International Journal of Biological Macromolecules

Nucleobindin 1 (NUCB1) is a multifunctional conserved protein located in Golgi luminal, nucleus, extracellular and cytosolic pools. NUCB1 is multidomain protein comprised of a signal peptide, a DNA-binding domain, a leucine zipper and Ca2+ -binding domain. The multiple domains and localization of NUCB1 potentiates its interactions with various partners, such as DNA, Gi3 protein, cyclooxygenase 2, LRP10 and RNA suggests its importance in the regulation of many cellular events. We revealed that NUCB1 contains three RNA-binding regions and able to interact with two RNA fragments. It was suggested possible variants of the participation of NUCB1 in the interaction of the two partially complementary RNAs. The RNA-binding properties of the NUCB1 were also confirmed in vivo experiments.

OXA-232, an OXA-48 like carbapenemase stands amongst newly identified beta-lactamases that causes of the extensive of beta-lactam resistance. While active-site residues are well characterised, the contributions of conserved non-active-site residues in exerting enzymatic activity remain unexplored, limiting our understanding about the roles of these residues in the overall OXA-232 function. To address these gaps, the conserved residues S118, V120, L158, and D159 of OXA-232 positioned adjacent to the active-site motifs and within the omega-like loop were substituted with alanine. Substitutions of S118A and D159A rendered the expressing cells susceptible to penicillins, cephalosporins, and carbapenems, whereas the cells harbouring OXA-232V120A and OXA-232L158A proteins exhibited substrate-selective susceptibility changes. Kinetic analysis with purified proteins revealed the reduction in catalytic efficiency of all the mutants compared to wild-type protein. Though the L158A and D159A mutated proteins become deacylation-deficient, the mutations S118A and V120A exhibited selective acylation defects without trapping intermediates. It is evident from circular dichroism spectroscopy and molecular dynamics simulations that OXA-232S118A, OXA-232V120A, and OXA-232L158A nearly retained their secondary structures and compactness, except for OXA-232D159A, which presumably triggered a misfolding leading to destabilisation of the omega-loop. Interestingly, bicarbonate supplementation partially rescued the lost activities in soluble mutants, underscoring the carbamylation dependence. Taken together, these findings establish S118 and D159 as essential for core catalysis and structural integrity, with V120 and L158 modulating substrate-specific turnover and orientation. The current study reappraised the mechanistic insights of OXA-48-like carbapenemases, providing significant resources in rationally designing future therapeutics to combat carbapenem resistance.

In wheat, weak seed dormancy (SD) is related to an increased tendency for pre-harvest sprouting (PHS), which reduces yield and quality. However, the molecular mechanism underlying SD remains elusive. Here, we identified a wheat R2R3-MYB transcription factor (TaMYB83-7B) related to SD. Expression analysis showed that TaMYB83-7B was highly expressed in wheat seeds, and was more highly expressed in strong-dormancy varieties than in weak-dormancy varieties. Sequence and association analysis indicated that T/C mutations at -907 bp and -1133 bp in the TaMYB83-7B promoter were significantly associated with wheat SD, with C at both sites related to strong dormancy. Dual-luciferase reporter assays demonstrated that the transcriptional activity of the TaMYB83-7B promoter was significantly higher in strong-dormancy varieties than in weak-dormancy varieties. Further analyses indicated that TaMYB83-7B functions as a transcriptional inhibitor. Germination experiments revealed that overexpression of TaMYB83-7B significantly enhanced SD, while its loss-of-function reduced SD. Finally, TaMYB83-7B was found to regulate SD by influencing the balance between abscisic acid (ABA) and gibberellin (GA) in wheat seeds. Overall, the results of this study enhance our understanding of the complex regulatory mechanism underlying SD, and provide gene targets and molecular markers for the genetic improvement of PHS resistance in wheat.

