Unleashing Bacillus species as versatile antagonists: Harnessing the biocontrol potentials of the plant growth-promoting rhizobacteria to combat Macrophomina phaseolina infection in Gloriosa superba.

Charcoal rot caused by Macrophomina phaseolina is one of the most devastating diseases that cause severe yield loss in Gloriosa superba cultivation. Plant growth-promoting rhizobacteria (PGPR) are extensively harnessed as biocontrol agents due to their effectiveness in combating a wide array of plant pathogens through a multifaceted approach. The present study delved into the mechanisms underlying its ability to inhibit root rot pathogen and its capacity to promote plant growth in G. superba, commonly known as glory lily. PGPR isolated from the rhizosphere of glory lily were subjected to in vitro assessments using the dual plate technique. The isolated Bacillus subtilis BGS-10 and B. velezensis BGS-21 showed higher mycelial inhibition (61%) against M. phaseolina. These strains also promote plant growth by producing indole-3-acetic acid, siderophore, ammonia, amylase, cellulase, pectinase, xylanase, and lipase chemicals. Genome screening of BGS-10 and BGS-21 revealed the presence of antimicrobial peptide genes such as Iturin (ituD gene), surfactin (srfA and sfp genes) along with the mycolytic enzyme ß-1,3-glucanase. Further, the presence of secondary metabolites in the bacterial secretome was identified through gas chromatography-mass spectrometry (GC/MS) analysis. Notably, pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl), 9 H-pyrido[3,4-b] indole and L-leucyl-D-leucine exhibited the highest docking score against enzymes responsible for pathogen growth and plant cell wall degradation. Under glasshouse conditions, tuber treatment and soil application of talc-based formulation of B. subtilis BGS-10 and B. velezensis BGS-21 suppress the root rot incidence with a minimal disease incidence of 27.78% over untreated control. Concurrently, there was a notable induction of defense-related enzymes, including peroxidase (PO), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL), in glory lily. Therefore, it can be concluded that plant growth-promoting Bacillus strains play a significant role in fortifying the plant's defense mechanisms against the root rot pathogen.

Promiscuity and Quantitative Contribution of UGT2B17 in Drug and Steroid Metabolism Determined by Experimental and Computational Approaches.

Uridine 5'-diphospho-glulcuronosyltransferase 2B17 (UGT2B17) is important in the metabolism of steroids and orally administered drugs due to its high interindividual variability. However, the structural basis governing the substrate selectivity or inhibition of UGT2B17 remains poorly understood. This study investigated 76 FDA-approved drugs and 20 steroids known to undergo glucuronidation for their metabolism by UGT2B17. Specifically, we assessed the substrate selectivity for UGT2B17 over other UGT enzymes using recombinant human UGT2B17 (rUGT2B17), human intestinal microsomes, and human liver microsomes. The quantitative contribution of intestinal UGT2B17 in the glucuronidation of these compounds was characterized using intestinal microsomes isolated from UGT2B17 expressors and nonexpressors. In addition, a structure-based pharmacophore model for UGT2B17 substrates was built and validated using the studied pool of substrates and nonsubstrates. The results show that UGT2B17 could metabolize 23 out of 96 compounds from various chemical classes, including alcohols and carboxylic acids, particularly in the intestine. Interestingly, amines were less susceptible to UGT2B17 metabolism, though they could inhibit the enzyme. Three main pharmacophoric features of UGT2B17 substrates include (1) the presence of an accessible -OH or -COOH group near His35 residue, (2) a hydrophobic functional group at ∼4.5-5 Šfrom feature 1, and (3) an aromatic ring ∼5-7 Šfrom feature 2. Most of the studied compounds inhibited UGT2B17 activity irrespective of their substrate potential, indicating the possibility of multiple mechanisms. These data suggest that UGT2B17 is promiscuous in substrate selectivity and inhibition and has a high potential to produce significant variability in the absorption and disposition of orally administered drugs.

Identification of salivary proteins of the cowpea aphid Aphis craccivora by transcriptome and LC-MS/MS analyses.

