Loop Region Research Articles

The human Dicer helicase domain is capable of ATP hydrolysis and single-stranded nucleic acid binding

BackgroundVertebrates have one Dicer ortholog that generates both microRNAs (miRNAs) and small interfering RNAs (siRNAs), in contrast to the multiple Dicer-like proteins found in flies and plants. Here, we focus on the functions of the human Dicer (hDicer) helicase domain. The helicase domain of hDicer is known to recognize pre-miRNA substrates through interactions with their apical loop regions. Besides interacting with canonical substrates, the hDicer helicase domain has also been suggested to bind many different cellular RNAs; however, a comprehensive study of the biochemical activities and substrate specificity of the hDicer helicase domain towards different nucleic acids has yet to be undertaken.ResultsHere, we reveal that full-length hDicer, through its helicase domain, hydrolyzes ATP. The ATPase activity of hDicer can only be observed under low-turnover conditions. To the best of our knowledge, this is the first time this activity has been reported for vertebrate Dicers. We also show that the hDicer helicase domain binds single- but not double-stranded RNAs and DNAs and that this binding activity presumably is not nucleotide-dependent. Moreover, the hDicer helicase domain may influence the structure of the RNA to which it binds.ConclusionsPreservation of ATPase activity by hDicer suggests that this enzyme performs many more functions in the cell than is currently assumed. Our findings open new avenues for future studies aimed at defining the cellular activities of hDicer that may be associated with these newly described biochemical properties: ATP hydrolysis and single-stranded nucleic acid binding activities.

Genetic Characteristics of the Env Regions in HIV-1-Infected Subjects in Baoding City, Hebei Province, China.

The envelope glycoprotein (Env) on the surface of the human immunodeficiency virus (HIV-1) is a crucial protein that mediates binding to host cell receptors and subsequent membrane fusion. Env, as the sole target for neutralizing antibodies, holds unique importance in vaccine design. Therefore, analyzing the genetic characteristics of the Env region offers reference data for vaccine and drug design. From December 2021 to December 2022, 145 newly diagnosed, HIV-1-infected individuals in Baoding City were recruited into this study. The HIV-1 env gene sequence was successfully obtained from 142 of the 145 blood specimens, and the sequences were submitted to the Quality Control Tool (http//:HIV-DB Sequence Quality Control Tool (lanl.gov)) to analyze the viral subtype. The coreceptor tropism was predicted using the Geno2pheno web tool with falsepositive rate (FPR) values of 5%-15%, and the net charges of the third variable (V3) loop were calculated by Variable Region Characteristics (lanl.gov). The results showed that half of the patients were infected with the CCR5-tropic virus (50.0%, 71/142). In HIV-1 subtype CRF01_AE infection, the use of CXCR4 is expected to predominate, while in HIV-1 subtype CRF07_BC infection, CCR5 coreceptors are expected to be used predominantly. Sequence analysis of the V3 loop region revealed that subtypes CRF01_AE and CRF07_BC have similar median net charges (~3.0). Furthermore, GPGQ was found to be the major terminal tetrapeptide of the CRF07_ BC and CRF01_AE strains in this study. These findings enhance our understanding of the characteristics of the HIV-1 epidemic and provide important implications for HIV-1 vaccine design and clinical treatment.

Design, Synthesis, Computational Studies and Evaluation of Novel 2, 4, 5-Trisubstituted Pyrimidine Derivatives for Anticancer Activity against MCF-7 and A549 Cell Lines