Fucoidans have been widely reported to show SARS-CoV-2 antiviral activity. In this study, we observed a striking difference in the inhibitory potency between two commercially available fucoidans: Fucus vesiculosus crude (Fvc) and pure (Fvp). SEC-MALS analysis revealed two molecular weight populations for Fvc (1098 kDa, 58.58 kDa) and one for Fvp (40.48 kDa). At micromolar concentrations of fucoidans, the binding affinities (KDs) of Fvc_1098 (223 nM) and Fvc_58 (4.27 {micro}M) for the amine-biotinylated SARS-CoV-2 receptor binding domain (RBD) were higher than that of Fvp (76.5 {micro}M). At nanomolar concentrations, binding was observed only to the Avi-tag-, but not amine-biotinylated RBDs, suggesting better accessibility of their binding sites. The association rates (kon) were faster for Fvc than for Fvp. Similarly, affinities of Fvc_1098 (23.4 nM) and Fvc_58 (4.48 M) for ACE2 were greater than that of Fvp (66.8 M), indicating that Fvc can bind directly to both RBD and ACE2. Fvc demonstrated enhanced inhibitory potency (IC50 = 58 g/mL) compared to Fvp (IC50 > 239 g/mL) in the pseudovirus entry assay and did not induce cytotoxicity in HEK293T cells. In conclusion, crude fucoidan with high fucose content and high molecular weight shows promising antiviral activity.

In vegetarian diets, phytate is known to disrupt the adsorption of minerals. Fortifying foods with phytase, a therapeutic enzyme known to mitigate phytate, might increase the uptake of important nutrients. Phytase is susceptible to environmental stress such as heat and acidic conditions encountered during food processing. Therefore, we developed and optimized a core-shell microparticle composed of a phytase-chitosan core and a shell consisting of cross-linked alginate-{kappa}-carrageenan. Ethanol was used to precipitate the microparticles, and the ethanol concentration was optimized along with the chitosan and phytase ratio and the alginate-carrageenan concentration, to form stable core-shell microparticles. The optimized core-shell microparticles have a loading capacity of 32.7% with a high encapsulation efficiency of 80.3% and uniform micro-size with a diameter of 3.2 {micro}m and a poly-dispersity index of 0.178. Loaded phytase retained 62.7% enzymatic activity after heat treatment and digestion conditions. These results indicate that core-shell microparticles are suitable for retaining enzyme activity within the food matrix under typical food processing conditions. HighlightsO_LIDevelopment of size-controlled core-shell microparticles to protect phytase C_LIO_LIPhytase-chitosan microparticles are surrounded by an alginate-{kappa}-carrageenan shell C_LIO_LIOptimization achieved 32.7% loading capacity with a uniform size of 3.2 {micro}m C_LIO_LICore-shell microparticles retained 62.7% enzyme activity after heat and digestion C_LIO_LIPhytase powder (2 mg) is required for a single maize meal C_LI

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, can use host derived lipids as carbon and energy source for survival. Mammalian cell entry (Mce) associated membrane (Mam) proteins are important for the stability of lipid importing Mce complexes. Mtb has five homologs of Mam proteins referred as orphaned Mam (OmamA-E) proteins. A recent study suggested that OmamC (Rv1363c) is essential for the storage and utilization of lipids under starvation in Mtb. To understand the structure and interactions of OmamC, we generated a truncated soluble variant of OmamC (OmamC129-261). Here, we report on the challenges encountered during the crystallization and structure determination of OmamC129-261 and the strategies applied to overcome them. Despite the AlphaFold2 predicted model proving an initial molecular replacement solution, experimental phasing was necessary to determine the structure of OmamC129-261. Heat treatment of protein prior to crystallization setup removed partially unfolded protein present and played a critical role in enhancing the reproducibility and diffraction quality of OmamC129-261 crystals. Although reported earlier, it is not a widely used method. It is worth to try this method, especially, when faced with poor reproducibility and diffraction of crystals.

Rapid and specific diagnosis of viral and bacterial infections is a significant challenge in medicine and veterinary science, especially in the case of epidemically dangerous pathogens. The African swine fever virus (ASFV), for example, causes annual outbreaks among livestock, resulting in significant economic losses for farmers. DNA aptamers have been identified as a promising tool for point-of-care diagnostics, being highly specific to the target and stable ambient temperatures during storage. In this study, we describe the selection of DNA aptamers targeting the p54 viral protein using a single-round selection process. These aptamers were able to bind both to recombinant protein and inactivated ASFV viral particles. Analysis of the newly generated aptamers revealed a dependence of affinity and thermal stability on Ni2+ content, which was a dopant in the selection process. In some cases, the affinity increased 100 times, and melting temperature increased by 30{degrees}C. We have identify two novel DNA motifs that bound 2-3 Ni2+ or Zn2+ ions.

