3D Structure Research Articles

In Silico Design of Novel EpCAM-Binding Aptamers for Targeted Delivery of RNA Therapeutics.

Aptamers are short DNA or RNA sequences that adopt 3D structures and can bind to protein targets with high binding affinity and specificity. Aptamers exhibit excellent tissue penetration, are inexpensive to produce, and can be internalized by cells. Therefore, aptamers are attractive targeting ligands to direct the delivery of theranostic agents to the desired cells. Epithelial cell adhesion molecule (EpCAM) is a tumor-associated antigen that is aberrantly overexpressed on many epithelial-derived cancers, including on cholangiocarcinoma (CCA) cells. Its expression on treatment-resistant cancer stem cells, along with its abundance in the CCA tumor microenvironment, highlights the need to develop EpCAM-targeted therapies for CCA. Herein, an in silico approach was used to design and screen DNA aptamers capable of binding to the EpCAM monomer and homodimer. Two aptamers, PLD01 and PLD02, met the selection criteria and were validated in vitro. Both aptamers exhibited high affinity for EpCAM+ CCA cells, with negligible binding to EpCAM- leukemia cells. Modified versions of PLD01 and PLD02 were successfully incorporated into the membranes of milk-derived nanovesicles. PLD01-functionalized nanovesicles enabled EpCAM-targeted delivery of the therapeutic cargo to CCA cells. In summary, these EpCAM-targeting aptamers can be utilized to direct the delivery of theranostic agents to EpCAM-expressing cells.

Correlative X-ray micro-nanotomography with scanning electron microscopy at the Advanced Light Source.

Geological samples are inherently multi-scale. Understanding their bulk physical and chemical properties requires characterization down to the nano-scale. A powerful technique to study the three-dimensional microstructure is X-ray tomography, but it lacks information about the chemistry of samples. To develop a methodology for measuring the multi-scale 3D microstructure of geological samples, correlative X-ray micro- and nanotomography were performed on two rocks followed by scanning electron microscopy with energy-dispersive spectroscopy (SEM-EDS) analysis. The study was performed in five steps: (i) micro X-ray tomography was performed on rock sample cores, (ii) samples for nanotomography were prepared using laser milling, (iii) nanotomography was performed on the milled sub-samples, (iv) samples were mounted and polished for SEM analysis and (v) SEM imaging and compositional mapping was performed on micro and nanotomography samples for complimentary information. Correlative study performed on samples of serpentine and basalt revealed multiscale 3D structures involving both solid mineral phases and pore networks. Significant differences in the volume fraction of pores and mineral phases were also observed dependent on the imaging spatial resolution employed. This highlights the necessity for the application of such a multiscale approach for the characterization of complex aggregates such as rocks. Information acquired from the chemical mapping of different phases was also helpful in segmentation of phases that did not exhibit significant contrast in X-ray imaging. Adoption of the protocol used in this study can be broadly applied to 3D imaging studies being performed at the Advanced Light Source and other user facilities.

Linking structure to function: the connection between mesophyll structure and intrinsic water use efficiency.

Climate change-driven drought events are becoming unescapable in an increasing number of areas worldwide. Understanding how plants are able to adapt to these changing environmental conditions is a non-trivial challenge. Physiologically, improving a plant's intrinsic water use efficiency (WUEi ) will be essential for plant survival in dry conditions. Physically, plant adaptation and acclimatisation are constrained by a plant's anatomy. In other words, there is a strong link between anatomical structure and physiological function. Former research predominantly focused on using 2D anatomical measurements to approximate 3D structures based on the assumption of ideal shapes, such as spherical spongy mesophyll cells. As a result of increasing progress in 3D imaging technology, the validity of these assumptions is being assessed, and recent research has indicated that these approximations can contain significant errors. We suggest to invert the workflow and use the less common 3D assessments to provide corrections and functions for the more widely available 2D assessments. By combining these 3D and corrected 2D anatomical assessments with physiological measurements of WUEi , our understanding of how a plant's physical adaptation affects its function will increase and greatly improve our ability to assess plant survival.

Structures of Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus virions reveal species-specific tegument and envelope features.

