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At least 1,081 records · Page 60

BIPSPI+: Mining Type-Specific Datasets of Protein Complexes to Improve Protein Binding Site Prediction

Computational approaches for predicting protein-protein interfaces are extremely useful for understanding and modelling the quaternary structure of protein assemblies. In particular, partner-specific binding site prediction methods allow delineating the specific residues that compose the interface of protein complexes. In recent years, new machine learning and other algorithmic approaches have been proposed to solve this problem. However, little effort has been made in finding better training datasets to improve the performance of these methods. With the aim of vindicating the importance of the training set compilation procedure, in this work we present BIPSPI+, a new version of our original server trained on carefully curated datasets that outperforms our original predictor. We show how prediction performance can be improved by selecting specific datasets that better describe particular types of protein interactions and interfaces (e.g. homo/hetero). In addition, our upgraded web server offers a new set of functionalities such as the sequence-structure prediction mode, hetero- or homo-complex specialization and the guided docking tool that allows to compute 3D quaternary structure poses using the predicted interfaces. BIPSPI+ is freely available at https://bipspi.cnb.csic.es.

59 BASIC BIOLOGICAL SCIENCES↗

Asymmetric loading of TnsE regulates Tn7 targeting of DNA replication structures

Abstract Tn7 transposable elements are known for their sophisticated target-site selection mechanisms. For the prototypical Tn7 element, dedicated transposon-encoded proteins direct insertions to either a conserved site in the chromosome or replicating DNA structures in conjugal plasmids, ensuring the vertical and horizontal spread of the element. While the pathway targeting the attTn7 site in the bacterial chromosome has been extensively studied, the pathway targeting DNA replication structures remains poorly understood. We have used an integrative structural biology approach to elucidate how the Tn7-encoded protein TnsE recognizes replication sites. Using native mass spectrometry, we found that TnsE forms 1:1 and 2:1 (TnsE:DNA) complexes on 3′-recessed DNA, with gain-of-function TnsE variants favoring the formation of 2:1 complexes. Structural characterization confirms that two TnsE molecules bind to DNA with the C-terminal domain of the protein recognizing duplex DNA, leaving the N-terminal domain to impose DNA substrate specificity and recruit the core transposition machinery. Collectively, our work is consistent with a model where TnsE-mediated target-site selection relies on the formation of an asymmetric TnsE:DNA complex to recruit the Tn7 transposase to DNA replication structures.

Biochemistry & Molecular Biology↗

Genome-wide identification of novel flagellar motility genes in Pseudomonas syringae pv. tomato DC3000

Pseudomonas syringaepv.tomatoDC3000 (PstDC3000) is a plant pathogenic bacterium that possesses complicated motility regulation pathways including a typical chemotaxis system. A significant portion of our understanding about the genes functioning inPstDC3000 motility is based on comparison to other bacteria. This leaves uncertainty about whether gene functions are conserved, especially since specific regulatory modules can have opposite functions in sets ofPseudomonas. In this study, we used a competitive selection to enrich for mutants with altered swimming motility and used random barcode transposon-site sequencing (RB-TnSeq) to identify genes with significant roles in swimming motility. Besides many of the known or predicted chemotaxis and motility genes, our method identified PSPTO_0406 (dipA), PSPTO_1042 (chrR) and PSPTO_4229 (hypothetical protein) as novel motility regulators. PSPTO_0406 is a homolog ofdipA, a known cyclic di-GMP degrading enzyme inP. aeruginosa. PSPTO_1042 is part of an extracytoplasmic sensing system that controls gene expression in response to reactive oxygen species, suggesting that PSPTO_1042 may function as part of a mechanism that enablesPstDC3000 to alter motility when encountering oxidative stressors. PSPTO_4229 encodes a protein containing an HD-related output domain (HDOD), but with no previously identified functions. We found that deletion and overexpression of PSPTO_4229 both reduce swimming motility, suggesting that its function is sensitive to expression level. We used the overexpression phenotype to screen for nonsense and missense mutants of PSPTO_4229 that no longer reduce swimming motility and found a pair of conserved arginine residues that are necessary for motility suppression. Together these results provide a global perspective on regulatory and structural genes controlling flagellar motility inPstDC3000.

