprinid herpesvirus 2.

Displaying antigens on yeast surface as an oral vaccine has been widely explored, while its potential as an immersion vaccine has not been evaluated. Here, an immersion vaccine was prepared by displaying ORF25 of Cyprinid herpesvirus 2 (CyHV-2) on the surface of Saccharomyces cerevisiae. Carassius auratus gibelio was immersion immunized by 2 × 107 CFU/mL yeast for 2 h, and reinforce the immunity using the same method 14 days after the first immunization. The results showed that ORF25 specific antibody in immunized crucian carp serum was detected at a high level, and the mRNA expression level of IgM, IgT, IL-1ß, and IFN-1 in vaccinated head-kidney and spleen tissues were higher than the control group, indicating that innate and adaptive immunity were induced. Moreover, the immersion vaccination provided effective protection for fish against CyHV-2, leading to a relative percent survival of 50.2%. Meanwhile, immersion vaccination restrained virus replication and histological damage in CyHV-2 infected crucian carp. Our data suggested that immersion immunization of S. cerevisiae-displayed ORF25 could be served as a candidate vaccine to prevent CyHV-2 infection.

A comparison of differing organizational formats for teaching requesting skills to children with autism.

The selection of high-tech AAC for children diagnosed with autism spectrum disorder can be a challenging process due to the vast array of options available. One of the decisions that clinicians need to make involves how vocabulary will be organized on the display. This study aimed to compare a visual scene display (VSD) with a grid display using a multiple-probe design across participants with an embedded adapted alternating treatment design. Four young children with autism spectrum disorder who were beginning communicators were recruited and taught to request preferred items using two display formats: VSD and grid layout on a mainstream tablet with an AAC app. Two of the participants achieved criterion with both displays, the other two participants failed to achieve criterion in either display. For all participants, progress was similar in both displays. The results are discussed through the lens of each participant's characteristics with suggestions for clinical decision-making.

Isolation of camel single domain antibodies against Yersinia pestis V270 antigen based on a semi-synthetic single domain antibody library and development of a VHH-based lateral flow assay.

BACKGROUND: Antibodies have been proven effective as diagnostic agents for detecting zoonotic diseases. The variable domain of camel heavy chain antibody (VHH), as an antibody derivative, may be used as an alternative for traditional antibodies in existing immunodiagnostic reagents for detecting rapidly spreading infectious diseases. OBJECTIVES: To expedite the isolation of specific antibodies for diagnostic purposes, we constructed a semi-synthetic camel single domain antibody library based on the phage display technique platform (PDT) and verified the validity of this study. METHODS: The semi-synthetic single domain antibody sequences consist of two parts: one is the FR1-FR3 region amplified by RT-PCR from healthy camel peripheral blood lymphocytes (PBLs), and the other part is the CDR3-FR4 region synthesised as an oligonucleotide containing CDR3 randomised region. The two parts were fused by overlapping PCR, resulting in the rearranged variable domain of heavy-chain antibodies (VHHs). Y. pestis low-calcium response V protein (LcrV) is an optional biomarker to detect the Y. pestis infection. The semi-synthetic library herein was screened using recombinant (LcrV) as a target antigen. RESULTS: After four cycles of panning the library, four VHH binders targeting 1-270 aa residues of LcrV were isolated. The four VHH genes with unique sequences were recloned into an expression vector and expressed as VHH-hFc chimeric antibodies. The purified antibodies were identified and used to develop a lateral flow immunoassay (LFA) test strip using latex microspheres (LM) for the rapid and visual detection of Y. pestis infection. CONCLUSIONS: These data demonstrate the great potential of the semi-synthetic library for use in isolation of antigen-specific nanobodies and the isolated specific VHHs can be used in antigen-capture immunoassays.

An engineered ultrahigh affinity bi-paratopic uPAR targeting agent confers enhanced tumor targeting.

Urokinase-type plasminogen activator receptor (uPAR) is overexpressed on tumor cells in multiple types of cancer and contributes to disease progression and metastasis. In this work, we engineered a novel bi-paratopic uPAR targeting agent by fusing the binding domains of two native uPAR ligands: uPA and vitronectin, with a flexible peptide linker. The linker length was optimized to facilitate simultaneous engagement of both domains to their adjacent epitopes on uPAR, resulting in a high affinity and avid binding interaction. Furthermore, the individual domains were affinity-matured using yeast surface display and directed evolution, resulting in a bi-paratopic protein with affinity in the picomolar to femtomolar range. This engineered uPAR targeting agent demonstrated significantly enhanced tumor localization in mouse tumor models compared to the native uPAR ligand and warrants further investigation as a diagnostic and therapeutic agent for cancer.

A method for rapid and reliable quantification of VEGF-cell binding activity.

Vascular endothelial growth factor (VEGF) is a pleiotropic growth factor that binds a broad spectrum of cell types and regulates diverse cellular processes, including angiogenesis, growth and survival. However, it is technically difficult to quantify VEGF-cell binding activity because of reversible nature of ligand-receptor interactions. Here we used T7 bacteriophage display to quantify and compare binding activity of three human VEGF-A (hVEGF) isoforms, including hVEGF111, 165 and 206. All three isoforms bound equally well to immobilized aflibercept, a decoy VEGF receptor. hVEGF111-Phage exhibited minimal binding to immobilized heparan sulfate, whereas hVEGF206-Phage and hVEGF165-Phage had the highest and intermediate binding to heparan, respectively. In vitro studies revealed that all three isoforms bound to human umbilical vein endothelial cells (HUVECs), HEK293 epithelial and SK-N-AS neuronal cells. hVEGF111-Phage has the lowest binding activity, while hVEGF206-Phage has the highest binding. hVEGF206-Phage was the most sensitive to detect VEGF-cell binding, albeit with the highest background binding to SK-N-AS cells. These results suggest that hVEGF206-Phage is the best-suited isoform to quantify VEGF-cell binding even though VEGF165 is the most biologically active. Furthermore, this study demonstrates the utility of T7 phage display as a platform for rapid and convenient ligand-cell binding quantification with pros and cons discussed.