Discovered in 1964, Epstein-Barr virus (EBV) is the first identified human oncogenic virus and the founding member of the gammaherpesvirus subfamily. In 1994, another cancer-causing virus was discovered in lesions of AIDS patients and later named Kaposi's sarcoma-associated herpesvirus (KSHV), the second human gammaherpesvirus. Despite the historical importance of EBV and KSHV, technical difficulties with isolating large quantities of these viruses and the pleiomorphic nature of their envelope and tegument layers have limited structural characterization of their virions. In this study, we employed the latest technologies in cryogenic electron microscopy (cryoEM) and tomography (cryoET) supplemented with an artificial intelligence-powered data processing software package to reconstruct 3D structures of the EBV and KSHV virions. We uncovered unique properties of the envelope glycoproteins and tegument layers of both EBV and KSHV. Comparison of these features with their non-tumorigenic counterparts provides insights into their relevance during infection.

Identification of two novel variants, c.-35A>T and c.[-35A>T, 725T>G], in the FUT1 gene in a patient exhibiting the para-Bombay phenotype.

Reduced or absent H antigens on red cells with the (para-)Bombay phenotype can arise from FUT1 gene mutations, impacting the structure and function of 1,2-L-fucosyltransferase 1 (1,2-L-FucT1). Here, we identified the novel mutations in one patient displaying the para-Bombay phenotype and examined the potential molecular mechanisms underlying this phenotype. ABH antigens and antibodies were detected in patient's blood and saliva using serological methods. The genotypes of ABO, FUT1, and FUT2 were imputed using the genetic variations discovered in the whole exome sequencing data. Three-dimensional (3D) models of FUT1 variants were built using Deepmind's AlphaFold2 and HDOCK, and the possible effects of the variants were predicted to evaluate using DynaMut2 and Polyphen-2. Serological analysis confirmed the para-Bombay B phenotype producing anti-HI and exhibiting normal genotypes ABO*B.01/O.01.02 and FUT2*01.09/01.09. Remarkably, the phenotype is caused by a compound heterozygous genotype: one allele containing the novel c.-35A>T mutation and the known c.725T>G mutation (p.Leu242Arg) of FUT1, and the other allele containing the c.-35A>T mutation. From the computerized stimulation analysis, p.Arg242 of the FUT1 variant may be detrimental, destabilizing, and probably damaging to 1,2-L-FucT1, although not altering the 3D structure of the entire enzyme. The c.-35A>T promoter DNA left at the binding interface of both ZID and c-Rel transcription factors may enable regulation of 1,2-L-FucT1 function at gene promoters. We identified the two novel variants, c.-35A>T and c.[-35A>T, 725T>G], in the FUT1 causing the para-Bombay phenotype. This finding may clarify the molecular mechanisms and enhance blood transfusion safety.

Biodegradable Citrate‐Based Polymers Enable 5D Monitoring of Implant Evolution

AbstractBiodegradable tissue engineering scaffolds have garnered increasing interest for their role in providing mechanical support, promoting tissue regeneration, and eliminating the need for removal. However, the in vivo degradation processes remain challenging to track. Here, a novel biodegradable polymer, N‐methyldiethanolamine (MDEA) and Gadolinium(III) diethylenetriamine pentaacetate (Gd‐DTPA) modified biodegradable photoluminescent polymers (BPLPMGd), which combines near‐infrared (NIR) fluorescence and magnetic resonance (MR) dual‐modality imaging are introduced to monitor scaffold degradation in vivo. The chemical structure of BPLPMGd is characterized and its dual‐imaging properties in vitro are evaluated. Subsequently, non‐invasive dual‐modality imaging to track the degradation of implanted BPLPMGd scaffolds is performed in a rat model, comparing these results with histological data. This approach reveals that BPLPMGd enables reliable non‐invasive tracking of the degradation, where NIR fluorescence imaging offers a qualitative and quantitative analysis of scaffold mass loss, total volume and solid content changes, while magnetic resonance imaging (MRI) details structural and morphological changes, allowing for 5D monitoring of implant degradation, including 3D structure, location, mass, volume, and geometry. The combination of these imaging modalities provides a comprehensive view of scaffold degradation, where the synergistic use of both yields results greater than either modality alone, offering unprecedented 5D information of implantable devices. This innovative approach has potential applications in regenerative engineering and beyond.

Discovery of Lacto-N-Biosidases and a Novel N-Acetyllactosaminidase Activity in the CAZy Family GH20: Functional Diversity and Structural Insights.

