Protein Secondary Structure Research Articles

Secondary Structure Detection and Structure Modeling for Cryo-EM.

Rapid advancements in cryogenic electron microscopy (cryo-EM) have revolutionized the field of structural biology by enabling the determination of complex macromolecular structures at unprecedented resolutions. When cryo-EM density maps have a resolution around 3Å, the atomic structure can be modeled manually. However, as the resolution decreases, analyzing these density maps becomes increasingly challenging. For modeling structures in lower resolution maps, deep learning can be used to identify structural features in the maps to assist in structure modeling.Here, we present a suite of deep learning-based tools developed by our lab that enable structural biologists to work with cryo-EM maps of a wide range of resolutions. For cryo-EM maps at near-atomic resolution (5Å or better), DeepMainmast automatically models all-atom structures by tracing the main chain from local map features of amino acids and atoms detected by deep learning; DAQ score quantifies map-model fit and indicates potential misassignments in protein models. In intermediate resolution maps (5-10Å), Emap2sec and Emap2sec+ can accurately detect protein secondary structures and nucleic acids. These tools and more are available at our web server: https://em.kiharalab.org/ .

Coherent Raman microscopy visualizes ongoing cellular senescence through amide I peak shifts originating from β sheets in disordered nucleolar proteins

AbstractCellular senescence occurs through the accumulation of many kinds of stresses. Senescent cells in tissues also cause various age-related disorders. Therefore, detecting them without labeling is beneficial for medical research and developing diagnostic methods. However, existing biomarkers have limitations of requiring fixation and labeling, or their molecular backgrounds are uncertain. Coherent anti-Stokes Raman scattering (CARS) spectroscopic imaging is a novel option because it can assess and visualize molecular structures based on their molecular fingerprint. Here, we present a new label-free method to visualize cellular senescence using CARS imaging in nucleoli. We found the peak of the nucleolar amide I band shifted to a higher wavenumber in binuclear senescent cells, which reflects changes in the protein secondary structure from predominant α-helices to β-sheets originating from amyloid-like aggregates. Following this, we developed a procedure that can visualize the senescent cells by providing the ratios and subtractions of these two components. We also confirmed that the procedure can visualize nucleolar aggregates due to unfolded/misfolded proteins produced by proteasome inhibition. Finally, we found that this method can help visualize the nucleolar defects in naïve cells even before binucleation. Thus, our method is beneficial to evaluate ongoing cellular senescence through label-free imaging of nucleolar defects.

Structural diversity of Alzheimer-related protein aggregations revealed using photothermal ratio-metric micro-spectroscopy

The crucial link between pathological protein aggregations and lipids in Alzheimer’s disease pathogenesis is increasingly recognized, yet its spatial dynamics remain challenging for labeling-based microscopy. Here, we demonstrate photothermal ratio-metric infrared spectro-microscopy (PRISM) to investigate the in situ structural and molecular compositions of pathological features in brain tissues at submicron resolution. By identifying the vibrational spectroscopic signatures of protein secondary structures and lipids, PRISM tracks the structural dynamics of pathological proteins, including amyloid and hyperphosphorylated Tau (pTau). Amyloid-associated lipid features in major brain regions were observed, notably the enrichment of lipid-dissociated plaques in the hippocampus. Spectroscopic profiling of pTau revealed significant heterogeneity in phosphorylation levels and a distinct lipid-pTau relationship that contrasts with the anticipated lipid-plaque correlation. Beyond in vitro studies, our findings provide direct visualization evidence of aggregate-lipid interactions across the brain, offering new insights into mechanistic and therapeutic research of neurodegenerative diseases.

