Cytotoxic Activity of Non-specific Lipid Transfer Protein (nsLTP1) from Ajwain \u3cem\u3e(Trachyspermum ammi)\u3c/em\u3e Seeds

Background Trachyspermum ammi, commonly known as Ajwain, is a member of the Apiaceae family. It is a therapeutic herbal spice with diverse pharmacological properties, used in traditional medicine for various ailments. However, all previous studies were conducted using small molecule extracts, leaving the protein’s bioactivity undiscovered. Aim The current study aimed to demonstrate the cytotoxic activity of Ajwain non-specific lipid transfer protein (nsLTP1) in normal breast (MCF10A), breast cancer (MCF-7), and pancreatic cancer (AsPC-1) cell lines. Also, to evaluate its structural stability in human serum as well as at high temperature conditions. Methods The cytotoxic activity of Ajwain nsLTP1 was evaluated in MCF-7 and AsPC-1 cell lines using MTT assay. Annexin V-FITC and PI staining were used to detect the early apoptotic and late apoptotic cells. The role of nsLTP1 in inducing apoptosis was further studied by quantifying Bcl-2, Bax, Caspase-3, Survivin, EGFR, and VEGF genes expression using RT-PCR. CD spectroscopy analyzed the nsLTP1 conformational changes after thermal treatment for structure stability determination. The RP-HPLC was used to analyze the nsLTP1 degradation rate in human serum at different time intervals incubated at 37 °C. Results Ajwain nsLTP1 showed a potent cytotoxic effect in MCF-7 and AsPC-1. The IC50 value obtained in MCF-7 was 8.21 μM, while for AsPC-1 4.17 μM. The effect of nsLTP1 on stimulating apoptosis revealed that the proportions of apoptotic cells in both cell lines were relatively increased depending on the concentration. The apoptotic cells percentage at 20 μM was in MCF-7 71% (***P \u3c 0.001) and AsPC-1 88% (***P \u3c 0.001). These results indicate that nsLTP1 might efficaciously induce apoptosis in multiple types of cancerous cells. Genes expression in MCF-7 and AsPC-1 showed significant upregulation in Bax and Caspase-3 and downregulation in Bcl-2, Survivin, EGFR, and VEGF protein. The CD analysis of nsLTP1 showed a significant thermostable property. In serum, nsLTP1 showed a slow degradation rate, indicating high stability with a half-life of ~ 8.4 h. Conclusion Our results revealed the potential anticancer activity of Ajwain nsLTP1 and its mechanism in inducing apoptosis. It further exhibited thermostable properties at high temperatures and in human serum, which suggested this protein as a promising anticancer agent

The Use of Advanced Spectral Imaging to Reveal Nanoparticle Identity in Biological Samples

Nanoparticles (NPs) have been used in drug delivery therapies, medical diagnostic strategies, and as current Covid-19 vaccine carriers. Many microscope-based imaging systems have been introduced to facilitate detection and visualization of NPs. Unfortunately, none can differentiate the core and the shell of NPs. Spectral imaging has been used to distinguish a drug molecule and its metabolite. We have recently integrated this technology to a resolution of 9 nm by using artificial intelligence-driven analyses. Such a resolution allowed us to collect many robust datapoints for each pixel of an image. Our analyses could recognize 45 spectral points within a pixel to detect unlabeled Ag-NPs and Au-NPs in single live cells and tissues (liver, heart, spleen and kidneys). The improved resolution and software provided a more specific fingerprinting for each single molecule, allowing simultaneous analyses of 990 complex interactions from the 45 points for each molecule within a pixel of an image. This in turn allowed us to detect surface-functionalization of Ag-NPs to distinguish the core from the shell of Ag-NPs for the first time. Our studies were validated using various laborious and time-consuming conventional techniques. We propose that spectral imaging has tremendous potential to study NP localization and identification in biological samples at a high temporal and spatial resolution, based primarily on spectral identity information

Integrated omics and machine learning-assisted profiling of cysteine-rich-receptor-like kinases from three peanut spp. revealed their role in multiple stresses