Microbiology↗

Crystal structures and functional analysis of the ZnF5-WWE1-WWE2 region of PARP13/ZAP define a distinctive mode of engaging poly(ADP-ribose)

PARP13/ZAP (zinc-finger antiviral protein) acts against multiple viruses by promoting degradation of viral mRNA. PARP13 has four N-terminal zinc (Zn) fingers that bind CG-rich nucleotide sequences, a C-terminal ADP ribosyltransferase fold, and a central region with a fifth Zn finger and tandem WWE domains. The central PARP13 region, ZnF5-WWE1-WWE2, is implicated in binding poly(ADP-ribose); however, there are limited insights into its structure and function. We present crystal structures of ZnF5-WWE1-WWE2 from mouse PARP13 in complex with ADP-ribose and in complex with ATP. The crystal structures and binding studies demonstrate that WWE2 interacts with ADP-ribose and ATP, whereas WWE1 does not have a functional binding site. Binding studies with poly(ADP-ribose) ligands indicate that WWE2 serves as an anchor for preferential binding to the terminal end of poly(ADP-ribose) chains. The composite ZnF5-WWE1-WWE2 structure forms an extended surface to engage ADP-ribose chains, representing a distinctive mode of recognition that provides a framework for investigating the impact of poly(ADP-ribose) on PARP13 function.

59 BASIC BIOLOGICAL SCIENCES↗

Data Science-Driven Analysis of Substrate-Permissive Diketopiperazine Reverse Prenyltransferase NotF: Applications in Protein Engineering and Cascade Biocatalytic Synthesis of (-)-Eurotiumin A

Prenyltransfer is an early-stage carbon-hydrogen bond (C-H) functionalization prevalent in the biosynthesis of a diverse array of biologically active bacterial, fungal, plant, and metazoan diketopiperazine (DKP) alkaloids. Toward the development of a unified strategy for biocatalytic construction of prenylated DKP indole alkaloids, we sought to identify and characterize a substrate-permissive C2 reverse prenyltransferase (PT). As the first tailoring event within the biosynthesis of cytotoxic notoamide metabolites, PT NotF catalyzes C2 reverse prenyltransfer of brevianamide F. Solving a crystal structure of NotF (in complex with native substrate and prenyl donor mimic dimethylallyl S-thiolodiphosphate (DMSPP)) revealed a large, solvent-exposed active site, intimating NotF may possess a significantly broad substrate scope. To assess the substrate selectivity of NotF, we synthesized a panel of 30 sterically and electronically differentiated tryptophanyl DKPs, the majority of which were selectively prenylated by NotF in synthetically useful conversions (2 to > 99%). Quantitative representation of this substrate library and development of a descriptive statistical model provided insight into the molecular origins of NotF's substrate promiscuity. This approach enabled the identification of key substrate descriptors (electrophilicity, size, and flexibility) that govern the rate of NotF-catalyzed prenyltransfer, and the development of an "induced fit docking (IFD)-guided" engineering strategy for improved turnover of our largest substrates. We further demonstrated the utility of NotF in tandem with oxidative cyclization using flavin monooxygenase, BvnB. This one-pot, in vitro biocatalytic cascade enabled the first chemoenzymatic synthesis of the marine fungal natural product, (-)-eurotiumin A, in three steps and 60% overall yield.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Experimental evolution reveals nitrate tolerance mechanisms in Desulfovibrio vulgaris