Development of uniform ribosome display technology enabling easy and efficient identification of full-length proteins that interact with bioactive small and large molecules.

Identifying target proteins that interact with bioactive molecules is indispensable for understanding their mechanisms of action. In this study, we developed a uniform ribosome display technology using equal-length DNAs and mRNAs to improve molecular display principle for target identification. The equal-length DNAs were designed to contain various coding sequences for full-length proteins with molecular weights of up to 130 kDa and were used to synthesize equal-length mRNAs, which allowed the formation of full-length protein-ribosome-equal-length mRNA complexes. Uniform ribosome display selections of dihydrofolate reductase and haloalkane dehalogenase mutant were performed against methotrexate and chlorohexane, respectively. Quantitative changes of proteins after each selection indicated that the target protein-displaying ribosomal complexes were specifically selected through non-covalent or covalent interactions with the corresponding bioactive molecules. Furthermore, selection of full-length proteins interacting with methotrexate or anti-DDX46 antibody from protein pools showed that only the target proteins could be precisely identified even though the molar amounts of equal-length mRNAs encoding them were adjusted to 1/20,000 of the total equal-length mRNAs. Thus, the uniform ribosome display technology enabled efficient identification of target proteins that interact with bioactive small and large molecules through simplified operations without deep sequencing.

Implementation of Spore Display in Paenibacillus polymyxa with Different Hydrolytic Enzymes.

Biotechnological processes are essential for producing climate-friendly high-value chemicals or pharmaceutical compounds, which can include steps catalyzed by enzymes. Therefore, establishing new, robust, and cheap enzyme production processes is desirable. One possible way to enhance processes is through the use of the spore display method. Spore display can present heterologous proteins on the surface of bacterial spores, offering numerous advantages in a range of biotechnological applications. This study demonstrates the implementation of the spore display method in Paenibacillus polymyxa, achieved by modifying the spore surface, incorporating an anchoring protein, and attaching green fluorescent protein to it, allowing the visualization of fluorescent spores. Following the initial experiment, a native lipase (Lip3), a heterologous lipase (LipA) from Bacillus subtilis, a native esterase (PnbA) from P. polymyxa, and a lipoyl synthase were expressed during sporulation and displayed on the spore surface. The activity profiles were determined in the temperature range from 4 °C to 70 °C. The PnbA reached its optimum at 4 °C, whereas the LipA from B. subtilis showed 4.4-fold higher activity at 42 °C compared to the control. Furthermore, we explored a possible new technique for the purification of enzymes with the TEV cleavage site between the anchor and the protein of interest. Finally, we showed a not-yet-described side activity of the lipoyl synthase over a wide temperature range.

Rapidly Inducible Yeast Surface Display for Antibody Evolution with OrthoRep.

We recently developed "autonomous hypermutation yeast surface display" (AHEAD), a technology that enables the rapid generation of potent and specific antibodies in yeast. AHEAD pairs yeast surface display with an error-prone orthogonal DNA replication system (OrthoRep) to continuously and rapidly mutate surface-displayed antibodies, thereby enabling enrichment for stronger binding variants through repeated rounds of cell growth and fluorescence-activated cell sorting. AHEAD currently utilizes a standard galactose induction system to drive the selective display of antibodies on the yeast surface. However, achieving maximal display levels can require up to 48 h of induction. Here we report an updated version of the AHEAD platform that utilizes a synthetic ß-estradiol-induced gene expression system to regulate the surface display of antibodies and find that induction is notably faster in achieving surface display for both our AHEAD system and traditional yeast surface display from nuclear plasmids that do not hypermutate. The updated AHEAD platform was fully functional in repeated rounds of evolution to drive the rapid evolution of antibodies.

Review of Integrated Gate Driver Circuits in Active Matrix Thin-Film Transistor Display Panels.

Many advanced technologies have been employed in high-performance active matrix displays, including liquid crystal displays, organic light-emitting diode displays, and micro-light-emitting diode displays. On the other side, there exists a strong demand for cost reduction, and it is one of the low-cost schemes for integrating the driver circuit in a panel based on thin-film transistor technologies. This paper reviews the overall concept, operation principles, and various circuit approaches in shift registers for scanning pulse generation. In addition, it deals with the implementation of additional functionalities in gate drivers to support pixel compensation, multi-line driving, in-cell capacitive touch screen, pixel sensing, and adaptive scanning region control.

Structure-activity relationships of middle-size cyclic peptides, KRAS inhibitors derived from an mRNA display.

Cyclic peptides are attracting attention as therapeutic agents due to their potential for oral absorption and easy access to tough intracellular targets. LUNA18, a clinical KRAS inhibitor, was transformed-without scaffold hopping-from the initial hit by using an mRNA display library that met our criteria for drug-likeness. In drug discovery using mRNA display libraries, hit compounds always possess a site linked to an mRNA tag. Here, we describe our examination of the Structure-Activity Relationship (SAR) using X-ray structures for chemical optimization near the site linked to the mRNA tag, equivalent to the C-terminus. Structural modifications near the C-terminus demonstrated a relatively wide range of tolerance for side chains. Furthermore, we show that a single atom modification is enough to change the pharmacokinetic (PK) profile. Since there are four positions where side chain modification is permissible in terms of activity, it is possible to flexibly adjust the pharmacokinetic profile by structurally optimizing the side chain. The side chain transformation findings demonstrated here may be generally applicable to hits obtained from mRNA display libraries.