A proteomic analysis of Ixodes scapularis nymph saliva identified 252 proteins, including six tubular lipid-binding proteins (TULIPs). Comparing nymphs fed on mice that were uninfected or infected with Borrelia burgdorferi, twelve salivary proteins showed significant differences in the amounts detected, including XP_040079658.2, which we refer to as TULIP2. Considering the known immunity-related functions of some TULIPs, we expressed and purified TULIP2 from Escherichia coli and analyzed its interaction with B. burgdorferi lipids. The purification of TULIP2 from E. coli presented many obstacles, due to insolubility, which is consistent with previous reports from studies of other TULIP family members. The binding results showed specificity for B. burgdorferi lipids, with evidence for cholesteryl {beta}-galactoside as a major binding target. Molecular modeling of TULIP2 did not show any strong lipid binding sites. We used molecular dynamics simulation of TULIP2 to explore its conformational landscape by thermal unfolding. The earliest unfolding intermediate opened a hydrophobic pocket to which cholesteryl {beta}-galactoside was predicted to bind strongly. We propose that a specific lipid bilayer interaction with TULIP2 triggers the opening of the ligand-binding site.

The Rho family of small GTPases plays a critical role in regulating actin cytoskeleton dynamics during endocytic processes in E. histolytica, including phagocytosis, pinocytosis, and trogocytosis. These proteins act as molecular switches, transitioning between inactive GDP-bound and active GTP-bound states, with guanine nucleotide exchange factors (GEFs) catalyzing this transition. Among the GEFs, EhFP10--a FYVE-domain-containing protein harbouring Dbl homology (DH) and pleckstrin homology (PH) domain was observed in phagocytosis along with seven functionally characterized Rho GTPases (EhRho1, EhRho2, EhRho4, EhRho5, EhRho6, EhRho8, and EhRho13). To study the specificity of FP10, a combination of GEF activity, binding affinity, and molecular dynamics simulations was used to characterize the interactions between EhFP10 and seven Rho GTPases systematically. The results revealed EhRho2 as the most specific and high-affinity interactor of EhFP10, with the highest nucleotide exchange rate and lowest dissociation constant (KD = 0.58 {micro}M). Structural modeling, sequence alignment, and interaction mapping further demonstrated that EhRho2 retains critical contact residues--such as Glu33, Arg4, and Leu69--that are variably absent in other isoforms, correlating with decreased GEF responsiveness. Molecular dynamics simulations and cross-correlation analyses supported the presence of a stable and coordinated interaction interface in the EhFP10-EhRho2 complex, distinguishing it from less active complexes. These findings indicate a highly selective GEF-GTPase module in E. histolytica, analogous to those in higher eukaryotes. The results uncover a potential regulatory mechanism specific to pathogenic amoebae and present EhFP10-EhRho2 as a novel therapeutic target for disrupting cytoskeleton-mediated processes crucial to virulence.