Elevated nitrate in the environment inhibits sulfate reduction by important microorganisms of sulfate-reducing bacteria (SRB). Several SRB may respire nitrate to survive under elevated nitrate, but how SRB that lack nitrate reductase survive to elevated nitrate remains elusive. To understand nitrate adaptation mechanisms, we evolved 12 populations of a model SRB (i.e., Desulfovibrio vulgaris Hildenborough, DvH) under elevated NaNO 3 for 1000 generations, analyzed growth and acquired mutations, and linked their genotypes with phenotypes. Nitrate-evolved (EN) populations significantly (p < 0.05) increased nitrate tolerance, and whole-genome resequencing identified 119 new mutations in 44 genes of 12 EN populations, among which six functional gene groups were discovered with high mutation frequencies at the population level. In this work, we observed a high frequency of nonsense or frameshift mutations in nitrosative stress response genes (NSR: DVU2543, DVU2547, and DVU2548), nitrogen regulatory protein C family genes (NRC: DVU2394-2396, DVU2402, and DVU2405), and nitrate cluster (DVU0246-0249 and DVU0251). Mutagenesis analysis confirmed that loss-of-functions of NRC and NSR increased nitrate tolerance. Also, functional gene groups involved in fatty acid synthesis, iron regulation, and two-component system (LytR/LytS) known to be responsive to multiple stresses, had a high frequency of missense mutations. Additionally, mutations in those gene groups could increase nitrate tolerance through regulating energy metabolism, barring entry of nitrate into cells, altering cell membrane characteristics, or conferring growth advantages at the stationary phase. This study advances our understanding of nitrate tolerance mechanisms and has important implications for linking genotypes with phenotypes in DvH.

59 BASIC BIOLOGICAL SCIENCES↗

Longitudinal proteomics analysis in the immediate microenvironment of islet allografts during progression of rejection

The applicability and benefits of pancreatic islet transplantation are limited due to various issues including the need to avoid immune-mediated rejection. In the present study, we used our experimental platform of allogeneic islet transplant in the anterior chamber of the eye (ACE-platform) to longitudinally monitor the progress of rejection in mice and obtain aqueous humor samples representative of the microenvironment of the graft for accurately-timed proteomic analyses. LC-MS/MS-based proteomics performed on such mass-limited samples (~5 μL) identified a total of 1296 proteins. Various analyses revealed distinct protein patterns associated with the mounting of the inflammatory and immune responses and their evolution with the progression of the rejection. Pathway analyses indicated predominant changes in cytotoxic functions, cell movement, and innate and adaptive immune responses. Furthermore, network prediction analyses revealed transition from humoral to cellular immune response and exacerbation of pro-inflammatory signaling. One of the proteins identified by this localized proteomics as a candidate biomarker of islet rejection, Cystatin 3, was further validated by ELISA in the aqueous humor. This study provides (1) experimental evidence demonstrating the feasibility of longitudinal localized proteomics using small aqueous humor samples and (2) proof-of-concept for the discovery of biomarkers of impending immune attack from the immediate local microenvironment of ACE-transplanted islets.

59 BASIC BIOLOGICAL SCIENCES↗

The Mycobacterium tuberculosis protein O -phosphorylation landscape

Bacterial phosphosignaling has long been synonymous with the histidine kinases of the two component systems (TCSs), but many bacteria, including Mycobacterium tuberculosis (Mtb), also code for Ser/Thr protein kinases (STPKs). STPKs are the main phosphosignaling enzymes in eukaryotes, but the full extent of phosphorylation on protein Ser/Thr and Tyr (O-phosphorylation) in bacteria is unclear. Here, we explored the global signaling capacity of the STPKs in Mtb. We generated a panel of STPK loss- and gain-of-function strains and measured the resulting O-phosphorylation and transcriptional changes. This deep phosphoproteome shows that O-phosphorylation in Mtb is a vastly underexplored protein modification that affects >70% of the proteome. The substrate-kinase interactions of STPKs show an extensive interface with the transcriptional machinery, resulting in STPK regulation of gene expression of over 30% of Mtb genes. Finally, Mtb O-phosphorylation gives rise to an expansive, distributed, and cooperative network of a complexity that has previously only been associated with eukaryotic phosphosignaling networks.

