Genome-Wide Comparative Analysis of Five Amaranthaceae Species Reveals a Large Amount of Repeat Content.

Amaranthus is a genus of C4 dicotyledonous herbaceous plant species that are widely distributed in Asia, Africa, Australia, and Europe and are used as grain, vegetables, forages, and ornamental plants. Amaranth species have gained significant attention nowadays as potential sources of nutritious food and industrial products. In this study, we performed a comparative genome analysis of five amaranth species, namely, Amaranthus hypochondriacus, Amaranthus tuberculatus, Amaranthus hybridus, Amaranthus palmeri, and Amaranthus cruentus. The estimated repeat content ranged from 54.49% to 63.26% and was not correlated with the genome sizes. Out of the predicted repeat classes, the majority of repetitive sequences were Long Terminal Repeat (LTR) elements, which account for about 13.91% to 24.89% of all amaranth genomes. Phylogenetic analysis based on 406 single-copy orthologous genes revealed that A. hypochondriacus is most closely linked to A. hybridus and distantly related to A. cruentus. However, dioecious amaranth species, such as A. tuberculatus and A. palmeri, which belong to the subgenera Amaranthus Acnida, have formed their distinct clade. The comparative analysis of genomic data of amaranth species will be useful to identify and characterize agronomically important genes and their mechanisms of action. This will facilitate genomics-based, evolutionary studies, and breeding strategies to design faster, more precise, and predictable crop improvement programs.

Simple cryopreservation protocol for Luffa pollen: enhancing breeding efficiency.

This study aimed to develop a long-term pollen storage protocol for Luffa species (L. acutangula, L. cylindrica, L. echinata, and L. graveolens) and assess its potential for crop improvement. The optimal medium for in vitro pollen germination varied by species, with Brewbaker and Kwack (BK) medium with 10% sucrose suitable for L. acutangula, L. cylindrica, and L. echinata, and BK medium with 3% sucrose ideal for L. graveolens. Overestimation in staining tests compared to in vitro pollen germination was observed. The best results for cryopreservation were achieved with desiccation periods of 20, 30, and 40 min, maintaining moisture content between 14.04% and 18.55%. Pollen viability was negatively correlated with storage temperature (25, 4, and -20°C) and duration. Cryopreserved pollen at -196°C exhibited the highest viability over a prolonged period (2 months) and was comparable to fresh pollen in terms of germination, ovule fertilization, and fruit and seed set. This study presents a simple and reproducible pollen cryopreservation protocol applicable across Luffa species, facilitating long-term storage and its use in crop improvement efforts.

Transcriptome Profiling during Sequential Stages of Cryopreservation in Banana (Musa AAA cv Borjahaji) Shoot Meristem.

Cryopreservation approaches have been implemented in gene banks as a strategy to back up plant genetic resource collections that are vegetatively propagated. Different strategies have been employed to effectively cryopreserve plant tissue. There is little information on the cellular processes and molecular adjustments that confer resilience to the multiple stresses imposed during a cryoprotocol. In the present work, the cryobionomics of banana (Musa sp.), a non-model species, was investigated through the transcriptomic approach using RNA-Seq. Proliferating meristems of in vitro explants (Musa AAA cv 'Borjahaji') were cryopreserved using the droplet-vitrification technique. Transcriptome profiling analysis of eight cDNA libraries including the bio-replicates for T0 (stock cultures (control tissue), T1 (high sucrose pre-cultured), T2 (vitrification solution-treated) and T3 (liquid nitrogen-treated) meristem tissues was carried out. The raw reads obtained were mapped with a Musa acuminata reference genome sequence. A total of 70 differentially expressed genes (DEGs) comprising 34 upregulated and 36 downregulated were identified in all three phases as compared to control (T0). Among the significant DEGs (>log FC 2.0), during sequential steps, 79 in T1, 3 in T2 and the 4 in T3 were upregulated and 122 in T1, 5 in T2 and 9 in T3 were downregulated. Gene ontology (GO) enrichment analysis showed that these significant DEGs were involved in the upregulation of biological process (BP-170), cellular component (CC-10) and molecular function (MF-94) and downregulation of biological process (BP-61), cellular component (CC-3) and molecular function (MF-56). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that DEGs were involved in the biosynthesis of secondary metabolites, glycolysis/gluconeogenesis, MAPK signaling, EIN 3-lke 1 protein, 3-ketoacy-CoA synthase 6-like, and fatty acid elongation during cryopreservation. For the first time, a comprehensive transcript profiling during four stages of cryopreservation in banana were carried out, which will pave the way for devising an effective cryopreservation protocol.