RNA editing-induced structural and functional adaptations of NAD9 in Triticum aestivum under drought stress

IntroductionMitochondria are essential organelles in eukaryotic cells, producing ATP through the electron transport chain to supply energy for cellular activities. Beyond energy production, mitochondria play crucial roles in cellular signaling, stress responses, and the regulation of reactive oxygen species. In plants, mitochondria are one of the keys to responding to environmental stresses which can significantly affect crop productivity, particularly in crops like wheat. RNA editing, a post-transcriptional RNA modification process in mitochondria, is linked to regulating these stress responses.MethodsThis study explores RNA editing patterns in the nad9 gene of wheat drought-tolerant (Giza168) and drought-sensitive (Gemmiza10) wheat cultivars under drought stress to understand plant adaptation mechanisms. RNA-seq data for these cultivars were analyzed using CLC Genomic Workbench to identify RNA editing sites in the nad9 gene, examining subsequent amino acid changes and predicting secondary structure modifications. These RNA editing sites were validated using qRT-PCR on drought-treated seedlings at 0, 2, and 12 hours post-treatment. Protein models were generated using AlphaFold, with functional predictions and structure verification conducted using various bioinformatics tools to investigate the effect of RNA editing on protein level.ResultsThe results showed significant RNA editing events, especially C-to-T conversions, in the nad9 gene across different drought exposure times. Giza168 had 22 editing sites, while Gemmiza10 had 19, with several showing significant differences between control and stress conditions. RNA editing influenced the NAD9 protein's secondary structure, particularly beta sheets, and 3D modeling highlighted the structural impacts of these edits. The N-terminal region of NAD9 contained important regulatory motifs, suggesting a complex regulatory environment.DiscussionThis study reveals key editing sites that differ between drought-tolerant and sensitive wheat cultivars, impacting NAD9 protein structures and highlighting the role of RNA editing in enhancing drought resilience. Additionally, the study suggests potential regulatory mechanisms, including phosphorylation and ubiquitination that influence mitochondrial stability and function.

Influence of protein in low paste viscosities of Bambara groundnut flours from heat-treated Bambara groundnut seeds

Heat treatment of Bambara groundnut seeds has been reported to cause low paste viscosities in resulting flours. Structural changes in Bambara groundnut protein, due to heat treatment, causes the protein to encapsulate starch, making it unavailable to paste causing low paste viscosities. In this study, trypsin was used to hydrolyze proteins in the flour and the microstructure analysis confirmed the disappearance of aggregates. Flour microstructure analysis confirmed hydrolysis of protein from previously aggregated status and showed liberated individual starch granules. Following the treatment of flours by trypsin, Confocal laser scanning microscopy did not show a protein signal. Hydrolysing Bambara groundnut proteins significantly increased the flour paste viscosities (P < 0.05). The final viscosities for flours from 20 % moisture-conditioned and infrared heat-treated seeds for 5 min were 733.9 mPa s before protein hydrolysis and 2081.71 mPa s after protein hydrolysis. The gelatinization temperature (81 °C) did not show a significant change following protein hydrolysis. Sodium dodecyl-sulphate polyacrylamide gel electrophoresis band intensity increased indicative of disulfide bonding and protein polymerisation when microwave and infrared heat treatment were combined. There were changes in Bambara groundnut protein secondary structures such as an increase of 57 % in β-sheet along with a 60 % reduction in the α-helix as shown by the Fourier-transform infrared spectroscopy. The changes in secondary structure of Bambara groundnut protein were caused by microwave and infrared heating. Heat treatment of Bambara groundnut seeds is partly responsible for the reduction in paste viscosities of their flours.

MIPPIS: protein–protein interaction site prediction network with multi-information fusion