Arachis hypogaea (peanut) is a leading oil and protein-providing crop with a major food source in many countries. It is mostly grown in tropical regions and is largely affected by abiotic and biotic stresses. Cysteine-rich receptor-like kinases (CRKs) is a family of transmembrane proteins that play important roles in regulating stress-signaling and defense mechanisms, enabling plants to tolerate stress conditions. However, almost no information is available regarding this gene family in Arachis hypogaea and its progenitors. This study conducts a pangenome-wide investigation of A. hypogaea and its two progenitors, A. duranensis and A. ipaensis CRK genes (AhCRKs, AdCRKs, and AiCRKs). The gene structure, conserved motif patterns, phylogenetic history, chromosomal distribution, and duplication were studied in detail, showing the intraspecies structural conservation and evolutionary patterns. Promoter cis-elements, protein–protein interactions, GO enrichment, and miRNA targets were also predicted, showing their potential functional conservation. Their expression in salt and drought stresses was also comprehensively studied. The CRKs identified were divided into three groups, phylogenetically. The expansion of this gene family in peanuts was caused by both types of duplication: tandem and segmental. Furthermore, positive as well as negative selection pressure directed the duplication process. The peanut CRK genes were also enriched in hormones, light, development, and stress-related elements. MicroRNA (miRNA) also targeted the AhCRK genes, which suggests the regulatory association of miRNAs in the expression of these genes. Transcriptome datasets showed that AhCRKs have varying expression levels under different abiotic stress conditions. Furthermore, the multi-stress responsiveness of the AhCRK genes was evaluated using a machine learning-based method, Random Forest (RF) classifier. The 3D structures of AhCRKs were also predicted. Our study can be utilized in developing a detailed understanding of the stress regulatory mechanisms of the CRK gene family in peanuts and its further studies to improve the genetic makeup of peanuts to thrive better under stress conditions

Novel Hybrid Indole-Based Caffeic Acid Amide Derivatives as Potent Free Radical Scavenging Agents: Rational Design, Synthesis, Spectroscopic Characterization, In Silico and In Vitro Investigations

Antioxidant small molecules can prevent or delay the oxidative damage caused by free radicals. Herein, a structure-based hybridization of two natural antioxidants (caffeic acid and melatonin) afforded a novel hybrid series of indole-based amide analogues which was synthesized with potential antioxidant properties. A multiple-step scheme of in vitro radical scavenging assays was carried out to evaluate the antioxidant activity of the synthesized compounds. The results of the DPPH assay demonstrated that the indole-based caffeic acid amides are more active free radical scavenging agents than their benzamide analogues. Compared to Trolox, a water-soluble analogue of vitamin E, compounds 3a, 3f, 3h, 3j, and 3m were found to have excellent DPPH radical scavenging activities with IC50 values of 95.81 ± 1.01, 136.8 ± 1.04, 86.77 ± 1.03, 50.98 ± 1.05, and 67.64 ± 1.02 µM. Three compounds out of five (3f, 3j, and 3m) showed a higher capacity to neutralize the radical cation ABTS•+ more than Trolox with IC50 values of 14.48 ± 0.68, 19.49 ± 0.54, and 14.92 ± 0.30 µM, respectively. Compound 3j presented the highest antioxidant activity with a FRAP value of 4774.37 ± 137.20 μM Trolox eq/mM sample. In a similar way to the FRAP assay, the best antioxidant activity against the peroxyl radicals was demonstrated by compound 3j (10,714.21 ± 817.76 μM Trolox eq/mM sample). Taken together, compound 3j was validated as a lead hybrid molecule that could be optimized to maximize its antioxidant potency for the treatment of oxidative stress-related diseases

Comparative genomics and bioinformatics approaches revealed the role of CC-NBS-LRR genes under multiple stresses in passion fruit

Passion fruit is widely cultivated in tropical, subtropical regions of the world. The attack of bacterial and fungal diseases, and environmental factors heavily affect the yield and productivity of the passion fruit. The CC-NBS-LRR (CNL) gene family being a subclass of R-genes protects the plant against the attack of pathogens and plays a major role in effector-triggered immunity (ETI). However, no information is available regarding this gene family in passion fruit. To address the underlying problem a total of 25 and 21 CNL genes have been identified in the genome of purple (Passiflora edulis Sims.) and yellow (Passiflora edulis f. flavicarpa) passion fruit respectively. Phylogenetic tree was divided into four groups with PeCNLs present in 3 groups only. Gene structure analysis revealed that number of exons ranged from 1 to 9 with 1 being most common. Most of the PeCNL genes were clustered at the chromosome 3 and underwent strong purifying selection, expanded through segmental (17 gene pairs) and tandem duplications (17 gene pairs). PeCNL genes contained cis-elements involved in plant growth, hormones, and stress response. Transcriptome data indicated that PeCNL3, PeCNL13, and PeCNL14 were found to be differentially expressed under Cucumber mosaic virus and cold stress. Three genes were validated to be multi-stress responsive by applying Random Forest model of machine learning. To comprehend the biological functions of PeCNL proteins, their 3D structure and gene ontology (GO) enrichment analysis were done. Our research analyzed the CNL gene family in passion fruit to understand stress regulation and improve resilience. This study lays the groundwork for future investigations aimed at enhancing the genetic composition of passion fruit to ensure robust growth and productivity in challenging environments