HrpZ2, a harpin protein produced by Pseudomonas syringae, a gram-negative plant pathogenic bacterium, elicits hypersensitive response and pathogen defense in non-host plants. Harpins from various bacterial sources elicit varying responses in different non-host plants, due to its structural variations, their precise mechanisms of action are not yet completely understood. As per previous reports, harpins from diverse bacterial sources interact with distinctive members of integral membrane proteins, known as aquaporins. For example, harpin (Hpa1Xoo) interacts with OsPIP1;3 in rice, whereas, in Arabidopsis the harpins Hpa1 and HrpZ interacts with AtPIP1;4 and AtPIP1;3 respectively. Here, we conducted the first genome-wide computational screening of protein-protein interactions between HrpZ2 and all 47 members of tomato aquaporins. Molecular docking identified nine interactors across five subfamilies of aquaporins, with HrpZ2 N-terminal residues mediating these interactions. We validated these via molecular dynamics (MD) simulations, principal component analysis, and free energy landscape analysis, assessing the stability (RMSD, RMSF, radius of gyration), dynamics, and affinity (MM-GMSA). PIP complexes, especially PIP2;1 (-460.46 kcal/mol) and PIP1;7 (-303.82 kcal/mol), exhibited superior stability, compactness, and defined energy minima, confirming PIPs as primary sensors of harpins. Non-PIP aquaporins like TIP1;1 and NIP4;1 showed moderate stability, outperforming weaker interactors (SIP2;1, XIP1;5, XIP1;3). These findings provide robust evidence that HrpZ2 preferentially targets PIPs in tomato, while engaging TIPs and NIPs as auxiliary partners. This multifaceted interaction profile of harpins suggests complex plant-pathogen recognition, modulating aquaporin-mediated cellular responses like growth and stress management in plants.

Cryptosporidium parvum is a protozoan parasite responsible for cryptosporidiosis, significantly threatening immunocompromised individuals, particularly HIV/AIDS patients, by causing severe diarrhea and potential mortality. Current treatments are largely ineffective, prompting investigations into new therapeutic options. This study evaluated two antiparasitic drugs: Mebendazole, used for helminth infections, and Artemisinin, used for malaria. The SKSR gene family encodes virulence factors in C. parvum, and Calcium-dependent protein kinase1 (CpCDPK1) regulates the life cycle of C. parvum; targeting these proteins may reduce growth and infection in hosts. In the current study, molecular docking was conducted taking Mebendazole and Artemisinin drugs as ligands, SKSR gene family and CpCDPK1 proteins as drug targets. Results with SKSR showed binding energy of -4.9 kcal/mol, -6.72 kcal/mol for Mebendazole and Artemisinin, respectively. Whereas, with CpCDPK1, the binding energies were -6.44 kcal/mol, -9.18 kcal/mol for Mebendazole and Artemisinin, respectively. Docking of Nitazoxanide (an in-use drug for C. parvum) with SKSR and CpCDPK1 revealed binding energies -4.2 kcal/mol, -4.81 kcal/mol, respectively. The stability of the proteins (targets) upon binding to the ligands was assessed by performing all-atom MD simulations for 100ns using the GROMACS package. No major variations were observed upon binding of Artemisinin and Mebendazole to SKSR and CpCDPK1. The findings of MD simulations imply that both proteins maintain their stability upon binding of Artemisinin and Mebendazole. Molecular Docking and MD simulation studies suggest that Artemisinin and Mebendazole are potential candidates for repurposing in the treatment of C. parvum infections, with recommendations for in vitro studies to validate these findings.

Bovine tuberculosis (TB), caused by Mycobacterium bovis, has become a global concern over the last two decades. Bovine TB primarily affects cattle, but other domestic livestock are also affected and it is more common in less developed and developing countries. The significant loss of livestock leads to trade restrictions and economic crises. Zoonotic potential of bovine TB raises health concerns for the public. Currently, no effective treatment is available and animal slaughtering is usually undertaken to reduce the burden of it in the environment. Antibiotic therapy can be used on animals living in captivity, but it is not reliable for herd or free-grazing animals. The BCG vaccine is another option available for treating the disease, but it shows limited efficacy in cattle. The prevention of bovine TB is a long-term goal that can only be accomplished by developing a more effective vaccine than BCG and designing new drugs. In this research, we propose therapeutic drug targets and vaccine for treating bovine TB. The conceptual framework for vaccine developed in this study uses a number of bioinformatics approaches to identify potential vaccine candidates and construct an in-silico epitope-based vaccine. Our holistic framework identified potential therapeutic candidates by directly analysing the proteome of TB bacterial strains. Specifically, we performed a comparative proteomic analysis of 11 Mycobacterium bovis strains to cover the diversity and identify conserved proteins among those strains for developing the bovine TB vaccine. An extensive reverse vaccinology and immunoinformatics analysis provided 26 highly immunogenic, non-toxic and non-allergenic epitopes (CTL epitopes-8, HTL epitopes-2 and B-cell epitopes-16) for Mycobacterium bovis required for three-dimensional structure construction of TB vaccine. The constructed epitope-based vaccine showed a potent interaction inside the host, thus generating efficient cell-mediated and humoral immune responses. Next, a framework based on a novel subtractive proteomic approach was developed for identifying bovine TB drug targets. We performed this approach on the 11 Mycobacterium bovis strains and identified nine drug targets that are conserved, essential, antigenic and have unique metabolic pathways in Mycobacterium bovis. These drug targets could further help investigate therapeutic drugs for the treatment of bovine TB. Several bioinformatics prediction tools were used together to ensure checks and balances, aiming to reduce the chance of errors and provide accurate results. The vaccine and drug targets developed in this study can be tested experimentally with confidence for further validation as therapeutics with the potential to eradicate bovine TB globally. The strategies implemented in the study are generic and can be used for other zoonotic infectious diseases. This study would be a game changer in the field of bovine tuberculosis treatment.