Aphid salivary proteins mediate the interaction between aphids and their host plants. Moreover, these proteins facilitate digestion, detoxification of secondary metabolites, as well as activation and suppression of plant defenses. The cowpea aphid, Aphis craccivora, is an important sucking pest of leguminous crops worldwide. Although aphid saliva plays an important role in aphid plant interactions, knowledge of the cowpea aphid salivary proteins is limited. In this study, we performed transcriptomic and LC-MS/MS analyses to identify the proteins present in the salivary glands and saliva of A. craccivora. A total of 1,08,275 assembled transcripts were identified in the salivary glands of aphids. Of all these assembled transcripts, 53,714 (49.11%) and 53,577 (49.48%) transcripts showed high similarity to known proteins in the Nr and UniProt databases, respectively. A total of 2159 proteins were predicted as secretory proteins from the salivary gland transcriptome dataset, which contain digestive enzymes, detoxification enzymes, previously known effectors and elicitors, and potential proteins whose functions have yet to be determined. The proteomic analysis of aphid saliva resulted in the identification of 171 proteins. Tissue-specific expression of selected genes using RT-PCR showed that three genes were expressed only in the salivary glands. Overall, our results provide a comprehensive repertoire of cowpea aphid salivary proteins from the salivary gland and saliva, which will be a good resource for future effector functional studies and might also be useful for sustainable aphid management.

Characterization of terminal flowering cowpea (Vigna unguiculata (L.) Walp.) mutants obtained by induced mutagenesis digs out the loss-of-function of phosphatidylethanolamine-binding protein.

Cowpea (Vigna unguiculata (L.) Walp) is one of the major food legume crops grown extensively in arid and semi-arid regions of the world. The determinate habit of cowpea has many advantages over the indeterminate and is well adapted to modern farming systems. Mutation breeding is an active research area to develop the determinate habit of cowpea. The present study aimed to develop new determinate habit mutants with terminal flowering (TFL) in locally well-adapted genetic backgrounds. Consequently, the seeds of popular cowpea cv P152 were irradiated with doses of gamma rays (200, 250, and, 300 Gy), and the M1 populations were grown. The M2 populations were produced from the M1 progenies and selected determinate mutants (TFLCM-1 and TFLCM-2) from the M2 generation (200 Gy) were forwarded up to the M5 generation to characterize the mutants and simultaneously they were crossed with P152 to develop a MutMap population. In the M5 generation, determinate mutants (80-81 days) were characterized by evaluating the TFL growth habit, longer peduncles (30.75-31.45 cm), erect pods (160°- 200°), number of pods per cluster (4-5 nos.), and early maturity. Further, sequencing analysis of the VuTFL1 gene in the determinate mutants and MutMap population revealed a single nucleotide transversion (A-T at 1196 bp) in the fourth exon and asparagine (N) to tyrosine (Y) amino acid change at the 143rd position of phosphatidylethanolamine-binding protein (PEBP). Notably, the loss of function PEPB with a higher confidence level modification of anti-parallel beta-sheets and destabilization of the protein secondary structure was observed in the mutant lines. Quantitative real-time PCR (qRT-PCR) analysis showed that the VuTFL1 gene was downregulated at the flowering stage in TFL mutants. Collectively, the insights garnered from this study affirm the effectiveness of induced mutation in modifying the plant's ideotype. The TFL mutants developed during this investigation have the potential to serve as a valuable resource for fostering determinate traits in future cowpea breeding programs and pave the way for mechanical harvesting.

Exploring bixin from Bixa orellana L. seeds: quantification and in silico insights into its anti-cancer potential.

Bixin, the key pigment of Bixa orellana L., is an apo-carotenoid found in the seed arils. The present study aimed to quantitatively determine the bixin content of seeds and explore its anti-cancer activity through in silico studies. The bixin content from the seeds of the local genotype, TNMTP8, quantified by RP-HPLC was 4.58 mg per gram. The prediction of pharmacological activity suggested that bixin may serve as a BRAF, MMP9, TNF expression inhibitors, and TP53 expression enhancer. According to molecular docking analysis, bixin interacted with eight different skin cancer targets and had the lowest binding energy compared to the standard drug, 5-fluorouracil. The binding score between bixin and the targets ranged from -4.7 to -8.7 kcal/mol. The targets BRAF and SIRT3 interacted well with bixin, with binding energies as low as -8.3 and -8.7 kcal/mol, respectively. Hence, the dynamic behavior of these two docked complexes throughout a 500 ns trajectory run was investigated further. The Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF) values, and total contacts as a function of time recorded during scrutiny suggest that both complexes were stable. This was validated by post-molecular dynamics analysis using Molecular Mechanics Generalized Born Surface Area (MM-GBSA). Principal component analysis (PCA) was used to analyze the significant differences in motion exhibited by BRAF-Bixin and SIRT3-Bixin. The results showed that bixin is a promising source for potential treatment interventions in skin cancer therapies.Communicated by Ramaswamy H. Sarma.

Human beta defensin-3 mediated activation of ß-catenin during human respiratory syncytial virus infection: interaction of HBD3 with LDL receptor-related protein 5.