The glycoside hydrolase family 20 (GH20) predominantly features N-acetylhexosaminidases (EC 3.2.1.52), with only few known lacto-N-biosidases (EC 3.2.1.140; LNBases). LNBases catalyze the degradation of lacto-N-tetraose (LNT), a prominent component of human milk oligosaccharides, thereby supporting a healthy infant gut microbiome development. We investigated GH20 diversity to discover novel enzymes that release disaccharides such as lacto-N-biose (LNB). Our approach combined peptide clustering, sequence analysis, and 3D structure model evaluation to assess active site topologies, focusing on the presence of a subsite -2. Five LNBases were active on pNP-LNB and four showed activity on LNT. One enzyme displayed activity on both pNP-LacNAc and pNP-LNB, establishing the first report of N-acetyllactosaminidase (LacNAcase) activity. Exploration of this enzyme cluster led to the identification of four additional enzymes sharing this dual substrate specificity. Comparing the determined crystal structure of a specific LNBase (TrpyGH20) and the first crystal structure of an enzyme with dual LacNAcase/LNBase activity (TrdeGH20) revealed a highly conserved subsite -1, common to GH20 enzymes, while the -2 subsites varied significantly. TrdeGH20 had a wider subsite -2, accommodating Gal with both β1,4- and β1,3-linkages to the GlcNAc in subsite -1. Biotechnological applications of these enzymes may include structural elucidation of complex carbohydrates and glycoengineering.

IsRNAcirc: 3D structure prediction of circular RNAs based on coarse-grained molecular dynamics simulation.

As an emerging class of RNA molecules, circular RNAs play pivotal roles in various biological processes, thereby determining their three-dimensional (3D) structure is crucial for a deep understanding of their biological significances. Similar to linear RNAs, the development of computational methods for circular RNA 3D structure prediction is challenging, especially considering the inherent flexibility and potentially long length of circular RNAs. Here, we introduce an extension of our previous IsRNA2 model, named IsRNAcirc, to enable circular RNA 3D structure predictions through coarse-grained molecular dynamics simulations. The workflow of IsRNAcirc consists of four main steps, including input preparation, end closure, structure prediction, and model refinement. Our results demonstrate that IsRNAcirc can provide reasonable 3D structure predictions for circular RNAs, which significantly reduce the locally irrational elements contained in the initial input. Moreover, for a validation test set comprising 34 circular RNAs, our IsRNAcirc can generate 3D models with better scores than the template-based 3dRNA method. These findings demonstrate that our IsRNAcirc method is a promising tool to explore the structural details along with intricate interactions of circular RNAs.

Multiple Acquisitions of XopJ2 Effectors in Populations of Xanthomonas perforans.

Type III effectors (T3Es) are major determinants of Xanthomonas virulence and targets for resistance breeding. XopJ2 (synonym AvrBsT) is a highly conserved YopJ-family T3E acquired by X. perforans, the pathogen responsible for bacterial spot disease of tomato. In this study, we characterized a new variant (XopJ2b) of XopJ2, which is predicted to have a similar three-dimensional (3D) structure as the canonical XopJ2 (XopJ2a) despite sharing only 70% sequence identity. XopJ2b carries an acetyltransferase domain and the critical residues required for its activity, and the positions of these residues are predicted to be conserved in the 3D structure of the proteins. We demonstrated that XopJ2b is a functional T3E and triggers a hypersensitive response (HR) when translocated into pepper cells. Like XopJ2a, XopJ2b triggers HR in Arabidopsis that is suppressed by the deacetylase, SOBER1. We found xopJ2b in genome sequences of X. euvesicatoria, X. citri, X. guizotiae, and X. vasicola strains, suggesting widespread horizontal transfer. In X. perforans, xopJ2b was present in strains collected in North America, Africa, Asia, Australia, and Europe, whereas xopJ2a had a narrower geographic distribution. This study expands the Xanthomonas T3E repertoire, demonstrates functional conservation in T3E evolution, and further supports the importance of XopJ2 in X. perforans fitness on tomato. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

3D-printable Kresling-embedded honeycomb metamaterials with optimized energy absorption capability