BackgroundThe prediction of protein–protein interaction sites plays a crucial role in biochemical processes. Investigating the interaction between viruses and receptor proteins through biological techniques aids in understanding disease mechanisms and guides the development of corresponding drugs. While various methods have been proposed in the past, they often suffer from drawbacks such as long processing times, high costs, and low accuracy.ResultsAddressing these challenges, we propose a novel protein–protein interaction site prediction network based on multi-information fusion. In our approach, the initial amino acid features are depicted by the position-specific scoring matrix, hidden Markov model, dictionary of protein secondary structure, and one-hot encoding. Simultaneously, we adopt a multi-channel approach to extract deep-level amino acids features from different perspectives. The graph convolutional network channel effectively extracts spatial structural information. The bidirectional long short-term memory channel treats the amino acid sequence as natural language, capturing the protein’s primary structure information. The ProtT5 protein large language model channel outputs a more comprehensive amino acid embedding representation, providing a robust complement to the two aforementioned channels. Finally, the obtained amino acid features are fed into the prediction layer for the final prediction.ConclusionCompared with six protein structure-based methods and six protein sequence-based methods, our model achieves optimal performance across evaluation metrics, including accuracy, precision, F1, Matthews correlation coefficient, and area under the precision recall curve, which demonstrates the superiority of our model.

Identification of Laccase Family of Auricularia auricula-judae and Structural Prediction Using Alphafold.

Laccase is an enzyme that plays an important role in fungi, including lignin degradation, stress defense, and formation of fruiting bodies. Auricularia auricula-judae is a white-rot fungus in the Basidiomycota phylum, capable of delignifying wood. In this study, seven genes belonging to the laccase family were identified through de novo sequencing, containing Cu-Oxidase, Cu-Oxidase_2, and Cu-Oxidase_3 domains. Subsequently, the physical characteristics, phylogenetic relationships, protein secondary structure, and tertiary structure of the laccase family (AaLac1-AaLac7) were analyzed. Prediction of N-glycosylation sites identified 2 to 10 sites in the laccase family, with AaLac7 having the highest number of sites at 10. Sequence alignment and analysis of the laccase family showed high consistency in signature sequences. Phylogenetic analysis confirmed the relationship among laccases within the family, with AaLac3-AaLac4 and AaLac5-AaLac6 being closely positioned on the tree, exhibiting high similarity in tertiary structure predictions. This study identified and analyzed laccase family genes in Auricularia auricula-judae using de novo sequencing, offering a simple method for identifying and analyzing the laccase family in organisms with unknown genetic information.

Quality variation analysis and rehydration kinetics modeling of yuba subjecting to three different drying process

This study investigated the effects of hot air drying (HAD), microwave vacuum drying (MVD), and vacuum frying drying (VFD) on the quality of yuba and utilized mathematical models (Peleg model, Weibull equation, first-order kinetic model, and exponential association model) to explore the rehydration kinetics of yuba. The results indicated that, compared to HAD, MVD significantly increased yuba's L* value and sensory score, while VFD notably reduced the cooking loss rate and enhanced moisture retention. Significant changes were observed in the texture of yuba before and after cooking, with distinct differences noted under microscopic observation, and a pronounced change in protein secondary structure. The Peleg model can effectively fit the rehydration processes of HAD and VFD yuba, while the first-order kinetic model can effectively fit the rehydration processes of MVD yuba. This study provides a valuable reference for yuba's drying method and rehydration Kinetics model.

Inhibitory mechanism of apigenin, quercetin, and phloretin on α-glucosidase

Flavonoids present promising potential as α-glucosidase inhibitors, with potential benefits in lowering blood glucose levels. In our previous study, apigenin, quercetin, and phloretin obtained from whole-grain highland barley demonstrated notable efficacy as α-glucosidase inhibitors. Therefore, in this study, we used enzyme kinetics, fluorescence spectroscopy, circular dichroism, and molecular docking to explore the interactions between apigenin, quercetin, and phloretin, and α-glucosidase. With the addition of three flavonoids, the inhibitory effect of all the flavonoids on α-glucosidase activity was reversible and exhibited a mixed-type pattern. Molecular docking analysis revealed that the three flavonoids predominantly bind to yeast-derived α-glucosidase mainly through hydrophobic interactions and van der Waals forces, whereas the interaction with human-derived α-glucosidase involved hydrophobic interactions. Further experiments revealed that the flavonoids quenched the fluorescence of α-glucosidase due to the complex formation between the flavonoids and α-glucosidase, leading to changes in the protein secondary structure and conformational changes, which was further supported by molecular dynamics simulations. These results provided insights into the mechanism of the flavonoid inhibition of α-glucosidase. These findings could promote the consumption of whole-grain highland barley and the development of hypoglycemic foods rich in flavonoids.