Respiratory Syncytial Virus (RSV) is a non-segmented negative-sense RNA virus belonging to the paramyxovirus family. RSV infects the respiratory tract to cause pneumonia and bronchiolitis in infants, elderly, and immunocompromised patients. Effective clinical therapeutic options and vaccines to combat RSV infection are still lacking. Therefore, to develop effective therapeutic interventions, it is imperative to understand virus-host interactions during RSV infection. Cytoplasmic stabilization of ß-catenin protein results in activation of canonical Wingless (Wnt)/ß-catenin signaling pathway that culminates in transcriptional activation of various genes regulated by T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors. This pathway is involved in various biological and physiological functions. Our study shows RSV infection of human lung epithelial A549 cells triggering ß-catenin protein stabilization and induction of ß-catenin mediated transcriptional activity. Functionally, the activated ß-catenin pathway promoted a pro-inflammatory response during RSV infection of lung epithelial cells. Studies with ß-catenin inhibitors and A549 cells lacking optimal ß-catenin activity demonstrated a significant loss of pro-inflammatory chemokine interleukin-8 (IL-8) release from RSV-infected cells. Mechanistically, our studies revealed a role of extracellular human beta defensin-3 (HBD3) in interacting with cell surface Wnt receptor LDL receptor-related protein-5 (LRP5) to activate the non-canonical Wnt independent ß-catenin pathway during RSV infection. We showed gene expression and release of HBD3 from RSV-infected cells and silencing of HBD3 expression resulted in reduced stabilization of ß-catenin protein during RSV infection. Furthermore, we observed the binding of extracellular HBD3 with cell surface localized LRP5 protein, and our in silico and protein-protein interaction studies have highlighted a direct interaction of HBD3 with LRP5. Thus, our studies have identified the ß-catenin pathway as a key regulator of pro-inflammatory response during RSV infection of human lung epithelial cells. This pathway was induced during RSV infection via a non-canonical Wnt-independent mechanism involving paracrine/autocrine action of extracellular HBD3 activating cell surface Wnt receptor complex by directly interacting with the LRP5 receptor.

Melatonin Enhances the Photosynthesis and Antioxidant Enzyme Activities of Mung Bean under Drought and High-Temperature Stress Conditions.

Mung bean, a legume, is sensitive to abiotic stresses at different growth stages, and its yield potential is affected by drought and high-temperature stress at the sensitive stage. Melatonin is a multifunctional hormone that plays a vital role in plant stress defense mechanisms. This study aimed to evaluate the efficiency of melatonin under individual and combined drought and high-temperature stress in mung bean. An experiment was laid out with five treatments, including an exogenous application of 100 µM melatonin as a seed treatment, foliar spray, and a combination of both seed treatment and foliar spray, as well as absolute control (ambient condition) and control (stress without melatonin treatment). Stresses were imposed during the mung bean's reproductive stage (31-40 DAS) for ten days. Results revealed that drought and high-temperature stress significantly decreased chlorophyll index, Fv/Fm ratio, photosynthetic rate, stomatal conductance, and transpiration rate through increased reactive oxygen species (ROS) production. Foliar application of melatonin at 100 µM concentration enhanced the activity of antioxidant enzymes such as superoxide dismutase, catalase, and ascorbate peroxidase and the concentration of metabolites involved in osmoregulation and ion homeostasis; thereby, it improves physiological and yield-related traits in mung bean under individual and combined stress at the reproductive stage.

Molecular basis for the recognition of 24-(S)-hydroxycholesterol by integrin αvß3.

A growing body of evidence suggests that oxysterols such as 25-hydroxycholesterol (25HC) are biologically active and involved in many physiological and pathological processes. Our previous study demonstrated that 25HC induces an innate immune response during viral infections by activating the integrin-focal adhesion kinase (FAK) pathway. 25HC produced the proinflammatory response by binding directly to integrins at a novel binding site (site II) and triggering the production of proinflammatory mediators such as tumor necrosis factor-α (TNF) and interleukin-6 (IL-6). 24-(S)-hydroxycholesterol (24HC), a structural isomer of 25HC, plays a critical role in cholesterol homeostasis in the human brain and is implicated in multiple inflammatory conditions, including Alzheimer's disease. However, whether 24HC can induce a proinflammatory response like 25HC in non-neuronal cells has not been studied and remains unknown. The aim of this study was to examine whether 24HC produces such an immune response using in silico and in vitro experiments. Our results indicate that despite being a structural isomer of 25HC, 24HC binds at site II in a distinct binding mode, engages in varied residue interactions, and produces significant conformational changes in the specificity-determining loop (SDL). In addition, our surface plasmon resonance (SPR) study reveals that 24HC could directly bind to integrin αvß3, with a binding affinity three-fold lower than 25HC. Furthermore, our in vitro studies with macrophages support the involvement of FAK and NFκB signaling pathways in triggering 24HC-mediated production of TNF. Thus, we have identified 24HC as another oxysterol that binds to integrin αvß3 and promotes a proinflammatory response via the integrin-FAK-NFκB pathway.