Abstract Kresling origami structure has attracted significant interest for achieving extraordinary mechanical properties. In this study, we proposed a new strategy to develop 3D-printable Kresling-embedded honeycombs (KEHs) based mechanical metamaterials and achieve optimized mechanical energy absorption capability. By exploiting the twisted deformation modes and boundary constraints, various KEH reinforced metamaterials were designed, where their deformation behaviors and energy absorption properties were investigated using finite element analysis and quasi-static compression tests. Effects of orientation twisting angle, boundary constraint and crease tilting angle on the deformation behaviors of these KEH reinforced metamaterials were studied to optimize their energy absorption properties. Finally, deformation behaviors and energy absorption properties of KEH reinforced metamaterials incorporated of KEH arrays in both 2D structure and 3D structures were studied. Both experimental and simulation results showed that the proposed KEH reinforced metamaterials achieved much more stable compression behaviors and higher energy absorption capabilities than those of the traditional honeycomb structures. This study provides a novel KEH reinforcement strategy for 3D printed metamaterials with optimized energy absorption capabilities to dramatically expand their practical applications.

Continuous Phase Separation Induced Tough Hydrogel Fibers with Ultrahigh Conductivity for Multidimensional Soft Electronics

AbstractConductive hydrogel fibers exhibit great potential in soft robots, bioelectronics, and human–machine interfaces due to the unique combination of electrical conductivity, high water content, tissue‐like mechanical properties, and 1D structure. Despite significant advances in hydrogel technologies, the typical conductive hydrogel fibers show low conductivity (<10 S cm−1), weak mechanical properties, and water stability, which makes it challenging to satisfy the requirements of practical applications. Here, a facile strategy is proposed to construct hydrogel fibers with ultrahigh conductivity and toughness by exploiting the synergistic effects of freezing‐thawing, salting‐out, and drying‐annealing. The continuous phase separation induced by the combined processes results in hierarchical structures, promoting the formation of interconnected conductive networks and increasing the fiber's crystallinity and crystal domain size. The prepared conductive hydrogel fibers exhibited ultrahigh conductivity (≈958 S cm−1), excellent mechanical properties (strength (≈6.2 MPa), stretchability (>300%), and toughness (≈10 MJ m−2)), high water content (≈75%), outstanding water stability, and fatigue resistance properties. In addition, the processibility of conductive hydrogel yarns and fabrics are demonstrated and their potential application in bioelectronics. Overall, this work presents a preparation strategy for conductive hydrogel fibers, which will facilitate the advancement of soft electronics and may inspire structural construction in other polymers.

High-pressure supercritical CO2-assisted fabrication of two-dimensional LaAlO3 and its room temperature ferromagnetism

Abstract Spintronics applications in two-dimensional (2D) magnetic materials can significantly contribute to the miniaturization and energy efficiency of semiconductor devices. However, current 2D ferromagnetic materials face challenges such as ferromagnetic instability and low Curie temperature (Tc), which limit their broader application. In this study, 2D room-temperature ferromagnetic LaAlO3 was successfully prepared with supercritical carbon dioxide (SC CO2). With the enhanced stress effect of CO2 molecules, the contents of oxygen vacancies OV, aluminium vacancies AlV, and AlO4 in the 2D structure are elevated, and then the ferromagnetic properties of LaAlO3 appeared to be significantly enhanced due to defects and spatial symmetry breaking of the octahedron. Notably, the 2D intrinsic ferromagnetic LaAlO3 exhibited a Tc above 350 K. Therefore, this work supplies a new means for SC CO2 to modulate the microstructure and ferromagnetic properties of 2D LaAlO3, which is conducive to expanding the applications of LaAlO3.

Hybrid Space Calibrated 3D Network of Diffractive Hyperspectral Optical Imaging Sensor

Diffractive multispectral optical imaging plays an essential role in optical sensing, which typically suffers from the image blurring problem caused by the spatially variant point spread function. Here, we propose a novel high-quality and efficient hybrid space calibrated 3D network “HSC3D” for spatially variant diffractive multispectral imaging that utilizes the 3D U-Net structure combined with space calibration modules of magnification and rotation effects to achieve high-accuracy eight-channel multispectral restoration. The algorithm combines the advantages of the space calibrated module and U-Net architecture with 3D convolutional layers to improve the image quality of diffractive multispectral imaging without the requirements of complex equipment modifications and large amounts of data. A diffractive multispectral imaging system is established by designing and manufacturing one diffractive lens and four refractive lenses, whose monochromatic aberration is carefully corrected to improve imaging quality. The mean peak signal-to-noise ratio and mean structural similarity index of the reconstructed multispectral images are improved by 3.33 dB and 0.08, respectively, presenting obviously improved image quality compared with a typical Unrolled Network algorithm. The new algorithm with high space calibrated ability and imaging quality has great application potential in diffraction lens spectroscopy and paves a new method for complex practical diffractive multispectral image sensing.