Synthesized novel trisubstituted pyrimidine derivatives (U 01-15) were tested for anti-cancer efficacy against two cancer cell line MCF-7 and A549. Several spectroscopic methods assisted to characterized and confirmed all synthesized compounds. Some of the synthesized pyrimidine derivatives showed strong effectiveness, with IC50 values comparable to the standard drug doxorubicin and gefitinib. Compounds U-02, U-04, U-07, U-10 and U-11 showed good anti-cancer activity for both cell lines having IC50 values in range from 12.06 µM to 22.45 µM, while compounds U-06 and U-03 showed moderate potency against both the cell lines, respectively. Using PyRx software (based on Auto Dock 4 and a genetic algorithm), all the synthesized compounds were put through a molecular docking with receptor tyrosine-protein kinase erbB-4 and docking score was compared to approved drugs, gefitinib and lapatinib. Interestingly almost all derivatives showed good docking score as compared to reference drugs. Molecular dynamics simulation of the U-11 protein-ligand complex over 100 ns revealed key insights into stability and conformational changes. RMSD analysis indicated stability between 10-20 ns with fluctuations up to 5.71 Å, while RMSF showed an average fluctuation of 1.74 Å, particularly in the loop regions. Molecular interactions, including hydrogen bonds, were observed with key residues such as ASP836, MET774, and LEU769.Future studies could focus on optimizing the most promising pyrimidine derivatives, such as U-02, U-04, U-07, U-10, and U-11, for enhanced potency and selectivity through advanced biological testing and structure-activity relationship studies may provide deeper insights into their therapeutic potential against cancer.

Super-slow Nonlinear Hysteresis Loop “Tracking” for Managing Energy Intake in the Forced Duffing Oscillator

Hysteresis loops are ubiquitous in harmonically (and not only) forced nonlinear oscillators. These loops result due to the well-known nonlinear bi-stability phenomenon, i.e., the co-existence of steady state responses, with the initial conditions dictating the attraction of a specific orbit by either of these steady states. This introduces an element of uncertainty in the forced dynamics. Hence, if one targets the excitation of a higher-amplitude co-existing steady state solution, e.g., to maximize the energy input into the forced oscillator, this sensitivity on initial conditions introduces uncertainty. Moreover, the role of such hysteresis loops in terms of nonlinear physics is not entirely clear; this contrasts with similar hysteresis loops appearing in, e.g., in cyclically loaded viscoelastic materials, which denote the energy dissipated per excitation cycle. Here a methodology is presented for removing the uncertainty of the forced dynamics on initial conditions, and, in the process, for better understanding and exploiting nonlinear hysteresis loops. Considering the forced Duffing oscillator, as an example, harmonic excitations with super-slowly modulated amplitudes are considered as a means of “tracking” the hysteresis loop during a super-slow cycle of the applied modulated force. For either single or repetitive cycles of super-slow force modulations one may tune the modulations to predictively maximize or minimize the energy intake into the forced oscillator depending on which regions of the hysteresis loop are tracked. Importantly, computational studies indicate that the forced dynamics become independent of the specific initial conditions, thus removing the uncertainty in the response due to bi-stability. These findings elucidate the physical significance of nonlinear hysteresis in terms of energy transfer in forced oscillators, and are applicable to a broad class of forced dynamical systems exhibiting nonlinear hysteresis.

Genetic characteristics and diversity of PDC variants of Pseudomonas aeruginosa and its clinical relevance.

Pseudomonas aeruginosa exhibits significant antibiotic resistance facilitated by both intrinsic and acquired mechanisms, prominently through Pseudomonas-derived cephalosporinase (PDC), serine Ambler class C β-lactamases encoded by the AmpC. AmpC, involved in the peptidoglycan recycling pathway, is regulated by genes such as ampD, ampR, and ampG, leading to increased expression and resistance to various beta-lactams. PDCs are classified into three main types: classical class C β-lactamases, extended-spectrum class C β-lactamases (ESAC β-lactamases), and inhibitor-resistant class C β-lactamases. This study aimed to identify prevalent PDC variants and its genetic characteristics in Indian and global P. aeruginosa isolates, focusing on their role in β-lactam resistance. Analyzing PDC sequences from 111 P. aeruginosa isolates collected at Christian Medical College (CMC), Vellore, we found the ESAC allele PDC-447 to be the most widespread among Indian isolates, present in 18 % of carbapenem-resistant and 11 % of carbapenem-susceptible strains. Global and Indian isolates PDC variants were validated using the NCBI PathogenWatch database, and the sequenced PDC region compared to PDC-1. PDC-398 and PDC-397 followed in prevalence among carbapenem-resistant isolates, while PDC-5 (ESAC) and PDC-1 (classical class C) were common in carbapenem-susceptible strains. A global analysis of 19,478 genomes revealed significant prevalence of ESAC variants such as PDC-3 (17.28 %) and PDC-5 (12.91 %), alongside classical class C beta-lactamases like PDC-8 (10.65 %). Indian isolates exhibited distinct patterns with PDC-3 and PDC-5 prevailing at 19.84 % and 10 %, respectively. Mutations in the omega loop, H-helix, and R2 region of PDCs were linked to enhanced antibiotic resistance, particularly the T105A mutation in the H-helix region. These findings underscore the complexity of antimicrobial resistance mechanisms in P. aeruginosa and highlight the need for novel therapeutic strategies and continuous surveillance to manage infections by this versatile pathogen. Understanding the prevalence and genetic characteristics of PDC variants is crucial for effective treatment strategies against P. aeruginosa and combating antibiotic resistance.