Allosteric modulation of α1ß3γ2 GABAA receptors by farnesol through the neurosteroid sites.

In mammalian cells, all-trans farnesol, a 15-carbon isoprenol, is a product of the mevalonate pathway. It is the natural substrate of alcohol dehydrogenase and a substrate for CYP2E1, two enzymes implicated in ethanol metabolism. Studies have shown that farnesol is present in the human brain and inhibits voltage-gated Ca2+ channels at much lower concentrations than ethanol. Here we show that farnesol modulates the activity of γ-aminobutyric acid type A receptors (GABAARs), some of which also mediate the sedative activity of ethanol. Electrophysiology experiments performed in HEK cells expressing human α1ß3γ2 or α6ß3γ2 GABAARs revealed that farnesol increased chloride currents through positive allosteric modulation of these receptors and showed dependence on both the alcoholic functional group of farnesol and the length of the alkyl chain for activity. In silico studies using long-timescale unbiased all-atom molecular dynamics (MD) simulations of the human α1ß3γ2 GABAA receptors revealed that farnesol modulates the channel by directly binding to the transmembrane neurosteroid-binding site, after partitioning into the surrounding membrane and reaching the receptor by lateral diffusion. Channel activation by farnesol was further characterized by several structural and dynamic variables, such as global twisting of the receptor's extracellular domain, tilting of the transmembrane M2 helices, radius, cross-sectional area, hydration status, and electrostatic potential of the channel pore. Our results expand the pharmacological activities of farnesol to yet another class of ion channels implicated in neurotransmission, thus providing a novel path for understanding and treatment of diseases involving GABAA receptor dysfunction.

Membrane Lipids Are an Integral Part of Transmembrane Allosteric Sites in GPCRs: A Case Study of Cannabinoid CB1 Receptor Bound to a Negative Allosteric Modulator, ORG27569, and Analogs.

A growing number of G-protein-coupled receptor (GPCR) structures reveal novel transmembrane lipid-exposed allosteric sites. Ligands must first partition into the surrounding membrane and take lipid paths to these sites. Remarkably, a significant part of the bound ligands appears exposed to the membrane lipids. The experimental structures do not usually account for the surrounding lipids, and their apparent contribution to ligand access and binding is often overlooked and poorly understood. Using classical and enhanced molecular dynamics simulations, we show that membrane lipids are critical in the access and binding of ORG27569 and its analogs at the transmembrane site of cannabinoid CB1 receptor. The observed differences in the binding affinity and cooperativity arise from the functional groups that interact primarily with lipids. Our results demonstrate the significance of incorporating membrane lipids as an integral component of transmembrane sites for accurate characterization, binding-affinity calculations, and lead optimization in drug discovery.

Development of ß-carotene, lysine, and tryptophan-rich maize (Zea mays) inbreds through marker-assisted gene pyramiding.

Maize (Zea mays L.) is the leading cereal crop and staple food in many parts of the world. This study aims to develop nutrient-rich maize genotypes by incorporating crtRB1 and o2 genes associated with increased ß-carotene, lysine, and tryptophan levels. UMI1200 and UMI1230, high quality maize inbreds, are well-adapted to tropical and semi-arid regions in India. However, they are deficient in ß-carotene, lysine, and tryptophan. We used the concurrent stepwise transfer of genes by marker-assisted backcross breeding (MABB) scheme to introgress crtRB1 and o2 genes. In each generation (from F1, BC1F1-BC3F1, and ICF1-ICF3), foreground and background selections were carried out using gene-linked (crtRB1 3'TE and umc1066) and genome-wide simple sequence repeats (SSR) markers. Four independent BC3F1 lines of UMI1200 × CE477 (Cross-1), UMI1200 × VQL1 (Cross-2), UMI1230 × CE477 (Cross-3), and UMI1230 × VQL1 (Cross-4) having crtRB1 and o2 genes and 87.45-88.41% of recurrent parent genome recovery (RPGR) were intercrossed to generate the ICF1-ICF3 generations. Further, these gene pyramided lines were examined for agronomic performance and the ß-carotene, lysine, and tryptophan contents. Six ICF3 lines (DBT-IC-ß1σ4-4-8-8, DBT-IC-ß1σ4-9-21-21, DBT-IC-ß1σ4-10-1-1, DBT-IC-ß2σ5-9-51-51, DBT-IC-ß2σ5-9-52-52 and DBT-IC-ß2σ5-9-53-53) possessing crtRB1 and o2 genes showed better agronomic performance (77.78-99.31% for DBT-IC-ß1σ4 population and 85.71-99.51% for DBT-IC-ß2σ5 population) like the recurrent parents and ß-carotene (14.21-14.35 µg/g for DBT-IC-ß1σ4 and 13.28-13.62 µg/g for DBT-IC-ß2σ5), lysine (0.31-0.33% for DBT-IC-ß1σ4 and 0.31-0.34% for DBT-IC-ß2σ5), and tryptophan (0.079-0.082% for DBT-IC-ß1σ4 and 0.078-0.083% for DBT-IC-ß2σ5) levels on par with that of the donor parents. In the future, these improved lines could be developed as a cultivar for various agro-climatic zones and also as good genetic materials for maize nutritional breeding programs.