Mechanical unfolding of RNA molecules using a knowledge-based model.

We revisit a coarse-grained model to study the dynamics of ribonucleic acid (RNA). In our model, each nucleotide is replaced by an interaction center located at the center of mass. The interaction between nucleotides is carried out by a series of effective pair potentials obtained from the statistical analysis of 501 RNA molecules of high molecular weight from the Protein Data Bank. In addition to the Watson-Crick interactions, we also include non-canonical interactions, which provide stability to the three-dimensional (3D) structure of the molecule. The resulting knowledge-based interactions for the nucleotides (KIN) model allow us to perform efficient Brownian dynamics simulations under different conditions. First, we simulate the stretch of a set of hairpins at a loading rate similar to the values employed in unfolding experiments near equilibrium using optical tweezers. Additionally, we explore unfolding a set of pseudoknots under conditions farther from equilibrium, namely, at loading rates higher than the experimental equilibrium values. The results of our simulations are compared with those obtained from experimental measurements and theoretical models intended to estimate transition states and activation energies. Our KIN model is able to reproduce the intermediate states observed during mechanical unfolding experiments. Moreover, the results of the KIN model are in good agreement with the measured data.

V-Shaped Heterostructure Nanocavities Array with CM and EM Coupled Enhancement for Ultra-Sensitive SERS Substrate.

The field of semiconductor surface-enhanced Raman scattering (SERS) substrates has experienced significant advancements, leading to a wide range of applications in several fields. However, the quest for new ultra-sensitive semiconductor SERS materials is still of utmost importance. In this regard, an efficient and novel substrate, F4TCNQ/MoS2 heterostructure is introduced, assisted by V-shaped aluminum anodic oxide (AAO) nanocavities with different depths. Utilizing the efficient charge transfer of organic/inorganic semiconducting heterostructure and the photoconfinement capability of the nanocavity structure of the AAO nanotemplate, excellent stability, fast sensing, enhanced Raman, and photodegradation activities are achieved. Due to its unique 3D structure, the optimized F4TCNQ/MoS2/AAO with 1500 nm depth achieves ultra-high sensitivity detection of 9.0×10-16 M for conventional probe molecules. Furthermore, precise detection of water contaminants is observed for the first time with a V-shaped heterostructure due to combined organic/inorganic features that differ significantly from conventional MoS2 structures or other metal/inorganic or inorganic/inorganic semiconductors. This research presents a novel and versatile strategy for SERS and demonstrates its diverse potential performance in practical applications.

Exome sequencing identifies a novel FBN1 variant in a Chinese family with Marfan syndrome that includes aberrant right subclavian artery

Abstract Background Marfan syndrome (MFS) is an inherited autosomal dominant disorder that affects connective tissue with an incidence of about 1 in 5,000 to 10,000 people. Ninety percent of MFS is caused by mutations in the fibrillin-1 (fbn1) gene. We recruited a family with MFS phenotype in South China and identified a novel variant in fbn1 by whole exome sequencing (WES). The zebrafish share high genetic similarity to humans. This study aimed to generate this novel mutation in the fbn1 gene of zebrafish using the CRISPR/Cas9 technology and researched whether this variant is pathogenic. Methods A three-generation consanguineous family was recruited in this study, which was visited and interviewed for the clinical data and family history. Peripheral blood samples were collected from family members for DNA isolation and WES. The 3D structure of the protein was predicted by Alphafold. CRISPR/Cas9 was applied to generate an fbn1 nonsense mutation (fbn1+/−) in zebrafish. Morphological abnormalities were assessed in F2 fbn1+/− zebrafish by comparing with the wild-type (WT) controls at different development stages. Results Our study recruited a family with MFS phenotype in South China and identified a novel variant [NM_000138.5; c.7764C>G: p.(Y2588*)] in fbn1. Through Sanger sequencing, we found that family members Ⅲ-1 (son) and Ⅲ-2 (daughter) had the fbn1 variant segregated with MFS in the family. The p.Y2588* mutation resulted in the absence of 283 amino acids, thereby leading to the deficiency of 17 β-folded structures, 4 α-helix structures, and various loop structures. Compared with WT zebrafish, F2 fbn1+/− zebrafish exhibited aortic arches bleeding, abnormal angiogenesis, decreased cardiac volume, cardiac function defects, curly tail, and cartilage malformations. Conclusion A novel variant [NM_000138.5; c.7764C>G: p.(Y2588*)] was identified in fbn1 gene, which has not been reported in previous literature. The variant caused loss of function through premature protein truncation or nonsense-mediated mRNA decay. In zebrafish, we identified functional evidence of the detected nonsense mutation, which aligns with the clinical significance, suggesting a pathogenic variant of fbn1. Zebrafish as a novel, efficient tool to allow for the quick classification of unknown variants in fbn1 and improve the clinical diagnosis and treatment of MFS. It has brought the implementation of personal and precision medicine in the clinic within reach and made it possible to find patient-specific treatments.Marfanoid symptoms of the patient.Phenotypic Spectrum of fbn1+/− Zebrafish