The anti-fouling activity of solvothermally synthesized Nb2O5-coated Fe ship strip on optimal interactions at the targeted interfaces of barnacle attachments -An Insilico study

Abstract Fouling is a major issue occurring in water-going vessels, such as ships that cause increased surface roughness and drag resistance. The fouling organisms produce extracellular polymeric substances (EPS), which negatively impact water-going vessels. The settlement-inducing protein complex (SIPC) is a contact pheromone that promotes the gregarious settling of barnacle larvae (cyprids). The SIPC can be found in both adult barnacle cuticles and cyprids as transient adhesive secretions (footprints). The presence of SIPC in the footprints plays a critical role during the initial adhesion, which facilitates further settlement. The adsorption of of SIPC on Iron/Fe ship strip(FSS) surface was often found to be irreversible even after physical treatements. For the antifouling studies, Nb2O5 coated FSS were constructed and simulated to analyze the interaction of barnacles Aacp20K protein. For simulation studies, the homology model of barnacles Aacp20K protein is fabricated using the SWISS automated comparative modeling platform. The result of homology model showed a good 3D secondary structure of Aacp20K protein, especially 7q1y template protein. Adsorption location analysis results illustrate that the surface of the FSS coated with Nb2O5 film disfavour the binding of SIPC inhibiting the binding of barnacle cuticles and cyprids. For validating the simulation results, Nb2O5 nanostructure film was synthesized using a solvothermal process and characterized using XRD,SEM and EDS. Furthermore, the wetting behaviour was studied experimentally. The simulations and experimental results indicate Nb2O5-coated FSS as potent anti-fouling surfaces.

Monomer unfolding of a bacterial ESCRT-III superfamily member is coupled to oligomer disassembly.

The inner membrane associated protein of 30 kDa (IM30), a member of the endosomal sorting complex required for transport (ESCRT-III) superfamily, is crucially involved in the biogenesis and maintenance of thylakoid membranes in cyanobacteria and chloroplasts. In solution, IM30 assembles into various large oligomeric barrel- or tube-like structures, whereas upon membrane binding it forms large, flat carpet structures. Dynamic localization of the protein in solution, to membranes and changes of the oligomeric states are crucial for its in vivo function. ESCRT-III proteins are known to form oligomeric structures that are dynamically assembled from monomeric/smaller oligomeric proteins, and thus these smaller building blocks must be assembled sequentially in a highly orchestrated manner, a still poorly understood process. The impact of IM30 oligomerization on function remains difficult to study due to its high intrinsic tendency to homo-oligomerize. Here, we used molecular dynamics simulations to investigate the stability of individual helices in IM30 and identified unstable regions that may provide structural flexibility. Urea-mediated disassembly of the IM30 barrel structures was spectroscopically monitored, as well as changes in the protein's tertiary and secondary structure. The experimental data were finally compared to a three-state model that describes oligomer disassembly and monomer unfolding. In this study, we identified a highly stable conserved structural core of ESCRT-III proteins and discuss the advantages of having flexible intermediate structures and their putative relevance for ESCRT-III proteins.

High-intensity ultrasound-assisted alkaline extraction of soy protein: Optimization, modeling, physicochemical and functional properties