Unlocking the genetic diversity of Indian turmeric (Curcuma longa L.) germplasm based on rhizome yield traits and curcuminoids.

Turmeric is an important commercial crop widely grown in Asia due to its pharmacological and nutritional value. India is the centre of turmeric diversity and many turmeric accessions have good rhizome yield, varying curcuminoids content and are well-adapted to various agro-climatic zones. In the present study, we unravel the diversity among 200 Indian turmeric accessions based on rhizome yield traits and curcuminoids content. Clustering and correlation studies were also performed to group the turmeric accessions and to observe the relationship between the traits. Results revealed the presence of large variability among turmeric accessions including the major traits such as yield (24.77 g p-1 to 667.63 g p-1), dry recovery percentage (13.42% to 29.18%), curcumin (0.41% to 2.17%), demethoxycurcumin (0.38% to 1.45%), bisdemethoxycurcumin (0.37% to 1.24%) and total curcuminoid content (1.26% to 4.55%). The superior germplasm identified for curcuminoids content were as follows; curcumin (CL 157 - 2.17% and CL 272 - 2.13%), demethoxycurcumin (CL 253 - 1.45% and CL 157 - 1.31%), bisdemethoxycurcumin (CL 216 - 1.24% and CL 57 - 1.11%) and total curcuminoid content (CL 157 - 4.55% and CL 272 - 4.37%). Clustering based on dendrogram, grouped 200 accessions into seven clusters. Among seven clusters, the maximum number of accessions were grouped into cluster II while cluster VII showed maximum mean value for majority of the traits. Correlation analysis revealed a significant relationship between the traits where the total curcuminoid content is significantly and positively correlated with the primary rhizome core diameter and length of the secondary rhizome. The selection of these particular traits may result in the identification of germplasm with high total curcuminoid content. Taken together, it is the first report on the large screening of turmeric accessions for variation in the rhizome yield traits and curcuminoids content. The genetic diversity revealed in this study could be useful for further crop improvement programs in turmeric to develop new varieties with high rhizome yield coupled with high curcuminoids content.

Membrane-Facilitated Receptor Access and Binding Mechanisms of Long-Acting ß2-Adrenergic Receptor Agonists.

The drugs salmeterol, formoterol, and salbutamol constitute the frontline treatment of asthma and other chronic pulmonary diseases. These drugs activate the ß2-adrenergic receptors (ß2-AR), a class A G protein-coupled receptor (GPCR), and differ significantly in their clinical onset and duration of actions. According to the microkinetic model, the long duration of action of salmeterol and formoterol compared with salbutamol were attributed, at least in part, to their high lipophilicity and increased local concentrations in the membrane near the receptor. However, the structural and molecular bases of how the lipophilic drugs reach the binding site of the receptor from the surrounding membrane remain unknown. Using a variety of classic and enhanced molecular dynamics simulation techniques, we investigated the membrane partitioning characteristics, binding, and unbinding mechanisms of the ligands. The obtained results offer remarkable insight into the functional role of membrane lipids in the ligand association process. Strikingly, salmeterol entered the binding site from the bilayer through transmembrane helices 1 and 7. The entry was preceded by membrane-facilitated rearrangement and presentation of its phenyl-alkoxy-alkyl tail as a passkey to an access route gated by F193, a residue known to be critical for salmeterol's affinity. Formoterol's access is through the aqueous path shared by other ß2-AR agents. We observed a novel secondary path for salbutamol that is distinct from its primary route. Our study offers a mechanistic description for the membrane-facilitated access and binding of ligands to a membrane protein and establishes a groundwork for recognizing membrane lipids as an integral component in the molecular recognition process. SIGNIFICANCE STATEMENT: The cell membrane's functional role behind the duration of action of long-acting ß2-adrenergic receptor (ß2-AR) agonists such as salmeterol has been a subject of debate for a long time. This study investigated the binding and unbinding mechanisms of the three commonly used ß2-AR agonists, salmeterol, formoterol, and salbutamol, using advanced simulation techniques. The obtained results offer unprecedented insights into the active role of membrane lipids in facilitating access and binding of the ligands, affecting the molecular recognition process and thus their pharmacology.