Spatiotemporal visualization of instantaneous flame structure in a hydrogen-fueled axisymmetric supersonic combustor

Spatiotemporal visualization of instantaneous flame structures in a hydrogen-fueled axisymmetric supersonic combustor was investigated using multiview planar laser-induced fluorescence of the hydroxyl radical, coupled with high-speed photography and pressure measurement. The axisymmetric cavity generates a loop-shaped recirculation flow and shear layer that sustains the flame. An irregular and wrinkled flame loop with a central hole is formed near the loop-shaped region. Due to turbulent disturbances, multiple small-scale holes and fragmented flames are randomly distributed in the flame loop or near the wrinkled flame front. The combustion near the cavity shear layer is more likely to be stronger and sustained. As the thickness of the cavity shear layer increases along the axial direction, the flame loop is expanded toward the core flow and the cavity. The flame base anchors near the cavity leading edge with a low global equivalence ratio (GER). The increased GER expands the flame loop to compress the high-speed core flow dramatically, promoting the flame base to propagate upstream along the hydrogen jet wake. The flame base is unable to anchor near the thin boundary layer. Consequently, it propagates reciprocally to enhance the combustion oscillation that disturbs the flame structure dramatically. The flame structure becomes more complex and tendentially fragmented, which increases the fractal dimension, especially near the middle part of the combustor. In comparison, the flame structure near the ramp is more resistant to disturbances due to the dramatic expansion of local flame loop, extending the favorable combustion environment. Despite the instantaneous flame structure being severely wrinkled and even tendentially fragmented, it is primarily sustained within a relatively regular loop region near the cavity recirculation flow and the cavity shear layer.

CBL1/CIPK23 phosphorylates tonoplast sugar transporter TST2 to enhance sugar accumulation in sweet orange (Citrus sinensis).

Fruit taste quality is greatly influenced by the content of soluble sugars, which are predominantly stored in the vacuolar lumen. However, the accumulation and regulation mechanisms of sugars in most fruits remain unclear. Recently, we established the citrus fruit vacuole proteome and discovered the major transporters localized in the vacuole membrane. Here, we demonstrated that the expression of tonoplast sugar transporter 2 (CsTST2) is closely associated with sugar accumulation during sweet orange (Citrus sinensis) ripening. It was further demonstrated that CsTST2 had the function of transporting hexose and sucrose into the vacuole. Overexpression of CsTST2 resulted in an elevation of sugar content in citrus juice sac, calli, and tomato fruit, whereas the downregulation of its expression led to the reduction in sugar levels. CsTST2 was identified as interacting with CsCIPK23, which binds to the upstream calcium signal sensor protein CsCBL1. The phosphorylation of the three serine residues (Ser277, Ser337, and Ser354) in the loop region of CsTST2 by CsCIPK23 is crucial for maintaining the sugar transport activity of CsTST2. Additionally, the expression of CsCIPK23 is positively correlated with sugar content. Genetic evidence further confirmed that calcium and CsCIPK23-mediated increase in sugar accumulation depends on CsTST2 and its phosphorylation level. These findings not only unveil the functional mechanism of CsTST2 in sugar accumulation, but also explore a vital calcium signal regulation module of CsCBL1/CIPK23 for citrus sweetness quality.