59 BASIC BIOLOGICAL SCIENCES↗

Hierarchical Nanomaterials Assembled from Peptoids and Other Sequence-Defined Synthetic Polymers

In nature, the self-assembly of sequence-specific biopolymers into hierarchical structures plays an essential role in the construction of functional biomaterials. To develop synthetic materials that can mimic and surpass the function of these natural counterparts, various sequence-defined bio- and biomimetic polymers have been developed and exploited as building blocks for hierarchical self-assembly. This review summarizes the recent advances in the molecular self-assembly of hierarchical nanomaterials based on peptoids (or poly-N-substituted glycines) and other sequence-defined synthetic polymers. Modern techniques to monitor the assembly mechanisms and characterize the physicochemical properties of these self-assembly systems are highlighted. In addition, discussions about their potential applications in biomedical sciences and renewable energy are also included. Furthermore, this review aims to highlight essential features of sequence-defined synthetic polymers (e.g., high stability and protein-like high-information content) and how these unique features enable the construction of robust biomimetic functional materials with high programmability and predictability, with an emphasis on peptoids and their self-assembled nanomaterials.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Structural analysis of the PTEN:P-Rex2 signaling complex reveals how cancer-associated mutations coordinate to hyperactivate Rac1

The dual-specificity phosphatase PTEN functions as a tumor suppressor by hydrolyzing PI(3,4,5)P 3 to PI(4,5)P 2 to inhibit PI3K-AKT signaling and cellular proliferation. P-Rex2 is a guanine nucleotide exchange factor for Rho GTPases and can be activated by Gβγ subunits downstream of G protein–coupled receptor signaling and by PI(3,4,5)P 3 downstream of receptor tyrosine kinases. The PTEN:P-Rex2 complex is a commonly mutated signaling node in metastatic cancer. Assembly of the PTEN:P-Rex2 complex inhibits the activity of both proteins, and its dysregulation can drive PI3K-AKT signaling and cellular proliferation. Here, using cross-linking mass spectrometry and functional studies, we gained mechanistic insights into PTEN:P-Rex2 complex assembly and coinhibition. We found that PTEN was anchored to P-Rex2 by interactions between the PDZ-interacting motif in the PTEN C-terminal tail and the second PDZ domain of P-Rex2. This interaction bridged PTEN across the P-Rex2 surface, preventing PI(3,4,5)P 3 hydrolysis. Conversely, PTEN both allosterically promoted an autoinhibited conformation of P-Rex2 and blocked its binding to Gβγ. In addition, we observed that the PTEN-deactivating mutations and P-Rex2 truncations combined to drive Rac1 activation to a greater extent than did either single variant alone. These insights enabled us to propose a class of gain-of-function, cancer-associated mutations within the PTEN:P-Rex2 interface that uncouple PTEN from the inhibition of Rac1 signaling.

D’Andrea, Laura↗

Neurons die with heightened but functional macro- and chaperone mediated autophagy upon increased amyloid-ß induced toxicity with region-specific protection in prolonged intermittent fasting