Serine-aspartate repeat-containing protein D (SdrD) is a Staphylococcus aureus cell wall-anchored, calcium-binding adhesin member of the MSCRAMM Sdr subfamily that may contribute to bacterial adhesion and virulence. S. aureus is the most common cause of periprosthetic joint infection (PJI). Population-level distribution and sequence diversity of SdrD among clinical PJI isolates have not been systematically characterized, and the SdrD binding mechanism is still not well understood. To address these gaps, sdrD alleles were queried across 156 newly sequenced PJI isolates and compared to publicly available S. aureus genomes, and nucleotide- and protein-level phylogenies of the sdrCDE locus constructed. The SdrD crystal structure from S. aureus JH1 was determined, with solution small-angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, and assessment of conformational changes with calcium depletion. Three dominant sdrD subtypes were defined, associating with USA300, JH1, and TCH60; the JH1 sdrD subtype was predominant among PJI isolates. Structural studies showed that the conformation of individual domains and interdomain organization of the multidomain SdrD have limited flexibility in solution, and that the calcium-binding B domain retains its core fold under conditions of calcium depletion. Together, the findings presented support functional diversification among Sdr family members in mediating host attachment and inform a re-evaluation of the ligand-binding mechanism previously proposed for SdrD. AUTHOR SUMMARYStaphylococcus aureus is the leading cause of infections that develop around joint implants (periprosthetic joint infection, PJI). This bacterium has a large arsenal of surface proteins that allow it to stick to human tissues and implanted devices. This work focused on one such protein, SdrD, which has been linked to implant-associated infections but the structure and diversity of which among patients with PJI had not been well characterized. The genetic sequences of SdrD were analyzed across thousands of bacterial genomes, including those from patients with PJI. Distinct genetic variants of the protein were found, one of which was particularly common with PJI. The three-dimensional structure of SdrD was determined at atomic resolution and solution small-angle X-ray scattering (SAXS) and molecular dynamics used to study how it moves and responds to changes in its environment. Contrary to what was previously described, SdrD was shown to be relatively rigid. These findings change how SdrDs mechanism of action should be considered, potentially informing design strategies to block bacterial attachment before infection takes hold.

Glioblastoma (GBM) presents significant therapeutic challenges due to its aggressive nature, complex microenvironment and the limitations of conventional drug delivery systems. In this study, hybrid nanoparticles were developed by combining synthetic liposomes with macrophage-derived extracellular vesicles (EVs) to harness the strengths of both platforms. Two distinct liposomal formulations, DPPC:Chol:DSPE-mPEG2000 (F1) and DPPC:DPPS:Chol:DSPE-mPEG2000 (F2), were used as the basis for the synthesis. EVs derived from J774 macrophages were integrated with F1 and F2 to create hybrid nanoparticles (H-F1 and H-F2). Doxorubicin (DOX) was encapsulated using a pH gradient and a remote loading procedure. The mean particle size of H-F1-DOX and H-F2-DOX was 158.2 {+/-} 1 nm and 162.8 {+/-} 9 nm, respectively. The polydispersity index (PDI) was 0.130 {+/-} 0.012 and 0.084 {+/-} 0.033, while the zeta potential values were -14.9 {+/-} 0.7 mV and -26.7 {+/-} 3.1 mV, respectively. H-F2-DOX exhibited the highest encapsulation efficiency (EE%), reaching 76.5{+/-}3.4%. The encapsulated hybrids remained stable up to one week, at +5{degrees}C. The release of DOX from H-F2-DOX in DMEM supplemented with 10% serum showed pH sensitivity, with total DOX release of 64.9 {+/-} 5.3% at pH 7.4 and 90.7 {+/-} 6.5% at pH 5.5. The cell viability assay demonstrated that all formulations exhibited strong cytotoxic effects against GBM cells under normoxic conditions, with H-F2-DOX showing the most potent effect under hypoxia-mimetic conditions.