Mechanisms of inducing cancer extracellular vesicles secretion and transfer for cancer malignancy

Assistant Professor Nao Nishida-Aoki, Waseda Institute for Advanced Study (WIAS), is investigating the role of extracellular vesicles (EVs) in physiological tumour tissue environments in order to improve cancer treatments. Communication tools like EVs are used by cancer cells to educate the surrounding cells and make them supportive to cancer cell growth and metastasis. Nishida-Aoki’s research focuses on understanding EV-mediated communication within the complex tissue environment of patient tumours, where they are secreted in densely packed cells and non-cellular components forming a 3D structure. She has developed ex vivo brain and lung metastatic models using organotypic tissue slice culture (TSC). This enables her to observe EV transfer within tissue architecture in real time, identify target cells affected by cancer EVs and determine how they are distributed. Nishida-Aoki is also exploring the mechanisms of inducible secretion of cancer extracellular vesicles upon cell recognition and contribution to cancer malignancy. Her research is investigating the molecular mechanics by which cancer cells regulate and the extent of interactions with neighbouring cells using EVs at metastatic initiation. She has hypothesised that cancer cells should increase communication frequency using EVs when they meet other types of cells in a new environment to benefit themselves. By elucidating the molecular basis of cancer-normal cell interactions mediated by EVs, Nishida-Aoki aims to develop novel cancer therapeutic strategies.

Advancing cardiac 3D organoid-on-chip: integrating iPSC-derived MELAS syndrome models to study electromechanical synchronicity and cardiac memory using contactless technology

Abstract Background Mitochondrial DNA mutations affect 1:5000 newborns, posing life-threatening risks, especially to high-energy organs such as the brain, heart, and muscles. Mortality rates can reach 71% when cardiomyopathy is present. MELAS syndrome (Mitochondrial Encephalopathy with Lactic Acidosis and Stroke-like Episodes), primarily affecting protein synthesis (MTTL1 gene, tRNALeu(UUR)), disrupts the electron transport chain complex I and is associated with cardiac conduction defects and hypertrophy. Manifesting symptoms typically appear between ages 3 and adulthood, presenting a complex clinical scenario. Human induced pluripotent stem cells (hiPSCs) offer a unique platform for studying cardiac manifestations of this disease when differentiated into cardiomyocytes. Purpose We adopted a highly adaptable in-vitro model, organized in 3D cardioids, to explore potential impairments in electromechanical coupling of cells. Methods We utilized hiPSCs carrying the MELAS mutation and their respective isogenic controls differentiated into cardiomyocytes, cultured in 3D cardioids. Electromechanical coupling was assessed using laser-poration technology combined with video kinematics evaluation under various stimulation patterns. Results Morphological analysis revealed a statistically significant reduction in the diameter of MELAS cardioids compared to the isogenic 3D structures (368±17.15 μm vs. 527±36.76 μm). In a spontaneous regimen, MELAS samples exhibited higher beating frequency (0.88±0.28 Hz vs. 0.17±0.05 Hz) and contractile speed (99.35±39.11 μm/s vs. 33.46±5.57 μm/s). Additionally, the response to different electric field stimulation protocols varied significantly, with the isogenic cell line showing better adaptation to sinusoidal stimuli than the pathological one, characterized by rectangular monophasic stimuli. Laser-poration technology, combined with MEA chips and video kinematic evaluation, revealed modulation of electromechanical coupling, affecting action potential upstroke and duration, reducing contraction velocity, and altering electromechanical delay. Furthermore, MELAS hiPSCs and primary cardiomyocytes exhibited reduced protein levels of various OXPHOS subunits (SDHB, ATPB, and COX IV) compared to isogenic controls. The mutated cell line also showed increased ROS production in non-stimulated samples compared to stimulated ones (1.3-fold vs. 1.2-fold), with this difference magnifying after 24 hours from stimulation (1.8-fold vs. 1.3-fold). Conclusions This study characterizes MELAS hiPSCs differentiated into cardiac organoids, offering insight into the cardiac aspect of this complex syndrome. Our findings deepen understanding of the syndrome's cardiac impact and response to extracellular stimuli, potentially benefiting affected tissues. This research defines critical physiological and kinematic parameters for MELAS, paving the way for future investigations.