Highlights: • Macroautophagy (MA) is strongly engaged upon APP overexpression and remains high over time. • APP colocalization with macroautophagy components remains increased until late, promoting continuous recruitment to the autophagy machinery. • Robust chaperone-mediated autophagy (CMA) engagement is observed in late APP overexpression. • Macroautophagic flux during APP overexpression is preserved, even at late time points, suggesting functional autophagy in the face of apoptosis onset. • Macroautophagy impairment increases markers of amyloidogenesis and further exacerbates the onset of apoptotic cell death. Alzheimer's disease (AD) is a devastating neurodegenerative condition with significant socio-economic impact that is exacerbated by the rapid increase in population aging, particularly impacting already burdened health care systems of poorly resourced countries. Accumulation of the amyloid-β (Aβ) peptide, generated through amyloid precursor protein (APP) processing, manifesting in senile plaques, is a well-established neuropathological feature. Aβ plays a key role in driving synaptic dysfunction, neuronal cell loss, glial cell activation and oxidative stress associated with the pathogenesis of AD. Thus, the enhanced clearance of Aβ peptide though modulation of the mechanisms that regulate intracellular Aβ metabolism and clearance during AD progression have received major attention. Autophagy, a lysosome-based major proteolytic pathway, plays a crucial role in intracellular protein quality control and has been shown to contribute to the clearance of Aβ peptide. However, to what extent autophagy activity remains upregulated and functional in the process of increasing Aβ neurotoxicity is largely unclear. Here, we investigated the extent of neuronal toxicity in vitro by characterising autophagic flux, the expression profile of key amyloidogenic proteins, and proteins associated with prominent subtypes of the autophagy pathway to dissect the interplay between the engagement of proteolytic pathways and cell death onset in the context of APP overexpression. Moreover, we assessed the neuroprotective effects of a caloric restriction regime in vivo on the modulation of autophagy in specific brain regions. Our results reveal that autophagy is upregulated in the presence of high levels of APP and Aβ and remains heightened and functional despite concomitant apoptosis induction, suggestive of a mismatch between autophagy cargo generation and clearance capacity. These findings were confirmed when implementing a prolonged intermittent fasting (IF) intervention in a model of paraquat-induced neuronal toxicity, where markers of autophagic activity were increased, while apoptosis onset and lipid peroxidation were robustly decreased in brain regions associated with neurodegeneration. This work highlights that especially caloric restriction mimetics and controlled prolonged IF may indeed be a highly promising therapeutic strategy at all stages of AD-associated pathology progression, for a cell-inherent and cell specific augmentation of Aβ clearance through the powerful engagement of autophagy and thereby robustly contributing to neuronal protection.

60 APPLIED LIFE SCIENCES↗

Computation of condition-dependent proteome allocation reveals variability in the macro and micro nutrient requirements for growth

Sustaining a robust metabolic network requires a balanced and fully functioning proteome. In addition to amino acids, many enzymes require cofactors (coenzymes and engrafted prosthetic groups) to function properly. Extensively validated resource allocation models, such as genome-scale models of metabolism and gene expression (ME-models), have the ability to compute an optimal proteome composition underlying a metabolic phenotype, including the provision of all required cofactors. Here we apply the ME-model for Escherichia coli K-12 MG1655 to computationally examine how environmental conditions change the proteome and its accompanying cofactor usage. We found that: (1) The cofactor requirements computed by the ME-model mostly agree with the standard biomass objective function used in models of metabolism alone (M-models); (2) ME-model computations reveal non-intuitive variability in cofactor use under different growth conditions; (3) An analysis of ME-model predicted protein use in aerobic and anaerobic conditions suggests an enrichment in the use of peroxyl scavenging acids in the proteins used to sustain aerobic growth; (4) The ME-model could describe how limitation in key protein components affect the metabolic state of E . coli . Genome-scale models have thus reached a level of sophistication where they reveal intricate properties of functional proteomes and how they support different E . coli lifestyles.