Magnaporthe oryzae, the rice blast fungus, plays a role as a model organism for molecular plant-microbe interaction research. Studies on the pathogenic mechanism of this fungus revealed many genes involved in signaling pathways. As multi-omics data are being available, genomic-level researches have been conducted to uncover the underlying biological processes during the pathogenesis of M. oryzae. Identifying the genome-wide protein-protein interaction (PPI) network is one of the omics-level approaches, which helps to understand signaling and regulatory pathways. However, existing biological network resources of M. oryzae are not sufficient to decipher pathogenesis mechanisms due to the abundance of false positives/negatives. In this study, a reliable PPI network database of M. oryzae, MagNet, was constructed with three methods, including homology-based Interolog search, co-expression network construction, and domain-domain interaction (DDI)-based prediction. With three approaches altogether, the pan-network with 5,600,976 interactions was generated, including 217,531 highly confident interactions supported by all three methods. Experimental data on M. oryzae PPIs supported that our PPI network can predict PPIs with higher accuracy compared to the previously constructed databases. MagNet would provide integrated biological network data, which can help to understand the molecular mechanisms of the rice blast fungus. The PPI data can be accessed via https:/magnet.scnu.ac.kr.

High-risk Human Papillomaviruses (HR-HPVs) are responsible for 5% of global cancers. While vaccines against HR-HPVs exist, there are no treatments available for individuals already infected. Cell-penetrating peptides (CPPs) have demonstrated antiviral properties against viruses by blocking viral entry and delivering antivirals into infected cells. Developing CPP-based therapies faces challenges including inefficient delivery of macromolecules and endosomal entrapment, which must be overcome for effective clinical application. This study identifies an HPV16 major capsid protein L1 derived cationic peptide as a potent CPP. Peptide uptake depended on both a cluster of cationic residues and the specific peptide sequence. Mechanistic studies showed peptide entry occurred via cell surface heparan sulfate-mediated, lipid-raft dependent endocytosis. The peptide efficiently delivered GFP into HaCaT keratinocytes, and associated with the Golgi apparatus, demonstrating endosomal escape. GFP fusion protein endocytosis relied on binding of the cationic peptide to cell surface heparan sulfates. Cell-penetrating ability was conserved among homologous regions of various HPV types. The peptide showed potent antiviral activity by inhibiting infection of HaCaT cells by several HR-HPV types collectively responsible for nearly all HPV-associated cancers. Excitingly, HPV18 L1-derived peptide from the homologous region exhibited potent antiviral activity against HPV16 by preventing viral internalization. Our findings characterize HPV-derived peptides as highly efficient CPPs with potential to deliver therapeutic agents into cells and assist in development of treatments for high-risk HPVs.