Optimizing and determining the click chemistry mediated Cu-64 radiolabeling and physiochemical characteristics of trastuzumab conjugates.

Over the last decade, 64Cu-labeling of monoclonal antibody (mAb) via inverse electron demand Diels-Alder click chemistry (IEDDA) have received much attention. Despite the tetrazine-transcyclooctene (Tz-TCO) click chemistry's convenience and efficiency in mAb labeling, there is limited information about the ideal parameters in the development of click chemistry mediated (radio)immunoconjugates. This encourages us to conduct a systematic optimization while concurrently determining the physiochemical characteristics of the model mAb, trastuzumab, and TCO conjugates. To accomplish this, we investigated a few critical parameters, first, we determined the degree of conjugations with varying molar equivalents (eq.) of TCO (3, 5, 10, and 15 eq.). Through analytical techniques like size exclusion chromatography, dynamic light scattering, and zeta potential, qualitative analysis were performed to determine the purity, degree of aggregation and net charge of the conjugates. We found that as the degree of conjugation increased the purity of intact mAb fraction is compromised and net charge of conjugates became less positive. Next, all trastuzumab-PEG4-TCO conjugates with varying molar ratio and quantity (30, 50, 100, 200, 250 µg) were radiolabeled with 64Cu-NOTA-PEG4-Tz via IEDDA click chemistry and radiochemical yields were determined by radio-thin layer chromatography. The radiochemical yields of trastuzumab conjugates improved with increased amount and molar ratio. Next, we investigated the effect of the radioprotectant ascorbic acid (AA) of varied concentrations (0.25, 0.5, 0.75, 1 mM) on radiochemical yields and subsequent pharmacokinetics. A concentration of 0.25 mM of AA was found to be optimal for click reaction and in vivo biodistribution. Finally, we investigated the indirect influence of bioconjugation buffers on radiochemical yields and biodistribution in NIH3T6.7 tumor models that resulted approximately ∼11 %ID/g tumor uptake.

Preclinical Evaluation of hnRNPA2B1 Antibody in Human Triple-Negative Breast Cancer MDA-MB-231 Cells via PET Imaging.

Triple-negative breast cancer (TNBC) does not express estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Because TNBC lacks the expression of commonly targeted receptors, it is challenging to develop a new imaging agent for this cancer subtype. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are RNA-protein complexes that have been linked to tumor development and progression. Considering the high expression of hnRNPA2B1, an hnRNP subtype, in TNBC MDA-MB-231 cells, this study aimed to develop a novel hnRNPA2B1 antibody-based nuclear imaging agent. The hnRNPA2B1-specific antibody was radiolabeled with 64Cu and evaluated in vitro and in vivo. The trans-cyclooctene (TCO) was functionalized on the antibody to obtain hnRNP-PEG4-TCO and reactive tetrazine (Tz) on the ultrastable bifunctional chelator PCB-TE2A-alkyne to yield PCB-TE2A-Tz for the inverse electron demand Diels-Alder reaction. The 64Cu-radiolabeled antibody was administered and imaged at 1-18 h time points for conventional imaging. Alternatively, the unlabeled antibody conjugate was administered, and 48 h later radiolabeled 64Cu-PCB-TE2A-Tz was administered to the same mice for the pretargeting strategy and imaged at the same time intervals for direct comparison. The tumor was successfully visualized in both strategies, and comparatively, pretargeting showed superior results. The 64Cu-PCB-TE2A-Tz was successfully clicked at the tumor site with hnRNP-PEG4-TCO and the non-clicked were concurrently eliminated. This led to increase the tumor uptake with extremely high tumor-to-background ratio manifested by positron emission tomography (PET) imaging and biodistribution studies.

In vivo detection of hydrogen sulfide in the brain of live mouse: application in neuroinflammation models.