59 BASIC BIOLOGICAL SCIENCES↗

Closing the Gap Between Modeling and Experiments in the Self-assembly of Biomolecules at Interfaces and in Solution

Molecular self-assembly is a powerful tool in materials design, wherein non-covalent interactions like electrostatic, hydrophobic, hydrogen bonding, and van der Waals can be exploited to produce supramolecular nanostructures that are functional and highly tunable. Biomolecules are attractive building blocks, as they are biocompatible, biodegradable and adopt a wide array of higher order structures. Moreover, naturally occurring protein systems display a manifold of structures and interactions that can be replicated in synthetic biomolecules. In this perspective, we highlight advances in multiscale simulation techniques across broad spatiotemporal scales that can aid in characterizing self-assembly of hybrid and hierarchical bionanomaterial systems, with an emphasis on physics-based simulation approaches currently employed to study biomolecules at mineral interfaces. The power of these approaches is highlighted across a few recent areas where molecular simulations have advanced our understanding of self-assembly spanning peptides to protein self-assembly. Looking forward, we discuss how in the near future emerging methods in statistical and machine learning will advance this research field in all areas from expanding the capabilities of physics-based simulation methods to enabling new analyses of high throughput experiments. These advances will pave the way for understanding the molecular recognition patterns in systems that are dictated by self-assembly - biomineralizing peptides, hierarchical peptoids, and large protein assemblies, and will aid in the development of a new synthesis science for achieving precise molecular control in materials design

Sampath, Janani↗

Design of Multicomponent Peptide Fibrils with Ordered and Programmable Compositional Patterns

Advanced applications of biomacromolecular assemblies require a stringent degree of control over molecular arrangement, which is a challenge to current synthetic methods. Here we used a neighbor-controlled patterning strategy to build multicomponent peptide fibrils with an unprecedented capacity to manipulate local composition and peptide positions. Eight peptides were designed to have regulable nearest neighbors upon co-assembly, which, by simulation, afforded 412 different patterns within fibrils, with varied compositions and/or peptide positions. The fibrils with six prescribed patterns were experimentally constructed with high accuracy. The controlled patterning also applies to functionalities appended to the peptides, as exemplified by arranging carbohydrate ligands at nanoscale precision for protein recognition. Importantly, this study offers a route to molecular editing of inner structures of peptide assemblies, prefiguring the uniqueness and richness of patterning-based material design.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Identification and Clinical Evaluation of Potential Biomarkers for Breast Cancer Resistance Protein ( BCRP / ABCG2 )

Clinical inhibition and genetic variation of the Breast Cancer Resistance Protein (BCRP/ABCG2) efflux transporter can significantly influence drug exposure, highlighting the need for reliable BCRP functional biomarkers. This study aimed to identify and evaluate biomarkers predictive of BCRP function in humans. A comprehensive analysis of metabolomic genome‐wide association studies (mGWAS) was conducted to discover potential BCRP biomarkers, followed by evaluation inin vitrotransporter assays and a clinical drug–drug interaction (DDI) study. Across multiple mGWAS datasets, plasma concentrations of three herbicide derivatives—4‐hydroxychlorothalonil (4HC), 3‐bromo‐5‐chloro‐2,6‐dihydroxybenzoic acid (BCDBA), and 3,5‐dichloro‐2,6‐dihydroxybenzoic acid (DCDBA)—were significantly elevated (P < 5E‐8) in individuals carrying reduced functionABCG2polymorphisms. These compounds were confirmed as novel BCRP substrates via transporter uptake assays and selected for clinical evaluation alongside riboflavin, a known BCRP substrate and potential BCRP biomarker. In a DDI study with 11 healthy subjects, eltrombopag, a BCRP inhibitor, increased rosuvastatin concentrations by approximately twofold (P = 0.002). No significant changes in the plasma concentrations of organic anion transporting polypeptide 1B (OATP1B) biomarkers (CP‐I and CP‐III) or potential BCRP biomarkers (4HC, BCDBA, DCDBA, or riboflavin) were observed. Notably, two subjects were heterozygous carriers for theABCG2p.Q141K variant and exhibited significantly higher baseline concentrations of 4HC (P = 0.004) and BCDBA (P = 0.0003), consistent with reduced BCRP function. These findings suggest that 4HC and BCDBA are promising biomarkers for baseline BCRP function in specific populations, such as those harboring reduced function genetic polymorphisms, but do not appear suitable for detecting acute BCRP inhibition.

