Phylogenetic analysis of phytochrome A gene from Lablab purpureus (L.) Sweet

Background Phytochromes are the best characterized photoreceptors that perceive Red (R)/Far-Red (FR) signals and mediate key developmental responses in plants. It is well established that photoperiodic control of flowering is regulated by PHY A (phytochrome A) gene. So far, the members of PHY A gene family remains unexplored in Lablab purpureus, and therefore, their functions are still not deciphered. PHYA3 is the homologue of phytochrome A and known to be involved in dominant suppression of flowering under long day conditions by downregulating florigens in Glycine max. The present study is the first effort to identify and characterize any photoreceptor gene (PHYA3, in this study) in Lablab purpureus and decipher its phylogeny with related legumes. Results PHYA3 was amplified in Lablab purpureus cv GNIB-21 (photo-insensitive and determinate) by utilizing primers designed from GmPHYA3 locus of Glycine max. This study was successful in partially characterizing PHYA3 in Lablab purpureus (LprPHYA3) which is 2 kb longer and belongs to exon 1 region of PHYA3 gene. Phylogenetic analysis of the nucleotide and protein sequences of PHYA genes through MEGA X delineated the conservation and evolution of Lablab purpureus PHYA3 (LprPHYA3) probably from PHYA genes of Vigna unguiculata, Glycine max and Vigna angularis. A conserved basic helix-loop-helix motif bHLH69 was predicted having DNA binding property. Domain analysis of GmPHYA protein and predicted partial protein sequence corresponding to exon-1 of LprPHYA3 revealed the presence of conserved domains (GAF and PAS domains) in Lablab purpureus similar to Glycine max. Conclusion Partial characterization of LprPHYA3 would facilitate the identification of complete gene in Lablab purpureus utilizing sequence information from phylogenetically related species of Fabaceae. This would allow screening of allelic variants for LprPHYA3 locus and their role in photoperiod responsive flowering. The present study could aid in modulating photoperiod responsive flowering in Lablab purpureus and other related legumes in near future through genome editing.

Development of Cobalt-Binding Peptide Chelate from Human Serum Albumin: Cobalt-Binding Properties and Stability

Radioactive isotopes are used as drugs or contrast agents in the medical field after being conjugated with chelates such as DOTA, NOTA, DTPA, TETA, CyDTA, TRITA, and DPDP. The N-terminal sequence of human serum albumin (HSA) is known as a metal binding site, such as for Co2+, Cu2+, and Ni2+. For this study, we designed and synthesized wAlb12 peptide from the N-terminal region of HSA, which can bind to cobalt, to develop a peptide-based chelate. The wAlb12 with a random coil structure tightly binds to the Co(II) ion. Moreover, the binding property of wAlb12 toward Co(II) was confirmed using various spectroscopic experiments. To identify the binding site of wAlb12, the analogs were synthesized by alanine scanning mutagenesis. Among them, H3A and Ac-wAlb12 did not bind to Co(II). The analysis of the binding regions confirmed that the His3 and α-amino group of the N-terminal region are important for Co(II) binding. The wAlb12 bound to Co(II) with Kd of 75 μM determined by isothermal titration calorimetry when analyzed by a single-site binding model. For the use of wAlb12 as a chelate in humans, its cytotoxicity and stability were investigated. Trypsin stability showed that the wAlb12 − Co(II) complex was more stable than wAlb12 alone. Furthermore, the cell viability analysis showed wAlb12 and wAlb12 + Co(II) to be non-toxic to the Raw 264.7 and HEK 293T cell lines. Therefore, a hot radioactive isotope such as cobalt-57 will have the same effect as a stable isotope cobalt. Accordingly, we expect wAlb12 to be used as a peptide chelate that binds with radioactive isotopes.

Structure Based Innovative Approach to Analyze Aptaprobe-GPC3 Complexes in Hepatocellular Carcinoma

Background Glypican-3 (GPC3), a membrane-bound heparan sulfate proteoglycan, is a biomarker of hepatocellular carcinoma (HCC) progression. Aptamers specifically binding to target biomolecules have recently emerged as clinical disease diagnosis targets. Here, we describe 3D structure-based aptaprobe platforms for detecting GPC3, such as aptablotting, aptaprobe-based sandwich assay (ALISA), and aptaoptical imaging analysis. Results For preparing the aptaprobe–GPC3 platforms, we obtained 12 high affinity aptamer candidates (GPC3_1 to GPC3_12) that specifically bind to target GPC3 molecules. Structure-based molecular interactions identified distinct aptatopic residues responsible for binding to the paratopic nucleotide sequences (nt-paratope) of GPC3 aptaprobes. Sandwichable and overlapped aptaprobes were selected through structural analysis. The aptaprobe specificity for using in HCC diagnostics were verified through Aptablotting and ALISA. Moreover, Aptaoptical imaging showed that the binding property of GPC3_3 and their GPC3 specificity were maintained in HCC xenograft models, which may indicate a new HCC imaging diagnosis.Conclusion Aptaprobe has the potential to be used as an affinity reagent to detect the target in vivo and in vitro diagnosing system.