Structural and Functional Characterization of an Amidase Targeting a Polyurethane for Sustainable Recycling.

Global plastic production exceeded 400 million tons in 2022, urgently demanding improved waste management and recycling strategies for a circular plastic economy. While the enzymatic hydrolysis of polyethylene terephthalate (PET) has become feasible on industrial scales, efficient enzymes targeting other hydrolysable plastic types such as polyurethanes (PURs) are lacking. Recently, enzymes of the amidase signature (AS) family, capable of cleaving urethane bonds in a polyether-PUR analog and a linear polyester-PUR, have been identified. Herein, we present high-resolution crystal structures of the AS enzyme UMG-SP3 in three states: ligand-free, bound with a suicidal inhibitor mimicking the transition state, and bound with a monomeric PUR degradation product. Besides revealing the conserved core and catalytic triad akin to other AS family members, the UMG-SP3 structures show remarkable flexibility of loop regions. Particularly, Arg209 in loop 3 adopts two induced-fit conformations upon ligand binding. Through structure-guided kinetic studies and enzyme engineering, we mapped structural key elements that determine the enhanced hydrolysis of urethane and amide bonds in various small molecules including a linear PUR fragment analog. Our findings contribute critical insights into urethanase activity, aiding PUR degradation campaigns and sustainable plastic recycling efforts in the future.

Structure-Guided Engineering Unveils Deeper Substrate Channel in Processive Endoglucanase EG5C-1 Contributing to Enhanced Catalytic Efficiency and Processivity.

Processive endoglucanases have generated significant interest due to their bifunctionality in the degradation of cellulose and low product inhibition. However, enhancing their catalytic efficiency through engineering remains a formidable challenge. To address this bottleneck, our engineering efforts targeted loop regions located in the substrate channel of processive endoglucanase EG5C-1. Guided by a comparative analysis of characteristic structural features of the substrate channels in cellobiohydrolase, endoglucanase, and processive endoglucanase, a highly active triple mutant CM6 (N105H/T205S/D233L) was generated that had a 5.1- and 4.7-fold increase in catalytic efficiency toward soluble substrate carboxymethyl cellulose-Na and insoluble substrate phosphoric acid-swollen cellulose (PASC), compared with wild-type EG5C-1. Furthermore, this mutant exhibited greater processivity compared to EG5C-1. Molecular dynamics simulations unveiled that the mutations in the loop regions reshaped the substrate channel, leading to a deeper cleft, resembling the closed channel configuration of cellobiohydrolases. The increased compactness of the substrate channel induced changes in the substrate binding mode and substrate deformation, thereby enhancing both binding affinity and catalytic efficiency. Moreover, metadynamics simulations demonstrated that the processive velocity of cellulose chain through the binding channel in mutant CM6 surpassed that observed in EG5C-1.

Mitogenome based adaptations and phylogeny of Beetal goats in India

Beetal goats, the second largest Indian goat breed, are known for their adaptation to hot arid tropical climates. Beyond their phylogenetic significance, recent evidence suggests that mitogenome modifications play a crucial role in the adaptation of animals to environmental stress factors. This study aims to characterize the mitogenome of Beetal goats at the molecular level, elucidate their mitogenomic adaptations and determine the maternal phylogenetic status of Indian Beetal goats. Whole genome sequencing of pooled DNA samples from five unrelated Beetal goats was carried out to obtain mitogenome sequence. The Beetal goat mitogenome comprised of a coding region with 37 genes: 22 transfer RNAs (tRNAs), 13 protein-coding genes (PCGs), and two ribosomal RNAs (rRNAs), as well as a non-coding hypervariable displacement loop (D-loop) region. We identified nine SNPs inclusive of seven synonymous and two nonsynonymous ones located in PCGs that too in respiratory complex I subunit genes, five of which were novel. Two nonsynonymous SNPs (10,229 A > G in ND4 and 13,964 G > A in ND6) were homoplasmic ones with marked influence on their mRNA and protein structures. Synonymous SNPs influenced the minimum free energy (MFE) and topology of predicted mRNA secondary structures, thereby affecting mRNA stability and translation efficiency. Phylogenetic analysis of the D-loop region classified Beetal goats into haplogroup B. Mitogenome analysis of Beetal goats, in comparison with other goat populations revealed the greatest genetic similarity to Southeast Asian goats. The comprehensive analysis of mitochondrial DNA in Beetal goats reveals significant genetic adaptations crucial for their survival in hot, arid environments. This study also highlights the complex matrilineal origins of Beetal goats.