This study examined the effect of high-intensity ultrasound-assisted alkaline extraction (HUAE) on the extraction yield and the physicochemical and functional properties of soy protein (SP) using the two-pot multivariate method for the first time. Plackett-Burman Design (PBD) coupled with Response Surface Methodology (RSM) was systematically utilized to select and subsequently optimize the HUAE parameters. Based on PBD results, the significant extraction factors were liquid to solid ratio (LSR), temperature, ultrasonic amplitude, and extraction time. The optimum conditions for the maximal extraction yield and minimal energy consumption were 50:1 mL/g LSR, 50 °C, 48 % ultrasonic amplitude, and 10 min extraction time. At optimum conditions, the extraction yield (35.28 %) was significantly improved compared to traditional extraction (26.39 %). Besides, HUAE resulted in modification of the protein secondary and tertiary structures due to the unfolding of protein molecules and the exposure of hydrophobic groups or regions as shown by FTIR spectroscopy, free sulfhydryl analysis, and scanning electron microscopy. These structural changes led to decreased solubility and emulsifying activity but improved emulsion stabilization and antioxidant properties. With future development, HUAE could potentially produce soy protein for targeted applications, broadening its utilization and meeting the need for more sustainable alternative processing.

Spectroscopic Studies on Maillard Reactions of Peanut Ara h 2.01 With Respective Reducing Sugars and Their Impacts on Ara h 2.01 Allergenicity.

The effects of the Maillard reactions of peanut Ara h 2.01 with respective glucose and xylose on its conformation and allergenicity were investigated. Circular dichroism (CD) spectral studies showed that short-term heating alone with reducing sugars did not change protein secondary structures, but after undergoing Maillard reactions, its secondary structures changed with some α-helices being transformed into β-sheets and random coils. Fluorescence spectral studies indicated that as temperature increased, protein became loose and extended induced by the reducing sugars. Long heat treatment at higher temperature caused protein collapse with the extrusion of its internal water molecules, which was hindered partially by the reducing sugars that entered the protein in Maillard reaction. Resonance light scattering (RLS) spectra confirmed that more xylose molecules entered protein due to its stronger interaction with the protein after Maillard reactions. ELISA assays exhibited that heat treatment at 100°C led to some allergenic epitopes being embedded inside protein molecules due to protein collapse and that partial epitopes were covered by the reducing sugars after Maillard reaction, resulting in decreases in its allergenicity. Also, xylose reduced the allergenicity of protein more efficiently owing to the stronger interaction between them. All above experimental results were explained reasonably by bioinformatic analysis and molecular docking.

Different nitrogen conditions regulating extracellular polymeric substances and erosion resistance of sewer sediment: Mechanism of microbial metabolism and molecular response

Nitrogen biotransformation plays a vital role in the metabolism of microbial communities in sewers. Extracellular polymeric substances (EPS) secreted by microbial communities can form gel-like sewer sediments, causing clogging of the sewer. However, knowledge on the effects of varying nitrogen conditions on the erosion resistance of sewer sediments and EPS produced by sewer microorganisms is limited. In this study, two typical organic/inorganic nitrogen ratios of sewage were reproduced in simulated sewer reactors, i.e., 3/7 (R1 group) and 7/3 (R2 group). Higher organic nitrogen (ON) concentrations were found to increase the critical erosion shear stress by 26.43%; this was ascribed to increased particle diameter, weakened electrostatic repulsion of sediments and stimulated EPS secretion in the R2 group. The protein and polysaccharide contents of the R2 group were 48.84% and 34.25% higher than those of the R1 group, respectively, which was supported by increased gene abundances for aromatic amino acid synthesis, general secretory pathways of protein, and synthesis of precursors and polysaccharides. Tightly-bound EPS in R2 group exhibited increased contents of hydrophobic protein secondary structures and intermolecular hydrogen bonds, thereby promoting the formation of gel-like sediment structures with enhanced erosion resistance. However, the microbial diversity and the abundance of key genes involved in EPS generation and secretion (e.g., tyrB, yajC, secB, gumF, and gumH) obviously decreased in the R1 group. Moreover, high ON concentrations increased microbial diversity and enhanced microbial glycolysis, tricarboxylic acid cycle and ammonium assimilation. This study reveals the formation mechanisms of EPS in sewer sediments under different nitrogen conditions and their effects on sediment erosion resistance, which contributed to improved sewer system operation and sewer sediment control.