Novel Hybrid Indole-Based Caffeic Acid Amide Derivatives as Potent Free Radical Scavenging Agents: Rational Design, Synthesis, Spectroscopic Characterization, In Silico and In Vitro Investigations

Antioxidant small molecules can prevent or delay the oxidative damage caused by free radicals. Herein, a structure-based hybridization of two natural antioxidants (caffeic acid and melatonin) afforded a novel hybrid series of indole-based amide analogues which was synthesized with potential antioxidant properties. A multiple-step scheme of in vitro radical scavenging assays was carried out to evaluate the antioxidant activity of the synthesized compounds. The results of the DPPH assay demonstrated that the indole-based caffeic acid amides are more active free radical scavenging agents than their benzamide analogues. Compared to Trolox, a water-soluble analogue of vitamin E, compounds 3a, 3f, 3h, 3j, and 3m were found to have excellent DPPH radical scavenging activities with IC50 values of 95.81 ± 1.01, 136.8 ± 1.04, 86.77 ± 1.03, 50.98 ± 1.05, and 67.64 ± 1.02 µM. Three compounds out of five (3f, 3j, and 3m) showed a higher capacity to neutralize the radical cation ABTS•+ more than Trolox with IC50 values of 14.48 ± 0.68, 19.49 ± 0.54, and 14.92 ± 0.30 µM, respectively. Compound 3j presented the highest antioxidant activity with a FRAP value of 4774.37 ± 137.20 μM Trolox eq/mM sample. In a similar way to the FRAP assay, the best antioxidant activity against the peroxyl radicals was demonstrated by compound 3j (10,714.21 ± 817.76 μM Trolox eq/mM sample). Taken together, compound 3j was validated as a lead hybrid molecule that could be optimized to maximize its antioxidant potency for the treatment of oxidative stress-related diseases

Development of new TAK-285 derivatives as potent EGFR/HER2 inhibitors possessing antiproliferative effects against 22RV1 and PC3 prostate carcinoma cell lines

AbstractEpidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) protein tyrosine kinases co-expressed in various cancers such as ovarian, breast, colon, and prostate subtypes. Herein, new TAK-285 derivatives (9a–h) were synthesised, characterised, and biologically evaluated as dual EGFR/HER2 inhibitors. Compound 9f exhibited IC50 values of 2.3 nM over EGFR and 234 nM over HER2, which is 38-fold of staurosporine and 10-fold of TAK-285 over EGFR. Compound 9f also showed high selectivity profile when tested over a small kinase panel. Compounds 9a–h showed IC50 values in the range of 1.0–7.3 nM and 0.8–2.8 nM against PC3 and 22RV1 prostate carcinoma cell lines, respectively. Cell cycle analysis, apoptotic induction, molecular docking, dynamics, and MM-GBSA studies confirmed the plausible mechanism(s) of compound 9f as a potent EGFR/HER2 dual inhibitor with an effective antiproliferative action against prostate carcinoma

Design, Synthesis, Biological Evaluation, and Molecular Dynamics Studies of Novel Lapatinib Derivatives

Co-expression of the epidermal growth factor receptor (EGFR, also known as ErbB1) and human epidermal growth factor receptor 2 (HER2) has been identified as a diagnostic or prognostic sign in various tumors. Despite the fact that lapatinib (EGFR/HER2 dual inhibitor) has shown to be successful, many patients do not respond to it or develop resistance for a variety of reasons that are still unclear. As a result, new approaches and inhibitory small molecules are still needed for EGFR/HER2 inhibition. Herein, novel lapatinib derivatives possessing 4-anilinoquinazoline and imidazole scaffolds (6a–l) were developed and screened as EGFR/HER2 dual inhibitors. In vitro and in silico investigations revealed that compound 6j has a high affinity for the ATP-binding regions of EGFR and HER2. All of the designed candidates were predicted to not penetrate the BBB, raising the expectation for the absence of CNS side effects. At 10 µM, derivatives possessing 3-chloro-4-(pyridin-2-ylmethoxy)aniline moiety (6i–l) demonstrated outstanding ranges of percentage inhibition against EGFR (97.65–99.03%) and HER2 (87.16–96.73%). Compound 6j showed nanomolar IC50 values over both kinases (1.8 nM over EGFR and 87.8 nM over HER2). Over EGFR, compound 6j was found to be 50-fold more potent than staurosporine and 6-fold more potent than lapatinib. A kinase selectivity panel of compound 6j showed poor to weak inhibitory activity over CDK2/cyclin A, c-MET, FGFR1, KDR/VEGFR2, and P38a/MAPK14, respectively. Structure–activity relationship (SAR) that were obtained with different substitutions were justified. Additionally, molecular docking and molecular dynamics studies revealed insights into the binding mode of the target compounds. Thus, compound 6j was identified as a highly effective and dual EGFR/HER2 inhibitor worthy of further investigation