PURPOSE: Hydrogen sulfide (H2S) plays important roles in brain pathophysiology. However, nuclear imaging probes for the in vivo detection of brain H2S in living animals have not been developed. Here, we report the first nuclear imaging probe that enables in vivo imaging of endogenous H2S in the brain of live mice. METHODS: Utilizing a bis(thiosemicarbazone) backbone, a fluorescent ATSM-FITC conjugate was synthesized. Its copper complex, Cu(ATSM-FITC) was thoroughly tested as a biosensor for H2S. The same ATSM-FITC ligand was quantitatively labeled with [64Cu]CuCl2 to obtain a radioactive [64Cu][Cu(ATSM-FITC)] imaging probe. Biodistribution and positron emission tomography (PET) imaging studies were performed in healthy mice and neuroinflammation models. RESULTS: The Cu(ATSM-FITC) complex reacts instantly with H2S to release CuS and becomes fluorescent. It showed excellent reactivity, sensitivity, and selectivity to H2S. Endogenous H2S levels in living cells were successfully detected by fluorescence microscopy. Exceptionally high brain uptake of [64Cu][Cu(ATSM-FITC)] (> 9% ID/g) was observed in biodistribution and PET imaging studies. Subtle changes in brain H2S concentrations in live mice were accurately detected by quantitative PET imaging. Due to its dual modality feature, increased H2S levels in neuroinflammation models were characterized at the subcellular level by fluorescence imaging and at the whole-body scale by PET imaging. CONCLUSION: Our biosensor can be readily utilized to study brain H2S function in live animal models and shows great potential as a novel imaging agent for diagnosing brain diseases.

Correction: A short PEG linker alters the in vivo pharmacokinetics of trastuzumab to yield high-contrast immuno-PET images.

Correction for 'A short PEG linker alters the in vivo pharmacokinetics of trastuzumab to yield high-contrast immuno-PET images' by Woonghee Lee et al., J. Mater. Chem. B, 2021, 9, 2993-2997, DOI: 10.1039/D0TB02911D.

Asymmetric Enamide-Imine Tautomerism in the Kinetic Resolution of Tertiary Alcohols.

An efficient protocol for kinetic resolution of tertiary alcohols has been developed through an unprecedented asymmetric enamide-imine tautomerism process enabled by chiral phosphoric acid catalysis. A broad range of racemic 2-arylsulfonamido tertiary allyl alcohols could be kinetically resolved with excellent kinetic resolution performances (with s-factor up to >200). This method is particularly effective for a series of 1,1-dialkyl substituted allyl alcohols, which produced chiral tertiary alcohols that would be difficult to access via other asymmetric methods. Facile and versatile transformations of the chiral α-hydroxy imine and enamide products, especially the efficient stereodivergent synthesis of all four stereoisomers of ß-amino tertiary alcohols using one enantiomer of the catalyst, demonstrated the value of this kinetic resolution method.

A short PEG linker alters the in vivo pharmacokinetics of trastuzumab to yield high-contrast immuno-PET images.

The prolonged blood circulation of the radiolabeled antibody conjugates is problematic when using immuno-PET imaging due to the increased radiation exposure and longer hospitalization required until sufficient contrast develops. In contrast to the prevailing belief that PEGylation prolongs blood retention time, we observed that a PEGylated antibody with a short PEG8 linker cleared much faster from the blood while maintaining tumor uptake compared to its non-PEGylated counterpart. Breast tumors were clearly visualized with a very high tumor-to-background ratio as early as 24 h after injection in immuno-positron emission tomography (PET) imaging.

Successful Application of CuAAC Click Reaction in Constructing 64Cu-Labeled Antibody Conjugates for Immuno-PET Imaging.