Sludge dewaterability improvement in microbial fuel cell powered electro-Fenton system (MFCⓅEFs) coupled with chitosan quaternary ammonium salt

As improved and expanded wastewater treatment facilities, sludge dewatering is the essential process and major challenge due to the complex and abundant organic matter. Microbial fuel cell powered electro-Fenton system (MFCⓅEFs) has been demonstrated to generate •OH and destroy hydrophilic extracellular polymeric substances (EPS) as an improved method of high efficiency and low energy consumption. Nevertheless, the smaller particle size of the treated sludge and the release of partial organic matter into the supernatant can affect the sludge-water separation process. Chitosan quaternary ammonium salt (CQAS) as a renewable and biodegradability cationic coagulant can be involved in the follow-up treatment. The sludge after combined treatment exhibited better dewaterability, where the water content of sludge cake (WCSC), capillary suction time (CST), and specific resistance filtration (SRF) were 61.21%, 15.6 s, and 1.02×1012 m/kg (26.47%, 80.72% and 84.28% reduction), respectively. The oxidation of the MFCⓅEFs destroyed the cellular and EPS structure and the bridging flocculation and charge neutralization of CQAS caused the fine particles to squeeze each other. The combined treatment fully reduced the content of negative charge and hydrophilic substances on the surface, which affected the intensity change of N-H bond and altered the protein secondary structure to make it looser, thus releasing more bound water while improving aggregation characteristics. Overall, combined treatment can significantly improve sludge dewaterability and owns operational advantages of high efficiency, low energy and consumption, safety, and non-toxicity.

A strategy for improving the mechanical properties of silk fibers through the combination of genetic manipulation and zinc ion crosslinking

Silk fiber is generally considered an excellent biological material due to its good biocompatibility, morphological plasticity and biodegradability. Previously, the construction of silkworm silk gland bioreactors based on the piggyBac transposon has been optimized. However, the inserted exogenous genes have problems such as position uncertainty, and expression is not strictly controlled. Here, we applied transcription activator-like effector nuclease (TALEN)-mediated homology-directed repair (HDR) to precisely insert histidine-rich cuticular protein (CP) into silkworm Sericin1 (Ser1) gene. The Ser1-CP fusion protein was successfully secreted into cocoon shell. Subsequently, based on the metal coordination ability of the histidine imidazole group, we crosslinked cocoon with metal ions in vitro. In this strategy, the mechanical properties of the fused silk fibers with crosslinked Zn2+ improved, and the maximum breaking stress of the crosslinked Zn2+-fused silk fibers was 23.5 % greater than that of the wild-type fibers. Analysis of the secondary structure of the silk protein showed that the fused silk fibers crosslinked with Zn2+ had more β-sheet structures. This study pioneered a method of improving the mechanical properties of silk fibers by crosslinking metal ions with fused exogenous proteins and expanded the application value of silk gland bioreactors in the development of novel biomaterials.

Research on the spoilage potential and metabolic patterns of specific spoilage bacteria in grouper (Epinephelus lanceolatus) during cold storage

Pseudomonas fragi and Serratia liquefaciens are key spoilage microorganisms in aerobically packed groupers during cold storage. Spoilage bacteria were inoculated into fillets to investigate their spoilage potential. The results showed that P. fragi had stronger degradation activity, and the P. fragi inoculated group had higher total volatile basic nitrogen (TVB-N) and K values, lower water holding capacity (WHC), and textural qualities. The P. fragi inoculated group had higher levels of carbonyl groups and lower levels of sulfhydryl groups, indicating higher levels of protein oxidation. The secondary and tertiary structure of myofibrillar protein (MP), scanning electron microscopy, and low-field nuclear magnetic resonance (LF-NMR) analyses showed that P. fragi degraded myofibrils more and induced water loss from the muscle. Metabolomics analyses indicated that purine metabolism and glycine, serine, and threonine metabolism were important pathways associated with P. fragi spoilage; linoleic acid metabolism and β-alanine metabolism were important pathways for S. liquefaciens spoilage.