Structure-based clustering and mutagenesis of bacterial tannases reveals the importance and diversity of active site-capping domains.

Tannins are critical plant defense metabolites, enriched in bark and leaves, that protect against microorganisms and insects by binding to and precipitating proteins. Hydrolyzable tannins contain ester bonds which can be cleaved by tannases-serine hydrolases containing so-called "cap" domains covering their active sites. However, comprehensive insights into the biochemical properties and structural diversity of tannases are limited, especially regarding their cap domains. We here present a code pipeline for structure prediction-based hierarchical clustering to categorize the whole family of bacterial tannases, and have used it to discover new types of cap domains and other structural insertions among these enzymes. Subsequently, we used two recently identified tannases from the gut/soil bacterium Clostridium butyricum as model systems to explore the biochemical and structural properties of the cap domains of tannases. We demonstrate using molecular dynamics and mutagenesis that the cap domain covering the active site plays a major role in enzyme substrate preference, inhibition, and activity-despite not directly interacting with smaller substrates. The present work provides deeper knowledge into the mechanism, structural dynamics, and diversity of tannases. The structure-based clustering approach presents a new way of classifying any other enzyme family, and will be of relevance for enzyme types where activity is influenced by variable loop or insert regions appended to a core protein fold.

Increasing thermostability of the key photorespiratory enzyme glycerate 3‐kinase by structure‐based recombination

SummaryAs global temperatures rise, improving crop yields will require enhancing the thermotolerance of crops. One approach for improving thermotolerance is using bioengineering to increase the thermostability of enzymes catalysing essential biological processes. Photorespiration is an essential recycling process in plants that is integral to photosynthesis and crop growth. The enzymes of photorespiration are targets for enhancing plant thermotolerance as this pathway limits carbon fixation at elevated temperatures. We explored the effects of temperature on the activity of the photorespiratory enzyme glycerate kinase (GLYK) from various organisms and the homologue from the thermophilic alga Cyanidioschyzon merolae was more thermotolerant than those from mesophilic plants, including Arabidopsis thaliana. To understand enzyme features underlying the thermotolerance of C. merolae GLYK (CmGLYK), we performed molecular dynamics simulations using AlphaFold‐predicted structures, which revealed greater movement of loop regions of mesophilic plant GLYKs at higher temperatures compared to CmGLYK. Based on these simulations, hybrid proteins were produced and analysed. These hybrid enzymes contained loop regions from CmGLYK replacing the most mobile corresponding loops of AtGLYK. Two of these hybrid enzymes had enhanced thermostability, with melting temperatures increased by 6 °C. One hybrid with three grafted loops maintained higher activity at elevated temperatures. Whilst this hybrid enzyme exhibited enhanced thermostability and a similar Km for ATP compared to AtGLYK, its Km for glycerate increased threefold. This study demonstrates that molecular dynamics simulation‐guided structure‐based recombination offers a promising strategy for enhancing the thermostability of other plant enzymes with possible application to increasing the thermotolerance of plants under warming climates.