Hotspots for mutations in the SARS-CoV-2 spike glycoprotein: a correspondence analysis

Spike glycoprotein (Sgp) is liable for binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to the host receptors. Since Sgp is the main target for vaccine and drug designing, elucidating its mutation pattern could help in this regard. This study is aimed at investigating the correspondence of specific residues to the SgpSARS-CoV-2 functionality by explorative interpretation of sequence alignments. Centrality analysis of the Sgp dissects the importance of these residues in the interaction network of the RBD-ACE2 (receptor-binding domain) complex and furin cleavage site. Correspondence of RBD to threonine500 and asparagine501 and furin cleavage site to glutamine675, glutamine677, threonine678, and alanine684 was observed; all residues are exactly located at the interaction interfaces. The harmonious location of residues dictates the RBD binding property and the flexibility, hydrophobicity, and accessibility of the furin cleavage site. These species-specific residues can be assumed as real targets of evolution, while other substitutions tend to support them. Moreover, all these residues are parts of experimentally identified epitopes. Therefore, their substitution may affect vaccine efficacy. Higher rate of RBD maintenance than furin cleavage site was predicted. The accumulation of substitutions reinforces the probability of the multi-host circulation of the virus and emphasizes the enduring evolutionary events.

A Commitment Scheme with Output Locality-3 Fit for the IoT Device

Low output locality is a property of functions, in which every output bit depends on a small number of input bits. In IoT devices with only a fragile CPU, it is important for many IoT devices to cooperate to execute a single function. In such IoT’s collaborative work, a feature of low output locality is very useful. This is why it is desirable to reconstruct cryptographic primitives with low output locality. However, until now, commitment with a constant low output locality has been constructed by using strong randomness extractors from a nonconstant-output-locality collision-resistant hash function. In this paper, we construct a commitment scheme with output locality-3 from a constant-output-locality collision-resistant hash function for the first time. We prove the computational hiding property of our commitment by the decisional M , δ -bSVP assumption and prove the computational binding property by the M , δ -bSVP assumption, respectively. Furthermore, we prove that the M , δ -bSVP assumption can be reduced to the decisional M , δ -bSVP assumption. We also give a parameter suggestion for our commitment scheme with the 128 bit security.

Temperature-Responsive Magnetic Nanoparticles for Bioanalysis of Lysozyme in Urine Samples

Highly selective multifunctional magnetic nanoparticles containing a thermoresponsive polymer shell were developed and used in the sample pretreatment of urine for the assessment of lysozymuria in leukemia patients. Crosslinked poly(N-isopropylacrylamide-co-acrylic acid-co-N-tert-butylacrylamide) was grown onto silica-coated magnetic nanoparticles by reversible addition fragmentation chain transfer (RAFT) polymerization. The lysozyme binding property of the nanoparticles was investigated as a function of time, protein concentration, pH, ionic strength and temperature and their selectivity was assessed against other proteins. High-abundant proteins, like human serum albumin and γ-globulins did not interfere with the binding of lysozyme even at elevated concentrations characteristic of proteinuria. A sample cleanup procedure for urine samples has been developed utilizing the thermocontrollable protein binding ability of the nanoparticles. Method validation was carried out according to current bioanalytical method validation guidelines. The method was highly selective, and the calibration was linear in the 25 to 1000 µg/mL concentration range, relevant in the diagnosis of monocytic and myelomonocytic leukemia. Intra- and inter-day precision values ranged from 2.24 to 8.20% and 1.08 to 5.04%, respectively. Intra-day accuracies were between 89.9 and 117.6%, while inter-day accuracies were in the 88.8 to 111.0% range. The average recovery was 94.1 ± 8.1%. Analysis of unknown urine samples in comparison with a well-established reference method revealed very good correlation between the results, indicating that the new nanoparticle-based method has high potential in the diagnosis of lysozymuria.

Structure of Aedes aegypti procarboxypeptidase B1 and its binding with Dengue virus for controlling infection

Metallocarboxypeptidases play critical roles in the development of mosquitoes and influence pathogen/parasite infection of the mosquito midgut. Here, we report the crystal structure of Aedes aegypti procarboxypeptidase B1 (PCPBAe1), characterized its substrate specificity and mechanism of binding to and inhibiting Dengue virus (DENV). We show that the activated PCPBAe1 (CPBAe1) hydrolyzes both Arg- and Lys-substrates, which is modulated by residues Asp251 and Ser239. Notably, these residues are conserved in CPBs across mosquito species, possibly required for efficient digestion of basic dietary residues that are necessary for mosquito reproduction and development. Importantly, we characterized the interaction between PCPBAe1 and DENV envelope (E) protein, virus-like particles, and infectious virions. We identified residues Asp18A, Glu19A, Glu85, Arg87, and Arg89 of PCPBAe1 are essential for interaction with DENV. PCPBAe1 maps to the dimeric interface of the E protein domains I/II (Lys64–Glu84, Val238–Val252, and Leu278–Leu287). Overall, our studies provide general insights into how the substrate-binding property of mosquito carboxypeptidases could be targeted to potentially control mosquito populations or proposes a mechanism by which PCPBAe1 binds to and inhibits DENV.