Pharmacology & Pharmacy↗

Towards an Understanding of Oxidative Damage in an a-L-Arabinofuranosidase of Trichoderma reesei : a Molecular Dynamics Approach

Trichoderma reesei is a ‘workhorse’ fungus that produces glycosyl hydrolases (e.g., cellulases) at high titers for use in industrial bioprocessing. In this study, we focused on two side-chain hemicellulases, a-L-arabinofuranosidases and a-D-galactosidases, which are important for the treatment of lignocellulosic biomass, but susceptible to oxidative damage that can occur during industrial processing. The molecular details that render these enzymes inactive have not yet been identified. To approach this issue, we used proteomics to identify amino acid residues that were oxidized after a relevant oxidative treatment (Fenton reaction). These oxidative modifications were included in the 3D-protein structures, and using molecular dynamics simulations, we then studied the behaviors of non-modified and oxidized enzymes. These simulations showed significant alterations of the conformational stability of proteins when oxidized, as evidenced by changes in root mean square deviation (RMSD) and principal component analyses (PCA) trajectories. Likewise, enzyme-ligand interactions such as hydrogen bonds were greatly reduced in quantity and quality in the oxidized proteins. Finally, free energy landscape plots showed that there was a more rugged energy surface in the oxidized proteins, implying a less favorable reaction pathway. These results reveal the basis for loss of function in these carbohydrate active enzymes (CAZYs) in T. reesei.

60 APPLIED LIFE SCIENCES↗

Potent transmission-blocking monoclonal antibodies from naturally exposed individuals target a conserved epitope on Plasmodium falciparum Pfs230

Pfs230 is essential for Plasmodium falciparum transmission to mosquitoes and is the protein targeted by the most advanced malaria-transmission-blocking vaccine candidate. Prior understanding of functional epitopes on Pfs230 is based on two monoclonal antibodies (mAbs) with moderate transmission-reducing activity (TRA), elicited from subunit immunization. Here, we screened the B cell repertoire of two naturally exposed individuals possessing serum TRA and identified five potent mAbs from sixteen Pfs230 domain-1-specific mAbs. Structures of three potent and three low-activity antibodies bound to Pfs230 domain 1 revealed four distinct epitopes. Highly potent mAbs from natural infection recognized a common conformational epitope that is highly conserved across P. falciparum field isolates, while antibodies with negligible TRA derived from natural infection or immunization recognized three distinct sites. Our study provides molecular blueprints describing P. falciparum TRA, informed by contrasting potent and non-functional epitopes elicited by natural exposure and vaccination.

60 APPLIED LIFE SCIENCES↗

Neuromuscular Junction Model Optimized for Electrical Platforms

Neuromuscular junctions (NMJs), specialized synapses between motor neurons and muscle fibers, are essential for muscle activity. A simple and reproducible cell-based in vitro NMJ platform is needed to test the impact of chemicals on the neuron-muscle communication. Our platform utilizes genetically modified neurons and muscle cells, optimized culture conditions, and commercially available multielectrode array system for recording action potentials. Neuronal cells (NSC34) were optogenetically modified with channelrhodopsin chimera to allow for simultaneous, light-mediated, millisecond-precise activation of neuronal population. This signal is propagated through functional synapses to the muscle fibers. Muscle cells (C2C12) were modified by incorporating gap junction protein (Connexin-43) to improve intracellular communication without affecting muscle differentiation. This communication between muscle fibers resulted in better signal propagation and signal strength. Optimized culture medium facilitated the growth and differentiation of both cell types together. Our system was validated using vecuronium, a muscle relaxant, which abolished the muscle response. This in vitro model provides a unique tool for establishing a NMJ platform that is easy to record and analyze. Potential applications include nondestructive long-term screening of drugs affecting the NMJ.

59 BASIC BIOLOGICAL SCIENCES↗