Improvement of a Yairipok Chujak Maize Landrace from North Eastern Himalayan Region for ß-Carotene Content through Molecular Marker-Assisted Backcross Breeding.

In the North Eastern Himalayan region (NEHR) of India, maize is an important food crop. The local people cultivate the maize landraces and consume them as food. However, these landraces are deficient in ß-carotene content. Thus, we aimed to incorporate the crtRB1 gene from UMI285ß+ into the genetic background of the NEHR maize landrace, Yairipok Chujak (CAUM66), and thereby enhance the ß-carotene content through marker-assisted backcrossing (MABC). In this regard, we backcrossed and screened BC1F1 and BC2F1 plants possessing the heterozygous allele for crtRB1 and then screened with 106 polymorphic simple sequence repeat (SSR) markers. The plants having maximum recurrent parent genome recovery (RPGR) were selected in each generation and selfed to produce BC2F2 seeds. In the BC2F2 generation, four plants (CAUM66-54-9-12-2, CAUM66-54-9-12-11, CAUM66-54-9-12-13, and CAUM66-54-9-12-24) having homozygous crtRB1-favorable allele with maximum RPGR (86.74-90.16%) were selected and advanced to BC2F3. The four selected plants were selfed to produce BC2F3 and then evaluated for agronomic traits and ß-carotene content. The agronomic performance of the four lines was similar (78.83-99.44%) to that of the recurrent parent, and ß-carotene content (7.541-8.711 µg/g) was on par with the donor parent. Our study is the first to improve the ß-carotene content in NEHR maize landrace through MABC. The newly developed lines could serve as potential resources to further develop nutrition-rich maize lines and could provide genetic stock for use in breeding programs.

Enhancing ß-Carotene Concentration in Parental Lines of CO6 Maize Hybrid Through Marker-Assisted Backcross Breeding (MABB).

Vitamin A deficiency (VAD) is a global health problem; many people around the world, especially children and pregnant women, are VAD deficient or insufficient. Maize is known as an important source of provitamin A for humans. Hence, enhancement of provitamin A carotenoids (pVAC) in maize varieties through breeding or biofortification is a good option for alleviating VAD in developing countries, especially India. So far, numerous maize hybrids have been developed in India. Among them, CO6, derived from UMI1200 × UMI1230, is a popular maize hybrid and adapted to different agro-climatic zones of India, especially Tamil Nadu, a southern state of India. However, CO6 is deficient for pVAC carotenoid ß-carotene. Thus, the objectives of this study were to increase the ß-carotene concentration in UMI1200 and UMI1230 and generate the ß-carotene enriched hybrids through marker-assisted backcross breeding (MABB). For this purpose, the maize genotype HP467-15 was used as the donor for transferring the ß-carotene gene, crtRB1, into UMI1200 and UMI1230. In the MABB scheme, we used one gene-specific marker (crtRB1 3'TE) and 214 simples sequence repeat (SSR) markers for foreground and background selection, respectively. As a result, six improved lines with recurrent parent genome recovery (RPGR) ranging from 90.24 to 92.42%, along with good agronomic performance, were generated. The ß-carotene concentration of the improved lines ranged from 7.056 to 9.232 µg/g. Furthermore, five hybrid combinations were generated using improved lines and evaluated in a comparative yield trial (CYT) and multi-location trials (MLT) along with the original hybrid CO6 and commercial hybrids. It was revealed that ACM-M13-002 was a superior hybrid with a 7.3-fold increase in ß-carotene concentration and with a comparable yield to CO6. In summary, the improved maize inbreds can be used as possible donors for the development of ß-carotene-rich cultivars in maize breeding programs and the ß-carotene enriched hybrid developed in this study will hold great promise for food and nutritional security.

Marker aided introgression of opaque 2 (o2) allele improving lysine and tryptophan in maize (Zea mays L.).