Table1_Integrated omics and machine learning-assisted profiling of cysteine-rich-receptor-like kinases from three peanut spp. revealed their role in multiple stresses.DOCX

Arachis hypogaea (peanut) is a leading oil and protein-providing crop with a major food source in many countries. It is mostly grown in tropical regions and is largely affected by abiotic and biotic stresses. Cysteine-rich receptor-like kinases (CRKs) is a family of transmembrane proteins that play important roles in regulating stress-signaling and defense mechanisms, enabling plants to tolerate stress conditions. However, almost no information is available regarding this gene family in Arachis hypogaea and its progenitors. This study conducts a pangenome-wide investigation of A. hypogaea and its two progenitors, A. duranensis and A. ipaensis CRK genes (AhCRKs, AdCRKs, and AiCRKs). The gene structure, conserved motif patterns, phylogenetic history, chromosomal distribution, and duplication were studied in detail, showing the intraspecies structural conservation and evolutionary patterns. Promoter cis-elements, protein–protein interactions, GO enrichment, and miRNA targets were also predicted, showing their potential functional conservation. Their expression in salt and drought stresses was also comprehensively studied. The CRKs identified were divided into three groups, phylogenetically. The expansion of this gene family in peanuts was caused by both types of duplication: tandem and segmental. Furthermore, positive as well as negative selection pressure directed the duplication process. The peanut CRK genes were also enriched in hormones, light, development, and stress-related elements. MicroRNA (miRNA) also targeted the AhCRK genes, which suggests the regulatory association of miRNAs in the expression of these genes. Transcriptome datasets showed that AhCRKs have varying expression levels under different abiotic stress conditions. Furthermore, the multi-stress responsiveness of the AhCRK genes was evaluated using a machine learning-based method, Random Forest (RF) classifier. The 3D structures of AhCRKs were also predicted. Our study can be utilized in developing a detailed understanding of the stress regulatory mechanisms of the CRK gene family in peanuts and its further studies to improve the genetic makeup of peanuts to thrive better under stress conditions.</p

Table2_Integrated omics and machine learning-assisted profiling of cysteine-rich-receptor-like kinases from three peanut spp. revealed their role in multiple stresses.XLSX

Arachis hypogaea (peanut) is a leading oil and protein-providing crop with a major food source in many countries. It is mostly grown in tropical regions and is largely affected by abiotic and biotic stresses. Cysteine-rich receptor-like kinases (CRKs) is a family of transmembrane proteins that play important roles in regulating stress-signaling and defense mechanisms, enabling plants to tolerate stress conditions. However, almost no information is available regarding this gene family in Arachis hypogaea and its progenitors. This study conducts a pangenome-wide investigation of A. hypogaea and its two progenitors, A. duranensis and A. ipaensis CRK genes (AhCRKs, AdCRKs, and AiCRKs). The gene structure, conserved motif patterns, phylogenetic history, chromosomal distribution, and duplication were studied in detail, showing the intraspecies structural conservation and evolutionary patterns. Promoter cis-elements, protein–protein interactions, GO enrichment, and miRNA targets were also predicted, showing their potential functional conservation. Their expression in salt and drought stresses was also comprehensively studied. The CRKs identified were divided into three groups, phylogenetically. The expansion of this gene family in peanuts was caused by both types of duplication: tandem and segmental. Furthermore, positive as well as negative selection pressure directed the duplication process. The peanut CRK genes were also enriched in hormones, light, development, and stress-related elements. MicroRNA (miRNA) also targeted the AhCRK genes, which suggests the regulatory association of miRNAs in the expression of these genes. Transcriptome datasets showed that AhCRKs have varying expression levels under different abiotic stress conditions. Furthermore, the multi-stress responsiveness of the AhCRK genes was evaluated using a machine learning-based method, Random Forest (RF) classifier. The 3D structures of AhCRKs were also predicted. Our study can be utilized in developing a detailed understanding of the stress regulatory mechanisms of the CRK gene family in peanuts and its further studies to improve the genetic makeup of peanuts to thrive better under stress conditions.</p