A Facile Method in Fabricating Flexible Conductive Composites with Large-Size Segregated Structures for Electromagnetic Interference Shielding.

To resist the plastic deformation of polymer particles during hot press molding, high molecular weights, and moduli are required for composites with segregated structures, thus the prepared composites exhibit poor flexibility. Also, larger particle sizes can bring lower percolation thresholds while the ensuing greater deformation destroys the conductive network. Moreover, segregated composites still face preparation complexities. Herein, a facile method for developing flexible composites with large-size segregated structures is proposed. First, silver-coated polydopamine-modified reduced graphene oxide (Ag@PrGO), as conductive fillers, is prepared by electroless plating. Next, polydimethylsiloxane (PDMS)-coated polyolefin elastomer (POE) beads are put into a bag containing the fillers. After a simple shaking, the fillers are adhered to the POE surface as the cohesive property of cured PDMS. Finally, flexible composites with large-size segregated structures are obtained via hot pressing. Benefiting from the 2D structure of the Ag@PrGO and the ability to slip, the conductive networks possess adaptable deformability. The prepared composites exhibit excellent electrical conductivity (203.55 Scm-1) at filler volume fractions of 3.4 vol%. The EMI shielding effectiveness can reach 70dB in the X-band at a thickness of 1.9mm and remains stable after bending and rubbing damage. This work paves the way for constructing large-size segregated structures.

Design and development of dual targeting CAR protein for the development of CAR T-cell therapy against KRAS mutated pancreatic ductal adenocarcinoma using computational approaches

Mutant KRAS promotes the proliferation, metastasis, and aggressiveness of various cancers including pancreatic ductal adenocarcinoma (PDAC), non-small cell lung cancer (NSCLC), and colorectal adenocarcinoma (CRC) respectively. Mutant KRAS therapeutics are limited, while Sotorasib and Adagrasib were the only FDA-approved drugs for the treatment of KRASG12C mutated NSCLC. Chimeric antigen receptor (CAR) T-cell therapy has been emerged as an effective strategy against hematological malignancies and being extended towards solid cancers including PDAC. mesothelin (MSLN) and Carcinoembryonic Antigen (CEA) were reported to be highly overexpressed in KRAS-mutated PDAC. Meanwhile, in clinical trials, several CAR T-cell therapy studies are mainly focused towards these two cancer antigens in PDAC, however, the dual targeting of these two neoantigens is not reported. In the present study, we have designed and developed a novel dual-targeting CAR protein by employing various bioinformatics approaches such as functional analysis (antigenicity, allergenicity, antigen binding sites & signalling cascades), qualitative analysis (physicochemical, prediction, refinement & validation of 2D and 3D structures), molecular docking, and in silico cloning. Our results revealed that the designed CAR protein specifically binds with both MSLN & CEA with significant binding affinities, and was predicted to be stable & non-allergenic. Additionally, the protein–protein interaction network reveals the T-cell mediated antitumor responses of each domain in the designed CAR. Conclusively, we have designed and developed a dual targeting (MSLN & CEA) CAR protein towards KRAS-mutated PDAC using computational approaches. Alongside, we further recommend to engineer this designed CAR in T-cells and evaluating their therapeutic efficiency in in vitro and in vivo studies in the near future.