Maize is the predominant food source for the world population, but lack of lysine and tryptophan in maize endosperm cannot fulfill the nutritional requirements of humans. Hence, the improvement of lysine and tryptophan content is the ultimate goal of maize biofortification programs. In the present study, the marker-assisted backcross (MABC) breeding strategy was used to enhance the lysine and tryptophan content of the elite maize inbred line UMI1230 by introgressing opaque 2 (o2) gene from the VQL1. During the transfer of the gene into UMI1230, SSR marker umc1066 tightly associated with o2 used for foreground selection. Background recovery was estimated using 168 SSR markers. Phenotype screening for morphological traits was adopted to choose plants parallel to UMI1230. As a result, four BC2F3 improved lines (DBT5-1-14/25-5/25-8/25-8/25, DBT5-1-14/25-5/25-8/25-7/25, DBT5-1-14/25-5/25-8/25-10/25 and DBT5-1-14/25-5/25-8/25-12/25) with o2 were developed. The improved line's background genome recovery varied between 90.60 and 94.80%. Also, the improved lines had better agronomic performance along with increased lysine (0.311-0.331%) and tryptophan (0.040-0.048%) contents. In summary, the MABC breeding strategy has successfully improved the levels of lysine and tryptophan in UMI1230 without affecting agronomic performance. The improved line's hold great potential as donors in biofortification programs in maize.

Mechanisms of Herb-Drug Interactions Involving Cinnamon and CYP2A6: Focus on Time-Dependent Inhibition by Cinnamaldehyde and 2-Methoxycinnamaldehyde.

Information is scarce regarding pharmacokinetic-based herb-drug interactions (HDI) with trans-cinnamaldehyde (CA) and 2-methoxycinnamaldehyde (MCA), components of cinnamon. Given the presence of cinnamon in food and herbal treatments for various diseases, HDIs involving the CYP2A6 substrates nicotine and letrozole with MCA (KS = 1.58 µM; Hill slope = 1.16) and CA were investigated. The time-dependent inhibition (TDI) by MCA and CA of CYP2A6-mediated nicotine metabolism is a complex process involving multiple mechanisms. Molecular dynamic simulations showed that CYP2A6's active site accommodates two dynamic ligands. The preferred binding orientations for MCA and CA were consistent with the observed metabolism: epoxidation, O-demethylation, and aromatic hydroxylation of MCA and cinnamic acid formation from CA. The percent remaining activity plots for TDI by MCA and CA were curved, and they were analyzed with a numerical method using models of varying complexity. The best-fit models support multiple inactivator binding, inhibitor depletion, and partial inactivation. Deconvoluted mass spectra indicated that MCA and CA modified CYP2A6 apoprotein with mass additions of 156.79 (142.54-171.04) and 132.67 (123.37-141.98), respectively, and it was unaffected by glutathione. Heme degradation was observed in the presence of MCA (48.5% ± 13.4% loss; detected by liquid chromatography-tandem mass spectrometry). In the absence of clinical data, HDI predictions were made for nicotine and letrozole using inhibition parameters from the best-fit TDI models and parameters scaled from rats. Predicted area under the concentration-time curve fold changes were 4.29 (CA-nicotine), 4.92 (CA-letrozole), 4.35 (MCA-nicotine), and 5.00 (MCA-letrozole). These findings suggest that extensive exposure to cinnamon (corresponding to ≈ 275 mg CA) would lead to noteworthy interactions. SIGNIFICANCE STATEMENT: Human exposure to cinnamon is common because of its presence in food and cinnamon-based herbal treatments. Little is known about the risk for cinnamaldehyde and methoxycinnamaldehyde, two components of cinnamon, to interact with drugs that are eliminated by CYP2A6-mediated metabolism. The interactions with CYP2A6 are complex, involving multiple-ligand binding, time-dependent inhibition of nicotine metabolism, heme degradation, and apoprotein modification. An herb-drug interaction prediction suggests that extensive exposure to cinnamon would lead to noteworthy interactions with nicotine.

Functional analysis of thirty-four suspected pathogenic missense variants in ALDH5A1 gene associated with succinic semialdehyde dehydrogenase deficiency.

Deficiency of succinate semialdehyde dehydrogenase (SSADH; aldehyde dehydrogenase 5a1 (ALDH5A1), OMIM 271980, 610045), the second enzyme of GABA degradation, represents a rare autosomal-recessively inherited disorder which manifests metabolically as gamma-hydroxybutyric aciduria. The neurological phenotype includes intellectual disability, autism spectrum, epilepsy and sleep and behavior disturbances. Approximately 70 variants have been reported in the ALDH5A1 gene, half of them being missense variants. In this study, 34 missense variants, of which 22 novel, were evaluated by in silico analyses using PolyPhen2 and SIFT prediction tools. Subsequently, the effect of these variants on SSADH activity was studied by transient overexpression in HEK293 cells. These studies showed severe enzymatic activity impairment for 27 out of 34 alleles, normal activity for one allele and a broad range of residual activities (25 to 74%) for six alleles. To better evaluate the alleles that showed residual activity above 25%, we generated an SSADH-deficient HEK293-Flp-In cell line using CRISPR-Cas9, in which these alleles were stably expressed. This model proved essential in the classification as deficient for one out of the seven studied alleles. For 8 out of 34 addressed alleles, there were discrepant results among the used prediction tools, and/or in correlating the results of the prediction tools with the functional data. In case of diagnostic urgency of missense alleles, we propose the use of the transient transfection model for confirmation of their effect on the SSADH catalytic function, since this model resulted in fast and robust functional characterization for the majority of the tested variants. In selected cases, stable transfections can be considered and may prove valuable.