Molecular dynamics simulations reveal concentration-dependent blockage of graphene quantum dots to water channel protein openings

Graphene quantum dots (GQDs) have attracted significant attention across various scientific research areas due to their exceptional properties. However, studies on the potential toxicity of GQDs have yielded conflicting results. Therefore, a comprehensive evaluation of the toxicity profile of GQDs is essential for a thorough understanding of their biosafety. In this work, employing a molecular dynamics (MD) simulation approach, we investigate the interactions between GQDs and graphene oxide quantum dots (GOQDs) with the AQP1 water channel protein, aiming to explore the potential biological influence of GQDs/GOQDs. Our MD simulation results reveal that GQDs can adsorb to the loop region around the openings of AQP1 water channels, resulting in the blockage of these channels and potential toxicity. Interestingly, this blockage is concentration-dependent, with higher GQD concentrations leading to a greater likelihood of blockage. Additionally, GOQDs show a lower probability of blocking the openings of AQP1 water channels compared to GQDs, due to the hydrophobicity of the loop regions around the openings, which ultimately leads to lower interaction energy. Therefore, these findings provide new insights into the potential adverse impact of GQDs on AQP1 water channels through the blockage of their openings, offering valuable molecular insights into the toxicity profile of GQD nanomaterials.

Preferential cleavage of the coronavirus defective viral genome by cellular endoribonuclease with characteristics of RNase L

In testing whether coronavirus defective viral genome 12.7 (DVG12.7) with transcription regulating sequence (TRS) can synthesize subgenomic mRNA (sgmRNA) in coronavirus-infected cells, it was unexpectedly found by Northern blot assay that not only sgmRNA (designated sgmDVG 12.7) but also an RNA fragment with a size less than sgmDVG 12.7 was identified. A subsequent study demonstrated that the identified RNA fragment (designated clvDVG) was a cleaved RNA product originating from DVG12.7, and the cleaved sites were located in the loop region of stem‒loop structure and after UU and UA dinucleotides. clvDVG was also identified in mock-infected HRT-18 cells transfected with DVG12.7 transcript, indicating that cellular endoribonuclease is responsible for the cleavage. In addition, the sequence and structure surrounding the cleavage sites can affect the cleavage efficiency of DVG12.7. The cleavage features are therefore consistent with the general criteria for RNA cleavage by cellular RNase L. Furthermore, both the cleavage of rRNA and the synthesis of clvDVG were also identified in A549 cells. Because (i) the cleavage sites occurred predominantly after single-stranded UA and UU dinucleotides, (ii) the sequence and structure surrounding the cleavage sites affected the cleavage efficiency, (iii) the cleavage of rRNA is an index of the activation of RNase L, and (iv) the cleavage of both rRNA and DVG12.7 was identified in A549 cells, the results together indicated that the preferential cleavage of DVG12.7 is correlated with cellular endoribonuclease with the characteristics of RNase L and such cleavage features have not been previously characterized in coronaviruses.

A simple and enzyme-free method for sensitive p53 analysis based on DNAzyme-mediated signal amplification

There is an urgent demand for a simple yet extremely accurate biosensor to analyze tumorigenesis. Herein, we present a novel fluorescent and enzyme-free approach for detecting p53 gene cascading proximity ligation-mediated catalytic hairpin assembly and DNAzyme-assisted signal reaction. When the target p53 gene is present, the interaction between p53 and L1 and L2 chains initiates catalytic hairpin assembly and subsequently exposes DNAzyme in the P3 probe. The exposed DNAzyme binds with the loop region of the P4 probe and generates a nicking site, resulting in the release of a significant amount of ATMND that is conjugated in the stem section of P4. This leads to an amplified fluorescence response, which serves as a fluorescence signal for the detection of the p53 gene. This method allows for the accurate and sensitive identification of the p53 gene, exhibiting a linear reaction range of 1 fM to 1 nM, with a limit of detection as low as 0.23 fM. Furthermore, this fluorescent method has been utilized for the examination of clinical samples with a favorable recovery rate. Crucially, this versatile platform may be expanded to analyze different targets by changing the corresponding recognition unit, showing great potential for point-of-care testing in tumorigenesis analysis.

Reverse Vaccinology Integrated with Biophysics Techniques for Designing a Peptide-Based Subunit Vaccine for Bourbon Virus.