Cultivated meat is an emerging biotechnology that aims to produce edible tissues in an ethical and sustainable manner. However, the recreation of skeletal muscle tissue that replicates the protein composition and sensory characteristics of traditional meat is a major challenge. Skeletal muscle tissue engineering requires non-animal-based scaffolds which are inexpensive and food-safe, while meeting specific mechanical requirements with respect to viscosity, stress-relaxation and stiffness. While many of these characteristics can be fulfilled by alginate-based biomaterials, a key limitation of alginate is its lack of intrinsic attachment sites for animal cells, preventing efficient adhesion, differentiation and tissue formation. Here, we established a screening platform to evaluate extracellular matrix (ECM)-mimicking peptides as functionalisations of alginate scaffolds in 2D. Our platform enables high-throughput assessment of cell/peptide interactions, serving as a predictive tool for 3D tissue constructs. Our screen identified two RGD-containing sequences (vitronectin- and fibronectin-mimicking peptides) as most effective in promoting attachment and myogenic fusion of bovine satellite cells. Notably, these peptides outperformed more complex mixtures containing up to seven different ECM-mimicking peptides. Our findings provide a streamlined approach for optimising biomaterial functionalisations for cultivated meat applications, and lay the groundwork for future advancements in scalable, sustainable skeletal muscle tissue engineering.

Obtaining tomato plants with firm and intact fruit is one of the main goals in tomato breeding programs. Achieving these goals through conventional breeding is time-consuming and can lead to the loss of unwanted traits. In other hand, consumers are concerned about the presence of transgenic elements in plants acquired through RNA interference. The use of CRISPR/Cas9 technology has made it possible to overcome the above-mentioned shortcomings. In this study, the {beta}-D-N-acetylhexosaminidase ({beta}-hex) gene, which is involved in tomato fruit ripening, was knocked out using CRISPR/Cas9. In the resulting mutant plant genome, an indel mutation was found in exons 1 and 2 of the {beta}-hex gene. Plants with a mutation in their genome were observed to have increased fruit firmness and shelf life compared to control plants without affecting fruit quality.

Adeno-associated virus (AAV) vectors are widely used in gene therapy, whereas low manufacturing efficiency and a large proportion of empty capsids are major obstacles. This study focused on the Yin Yang 1 (YY1) binding motif (YY1-motif) and investigated the effect of its presence or insertion at upstream of the Replicase (Rep)/Capsid Cap) gene on AAV vector production. We found that the YY1-motif incidentally presented in a Rep/Cap plasmid was associated with high vector production. We then designed several modified Rep/Cap (RC2) constructs. The YY1-motif insertion at the upstream of Rep/Cap gene increased vector yield in a repeat-number-dependent manner, and similar effects were not observed with other promoters insertion. Furthermore, the insertion of the YY1-motif reduced the amount of Cap protein per the same amount of full particle in supernatants on multiple serotypes, indicating the improvement in the empty/full capsid ratio. The YY1-motif insertion did not affect the AAV vector infectivity. These results denote that the YY1-motif has a universal regulatory function that optimizes the Rep/Cap expression balance, and simultaneously improves the production efficiency and full particle formation of AAV vectors. This finding could contribute to the development of highly efficient and high-quality AAV manufacturing processes.

The binding of fluorescent dyes to nucleic acids and their fluorogenic properties are indispensable tools for nucleic acid detection, quantification, and imaging, yet the molecular structures of several widely used commercial dyes have remained unknown. Here, we de novo determined the molecular structures of RiboGreen and OliGreen and confirmed the previously proposed structure of PicoGreen using high-field NMR spectroscopy. All three dyes were identified as unsymmetric cyanine dyes, where a benzoxazole/benzothiazole moiety is linked to a 4-quinoline by a monomethine bridge. Complete 1H and 13C resonance assignments enabled us to expand the existing chemical shift reference set for this important class of dyes. Photophysical characterization with standardized single- and double-stranded DNA and RNA targets indicated that all dyes performed similarly upon binding despite being marketed towards different nucleic acid types. NMR spectroscopy and long-timescale molecular dynamics simulations showed that RiboGreen interacts with double-stranded DNA predominantly by two binding modes, electrostatic interactions with the phosphodiester backbone and {pi}-{pi} stacking with the ultimate and penultimate base pairs of the DNA molecule. These results establish the molecular structures of three widely used commercial dyes and provide a structural and mechanistic framework for understanding the fluorogenic properties of this class of dyes. HighlightsO_LIDetermination of the molecular structures of nucleic acid dyes RiboGreen, OliGreen, and PicoGreen C_LIO_LINMR spectroscopic characterization of all three dyes. C_LIO_LINMR and MD data indicate binding to be dominated by electrostatic and {pi}-{pi} stacking interactions C_LI