Molecular Dynamics Simulations Provide Insight into the Loading Efficiency of Proresolving Lipid Mediators Resolvin D1 and D2 in Cell Membrane-Derived Nanovesicles.

Resolvins D1 and D2 (RvDs) are structural isomers and metabolites of docosahexaenoic acid, an omega-3 fatty acid, enzymatically produced in our body in response to acute inflammation or microbial invasion. Resolvins have been shown to play an essential role in the resolution of inflammation, tissue repair, and return to homeostasis and thus are actively pursued as potential therapeutics in treating inflammatory disorders and infectious diseases. However, effective in vivo delivery of RvDs continues to be a challenging task. Recent studies demonstrated that RvD1 or RvD2 loaded in cell membrane-derived nanovesicles significantly increased therapeutic efficacy in treating murine peritonitis and ischemic stroke, respectively. The mechanistic details of how the subtle structural difference between RvD1 and RvD2 alters their molecular interactions with the membrane lipids of the nanovesicles and thus affects the loading efficiency remain unknown. Here, we report the encapsulation profiles of the neutral and ionized species of both RvD1 and RvD2 determined with the cell membrane-derived nanovesicles at pH values 5.4 and 7.4, respectively. Also, we performed microsecond time-scale all-atom molecular dynamics (MD) simulations in explicit water to elucidate the molecular interactions of both neutral and ionized species of RvD1 and RvD2 with the lipid bilayer using a model membrane system, containing 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol. We found that the differences in the position and chirality of hydroxyl groups in RvD1 and RvD2 affected their location, orientation, and conformations within the bilayer. Surprisingly, the deprotonation of their carboxyl group caused their orientation and conformation to change from a fully extended one that is oriented in parallel to the membrane plane to a J-shaped bent conformation that is oriented perpendicular to the bilayer plane. Our studies offer valuable insight into the molecular interactions of RvD1/D2 with the lipid bilayer in atomistic details and provide a mechanistic explanation for the observed differences in the encapsulation profiles of RvD1 and RvD2, which may facilitate the rational design of nanovesicle-based therapeutics for treating inflammatory diseases.

Marker-Assisted Selection to Pyramid the Opaque-2 (O2) and ß-Carotene (crtRB1) Genes in Maize.

Maize is an excellent nutritional source and is consumed as a staple food in different parts of the world, including India. Developing a maize genotype with a combination of higher lysine and tryptophan, along with ß-carotene, can help alleviate the problem of protein-energy malnutrition (PEM) and vitamin A deficiency (VAD). This study is aimed at improving lysine and tryptophan content by transferring opaque-2 (o2) gene from donor HKI163 to ß-carotene-rich inbred lines viz., UMI1200ß+ and UMI1230ß+. For this purpose, F1, BC1F1, BC2F1, BC2F2, and BC2F3 plants were developed using an o2 line HKI163 and two ß-carotene-rich inbred lines, UMI1200ß+ and UMI1230ß+, as the parents. Foreground selection using the associated marker umc1066 for the o2 gene and the marker crtRB1 3'TE for the crtRB1 gene was used to select the target genes. A total of 236 simple sequence repeat (SSR) markers distributed evenly across the maize genome were employed for the background selection. To fix the crtRB1 allele in the BC1F1 stage, individual plants homozygous at the crtRB1 locus and heterozygous at the o2 locus were selected and used for backcrossing to produce BC2F1 plants. Furthermore, the selected heterozygous BC2F1 plants from both crosses were selfed to obtain the BC2F2 plants, which were then selected for the target gene and selfed to generate the BC2F3 lines. From each cross, five improved lines with homozygous marker alleles for the crtRB1 and o2 genes with a recurrent parent genome (RPG) recovery ranging from 86.75 to 91.21% in UMI1200ß+×HKI163 and 80.00 to 90.08% in UMI1230ß+×HKI163 were identified. The improved lines had good agronomic performance and possessed high lysine (0.294-0.332%), tryptophan (0.073-0.081%), and ß-carotene (6.12-7.38 µg/g) content. These improved lines can be used as genetic resources for maize improvement.