Immuno-positron emission tomography (immuno-PET) is a rapidly growing imaging technique in which antibodies are radiolabeled to monitor their in vivo behavior in real time. However, effecting the controlled conjugation of a chelate-bearing radioactive atom to a bulky antibody without affecting its immunoreactivity at a specific site is always challenging. The in vivo stability of the radiolabeled chelate is also a key issue for successful tumor imaging. To address these points, a facile ultra-stable radiolabeling platform is developed by using the propylene cross-bridged chelator (PCB-TE2A-alkyne), which can be instantly functionalized with various groups via the click reaction, thus enabling specific conjugation with antibodies as per choice. The PCB-TE2A-tetrazine derivative is selected to demonstrate the proposed strategy. The antibody trastuzumab is functionalized with the trans-cyclooctene (TCO) moiety in the presence or absence of the PEG linker. The complementary 64Cu-PCB-TE2A-tetrazine is synthesized via the click reaction and radiolabeled with 64Cu ions, which then reacts with the aforementioned TCO-modified antibody via a rapid biorthogonal ligation. The 64Cu-PCB-TE2A-trastuzumab conjugate is shown to exhibit excellent in vivo stability and to maintain a higher binding affinity toward HER2-positive cells. The tumor targeting feasibility of the radiolabeled antibody is evaluated in tumor models. Both 64Cu-PCB-TE2A-trastuzumab conjugates show high tumor uptakes in biodistribution studies and enable unambiguous tumor visualization with minimum background noise in PET imaging. Interestingly, the 64Cu-PCB-TE2A-PEG4-trastuzumab containing an additional PEG linker displays a much faster body clearance compared to its counterpart with less PEG linker, thus affording vivid tumor imaging with an unprecedentedly high tumor-to-background ratio.

Chiral phosphoric acid-catalyzed stereodivergent synthesis of trisubstituted allenes and computational mechanistic studies.

Chiral molecules with multiple stereocenters are widely present in natural products and pharmaceuticals, whose absolute and relative configurations are both critically important for their physiological activities. In spite of the fact that a series of ingenious strategies have been developed for asymmetric diastereodivergent catalysis, most of these methods are limited to the divergent construction of point chirality. Here we report an enantioselective and diastereodivergent synthesis of trisubstituted allenes by asymmetric additions of oxazolones to activated 1,3-enynes enabled by chiral phosphoric acid (CPA) catalysis, where the divergence of the allenic axial stereogenicity is realized by modifications of CPA catalysts. Density functional theory (DFT) calculations are performed to elucidate the origin of diastereodivergence by the stacking- and stagger-form in the transition state (TS) of allene formation step, as well as to disclose a Münchnone-type activation mode of oxazolones under Brønsted acid catalysis.

Chiral Phosphoric Acid Catalyzed Kinetic Resolution of 2-Amido Benzyl Alcohols: Asymmetric Synthesis of 4H-3,1-Benzoxazines.

An efficient method for the asymmetric synthesis of 4H-3,1-benzoxazines was developed by kinetic resolution of 2-amido benzyl alcohols using chiral phosphoric acid catalyzed intramolecular cyclizations. A broad range of benzyl alcohols (both secondary and tertiary alcohols) were kinetically resolved with high selectivities, with an s factor of up to 94. Mechanistic studies were performed to elucidate the mechanism of these reactions, wherein the amide moieties reacted as the electrophiles. Gram-scale reaction and facile transformations of the chiral products demonstrate the potential of this method in asymmetric synthesis of biologically active chiral heterocycles.

Kinetic Resolution of Tertiary 2-Alkoxycarboxamido-Substituted Allylic Alcohols by Chiral Phosphoric Acid Catalyzed Intramolecular Transesterification.

A highly enantioselective kinetic resolution of tertiary 2-alkoxycarboxamido allylic alcohols has been achieved through a chiral phosphoric acid catalyzed intramolecular transesterification reaction. Both alkyl,aryl- and dialkyl-substituted tertiary allylic alcohols were resolved with excellent efficiencies, affording both the recovered tertiary alcohols and the carbamate products with high enantioselectivities (with s factors up to 164.6). A gram-scale reaction with 1 mol % catalyst loading and the facile conversion of the enantioenriched products into useful chiral building blocks, such as chiral oxazolidinones and ß-amino alcohols, demonstrate the value of this reaction.

Asymmetric Synthesis of ß-Indolyl Cyclopentanones and Cyclopentylamides with an All-Carbon Quaternary Stereocenter via Chiral Phosphoric Acid Catalyzed Friedel-Crafts Alkylation Reactions.

The asymmetric Friedel-Crafts alkylation reactions of indoles with ß-substituted cyclopentenimines enabled by chiral phosphoric acid catalysis has been developed, which affords adducts possessing an all-carbon stereocenter with high levels of enantioselectivities. Furthermore, the addition products could be readily converted into two types of useful but previously challenging chiral building blocks, such as ß-alkyl-ß-indolyl cyclopentanones and ß-alkyl-ß-indolyl cyclopentylamides, in one pot via in situ hydrolysis or reduction without erosion of chiral information.