Despite the seriousness of the disease carried by ticks, little is known about the Bourbon virus. Only three US states have recorded human cases of Bourbon virus (BRBV) infection; in all cases, a tick bite was connected with the onset of the illness. The Bourbon virus (BRBV) belongs to the Orthomyxoviridae family and Thogotovirus genus, originating in the states of the US, i.e., Kansas, Oklahoma and Missouri. The growing rates of BRBV infections in various parts of the US highlight the necessity for a thorough analysis of the virus's transmission mechanisms, vector types and reservoir hosts. Currently, there are no vaccines or efficient antiviral therapies to stop these infections. It is imperative to produce a vaccination that is both affordable and thermodynamically stable to reduce the likelihood of future pandemics. Various computational techniques and reverse vaccinology methodologies were employed to identify specific B- and T-cell epitopes. After thorough examination, the linker proteins connected the B- and T-cell epitopes, resulting in this painstakingly constructed vaccine candidate. Furthermore, 3D modeling directed the vaccine construct toward molecular docking to determine its binding affinity and interaction with TLR-4. Human beta-defensin was used as an adjuvant and linked to the N-terminus to boost immunogenicity. Furthermore, the C-IMMSIM simulation resulted in high immunogenic activities, with activation of high interferon, interleukins and immunoglobulin. The results of the in silico cloning process for E. coli indicated that the vaccine construct will try its utmost to express itself in the host, with a codon adaptation CAI value of 0.94. A net binding free energy of -677.7 kcal/mol obtained during docking showed that the vaccine has a high binding affinity for immunological receptors. Further validation was achieved via molecular dynamic simulations, inferring the confirmational changes during certain time intervals, but the vaccine remained intact to the binding site for a 100 ns interval. The thermostability determined using an RMSF score predicted certain changes in the mechanistic insights of the loop region with carbon alpha deviations, but no major changes were observed during the simulations. Thus, the results obtained highlight a major concern for researchers to further validate the vaccine's efficacy using in vitro and in vivo approaches.

An internal loop region is responsible for inherent target specificity of bacterial Cold-shock proteins.

Cold shock proteins (Csps), of around 70 amino acids, share a protein fold for the cold shock domain (CSD) that contains RNA binding motifs, RNP1 and RNP2, and constitute one family of bacterial RNA-binding proteins. Despite similar amino acid composition, Csps have been shown to individually possess inherent specific functions. Here we identify the molecular differences in Csps that allow selective recognition of RNA targets. Using chimeras and mutants of Escherichia coli CspD and CspA, we demonstrate that Lys43-Ala44 in an internal loop of CspD and the N-terminal portion with Lys4 of CspA are important for determining their target specificities. Pull-down assays suggest these distinct specificities reflect differences in the ability to act on the target RNAs rather than differences in binding to the RNA targets. A phylogenetic tree constructed from 1,573 Csps reveals that the Csps containing Lys-Ala in the loop form a monophyletic clade, and the members in this clade are shown to have target specificities similar to E. coli CspD. The phylogenetic tree also finds a small cluster of Csps containing Lys-Glu in the loop, and these exhibit different specificity than E. coli CspD. Examination of this difference suggests a role of the loop of CspD type proteins in recognition of specific targets. Additionally, each identified type of Csp shows a different distribution pattern among bacteria. Our findings provide a basis for subclassification of Csps based on target RNA specificity, which will be useful for understanding of the functional specialization of Csps.

Investigating the role of natural flavonoids in VEGFR inhibition: Molecular modelling and biological activity in A549 lung cancer cells

Genetics, lifestyle, and hormones influence lung cancer, the most common malignancy in the world. VEGFR plays a key role in tumor growth and metastasis by promoting angiogenesis. While anti-angiogenic therapies targeting VEGFR show potential, resistance remains a challenge, and elevated VEGFR levels are associated with aggressive tumors and poor outcomes. This research explores using natural flavonoids to inhibit VEGFR activity in lung cancer, focusing on computational studies and in-vitro assays with the A549 cell line. Molecular docking revealed that 18 flavonoids outperformed axitinib and 26 surpassed sorafenib in effectiveness. Molecular dynamics simulation showed system stabilization after 80 ns with RMSD between 2 and 4 Å RMSF analysis revealed fluctuations in the activation loop and hinge region. Hydrogen bonds, especially with GLU 149, were key to the complex's stability. PCA highlighted conformational changes, and MM/GBSA calculated a binding free energy of -34.72 kcal/mol. In particular, quercetin had a lower IC50 value (6.24 ± 0.42 µM) than axitinib (7.85 ± 0.59 µM), which means it had stronger effects on lung cancer cells. Other flavonoids like taxifolin and luteolin showed similar efficacy. The findings suggest potential for tailored therapies that could enhance personalized treatment approaches for lung cancer.

Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1) lncRNA Conformational Dynamics in Complex with RNA-Binding Protein with Serine-Rich Domain 1 (RNPS1) in the Pan-cancer Splicing and Gene Expression.

Alternative splicing events increase the transcriptomic and proteomic complexity in cancers. Overexpression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a highly conserved lncRNA, is widely known to promote cancer development, one mechanism for which may be through the regulation of alternative splicing and, thereby, gene expression. Its regulatory interactions with proteins have been a subject of much interest, yet little research has been carried out on the mechanisms adopted. It has been observed that MALAT1 binds to RNA-binding protein with serine-rich domain 1 (RNPS1), being colocalized in the nuclear speckles, and together, these two binding partners may regulate alternative splicing. Upregulated RNPS1 is predicted to play a key role in the pan-cancer development. Experimental tertiary structure of full-length MALAT1 is currently lacking despite the availability of the 3D structure of 3' expression and nuclear retention element. We hypothesize that the computationally modeled tertiary structures of the specific binding motifs in the M-region, E-region, and full-length structures of MALAT1 may adopt a modular structure and bind to the RNPS1 loop region of RS/P domain involved in exon skipping, interacting in a manner fully consistent with the biochemical experiments. Extensive observations using the powerful molecular dynamics (MD) simulations of MALAT1 regions bound to RNPS1 suggested that all three regions form interactive, yet stable complexes. The ranking of the MM-GBSA- and MM-PBSA-derived binding free energies between these complexes corroborated well in the MD simulations and experiments. Energy decomposition analyses suggested that arginines in the RNPS1 protein are among the major contributors toward the binding free energies as calculated by MM-GBSA present in the Amber package; while among the nucleotides, the major contributors were nucleotides with G and A nucleobases, with more contributory effect in comparison to arginines, across the bound M-region, E-region, and full-length MALAT1. This suggests that specific purines play a greater role in the complex formation, in a loop-specific manner, and the more proactive approach in complexation tilts toward MALAT1. To the best of our knowledge, our studies are the first studies taking a unique approach, utilizing the binding motifs to deduce a tertiary structure of MALAT1, toward our understanding of the lncRNA-protein interactions, stability, and binding on a structural basis. The therapeutic implications of targeting this complex formation to regulate splicing and hence, oncogenesis, is further envisaged.

In situ crystalline structure of the human eosinophil major basic protein-1.

Eosinophils are white blood cells that participate in innate immune responses and have an essential role in the pathogenesis of inflammatory and neoplastic disorders. Upon activation, eosinophils release cytotoxic proteins such as major basic protein-1 (MBP-1) from cytoplasmic secretory granules (SGr) wherein MBP-1 is stored as nanocrystals. How the MBP-1 nanocrystalline core is formed, stabilized, and subsequently mobilized remains unknown. Here, we report the in-situ structure of crystalline MBP-1 within SGrs of human eosinophils. The structure reveals a mechanism for intragranular crystal packing and stabilization of MBP-1 via a structurally conserved loop region that is associated with calcium-dependent carbohydrate binding in other C-type lectin (CTL) proteins. Single-cell and single-SGr profiling correlating real-space three-dimensional information from cellular montage cryo-electron tomography (cryo-ET) and microcrystal electron diffraction (MicroED) data obtained from non-activated and IL33-activated eosinophils revealed activation-dependent crystal expansion and extrusion of expanded crystals from SGr. These results suggest that MBP-1 crystals play a dynamic role in the release of SGr contents. Collectively, this research demonstrates the importance of in-situ macromolecular structure determination.