Effect of Succinylation on Oxidation-Aggregation of Low-Density Lipoprotein and Formation of Off-Flavors in Heated Egg Yolks.

This study examined the effect of succinylation on protein oxidation-aggregation and the formation of off-flavors in heated egg yolks (EYs). The sensory evaluation, content of volatile compounds, stability of low-density lipoprotein (LDL) particles, and oxidation of lipid and protein at six levels of succinylated EY (0.25%, 0.5%, 1%, 2%, 5%, and 10%, w/w) were determined. The results showed that the succinylated thermal EY's concentration of volatiles and off-flavors was reduced. Oil exudation and lipid and protein oxidation decreased with the improved succinylation degree. Succinylation also reduced the LDL particle size and changed the secondary structure (decreased the β-sheets and increased the α-helices) of protein in LDL particles. Meanwhile, succinylation could effectively change the thermal oxidation-aggregation of LDL protein by introducing succinyl groups with negative charges, thus increasing the stability of LDL particles in succinylated EY during heating. These results further revealed the relationship between the oxidation-aggregation of LDL and the formation of off-flavors in heated EY. These results also help improve the flavor quality of heat-treated EY and expands the application scope of egg products.

From Zebrafish To Humans: In Silico Comparative Study of RAD50 Sequences

DNA damage, particularly the occurrence of DNA double-strand breaks (DSBs), presents a significant hazard to the integrity and viability of cells. Improper repair of DSBs can result in chromosomal alterations, oncogenic changes, or cell demise. The MRE11-RAD50-NBS1 (MRN) complex plays a crucial role in DNA repair and signaling under the Ataxia Telangiectasia Mutated (ATM) kinase regulation. In this study, we employed comprehensive computational techniques to analyze the structure of RAD50 in Danio rerio (Zebrafish), utilized as a model organism. Additionally, we conducted in silico assessments of RAD50 from both Zebrafish and humans, comparing their characteristics. The substantial sequence resemblance between DrRAD50 and HsRAD50 suggests that DrRAD50 could potentially serve as a valuable model for HsRAD50. However, it is important to acknowledge that sequence similarity alone does not necessarily imply functional equivalence. Further functional studies are needed to confirm the extent of their functional similarities. By examining the secondary and tertiary protein structures of RAD50, we observed a notable likeness between Zebrafish and Human RAD50 proteins. In silico analysis demonstrated that the sequence of RAD50 in zebrafish shares 70% similarity with the human RAD50 protein.

A comprehensive and single-use foot-and-mouth disease sero-surveillance prototype employing rationally designed multiple viral antigens

Foot-and-mouth disease (FMD) is a great havoc in agri-business-based countries like Bangladesh, for which existing detection system limits the identification and differentiation of serotypes. In this study, an engineered platform was introduced incorporating serotype-specific FMDV VP1 (structural), serotype-independent VP2 (structural) and 3AB (non-structural) proteins for holistic detection. VP1 sequences were engineered combining sequences of BAN/TA/Dh-301/2016 (serotype O), BAN/CH/Sa-304/2016 (serotype A) and BAN/DH/Sa-318/2016 (serotype Asia1). Consensus 3AB sequence was constructed from the selected prevalent viral genomes. Both VP1 and 3AB along with designed VP2 sequences were optimized for codon usage bias, stable mRNA, secondary and tertiary protein structure. Proteins were synthesized in pET-21a ( +) plasmid vector followed by transformation of Escherichia coli BL21(DE3) and IPTG-induced- expression. The western blot analysis of engineered proteins showed that purified VP1 prominently bound to anti-VP1 antibodies in vaccinated sera, whereas 3AB and VP2 bound anti-3AB and anti-VP2 antibodies, respectively from infected cattle sera, all previously collected during epidemiological investigation. Furthermore, dot blot hybridization confirmed efficient antibody capture ability of the membrane-immobilized proteins. This holistic diagnostic platform justifies a comprehensive prototype diagnostic kit that would be cost-effective and efficient for serotype specific and non-specific FMDV sero-surveillance.