Regioselective and Enantioselective Synthesis of ß-Indolyl Cyclopentenamides by Chiral Anion Catalysis.

The regioselective and enantioselective synthesis of ß-indolyl cyclopentenamides, a versatile chiral building block, by asymmetric addition of indoles to α,ß-unsaturated iminium intermediates has been achieved through chiral anion catalysis. Key to the success of this methodology is the generation of a chiral anion-paired ketone-type α,ß-unsaturated iminium intermediate from α-hydroxy enamides. Preliminary mechanistic studies and DFT calculations are consistent with a mechanism involving multiple, concurrent pathways for isomerization of the initially formed azaallylcation into the key α,ß-unsaturated iminium intermediate, all mediated by the phosphoric acid catalyst.

Expedient synthesis of new cinnoline diones by Ru-catalyzed regioselective unexpected deoxygenation-oxidative annulation of propargyl alcohols with phthalazinones and pyridazinones.

Ruthenium-catalyzed simple, cascade and one-pot synthesis of cinnoline-fused diones has been carried out by the C-H activation of phthalazinones/pyridazinones accomplished by the unusual deoxygenation of propargyl alcohols. The bond selectivity is accredited to the traceless directing nature of the hydroxyl group of propargyl alcohol. A sequential C-H activation, insertion and deoxy-oxidative annulation has been proposed based on the preliminary mechanistic study.

Genetic diversity analysis of fungal pathogen Bipolaris sorghicola infecting Sorghum bicolor in India.

Bipolaris sorghicola (Lefebvre and Sherwin) is a well known and economically important seed-borne pathogen with the specific species of sorghum (Sorghum bicolor [L] Moench) as host. Thirty-two strains were obtained from different geographical area of sorghum growing places in India. Molecular characterization using three marker systems i.e., universal rice primers (URP), inter simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) was carried out. Molecular marker work revealed differences along with geographical origin clustering of various B. sorghicola strains which could not be revealed through conventional method of characterization. Out of 13 URPs, 20 ISSR and 50 RAPD primers screened, 8 primers each from URP and ISSR, and 10 primers from RAPD marker were found to result in reproducible banding pattern. One hundred per cent of polymorphic bands was recorded in all three molecular markers. Total number of bands was recorded 1986 with average of 248.25 in URP marker, and 2026 bands with average of 253.25 in ISSR marker and 2158 bands with average of 215.80 in RAPD markers. Maximum heterozygosity (Hn) was revealed by URP 17R (0.40), ISSR 10 (0.41) and RAPD marker OPC-5 (0.34). The polymorphism information content (PIC) values ranged between 5.89 to 8.28 in URP, 4.57 to 8.79 in ISSR and 4.44 to 9.64 in RAPD marker profiles. Maximum cophenetic correlation was found in URP (r = 0.910) followed by ISSR (r = 0.904) and RAPD (r = 0.870). The combined analysis of all three marker systems showed high cophenetic correlation (r = 0.911), which indicated a very good fit of the data for genetic diversity analysis. To best of our knowledge, this is a first report of genetic characterization of B. sorghicola. Hence, combined use of three marker systems would be more sensitive and reliable in characterizing genetic variability in B. sorghicola strains.

Ru(II)-Catalyzed ß-Carboline Directed C-H Arylation and Isolation of Its Cycloruthenated Intermediates.

A Ru(II)-catalyzed C-H arylation approach has been developed utilizing ß-carboline alkaloids as the directing group. Selective formations of diarylated products from moderate to excellent yields were accomplished. Broad substrate scope with excellent functional group tolerance for C1-phenyl/thienyl/PAHs-ß-carbolines was demonstrated. X-ray crystal structure of cycloruthenated complex 2cr and no arylation reaction with model substrate 13 strongly suggests that N2 is the directing group than N9 in C1-aryl-ß-carbolines. Catalytic properties and stability of the cycloruthenated complexes have been explored. Library of biologically relevant new ß-carboline derivatives and isolation of its cycloruthenated intermediates are the highlights of this work.

Expressed sequence tags and molecular cloning and characterization of gene encoding pinoresinol/lariciresinol reductase from Podophyllum hexandrum.

Podophyllotoxin, an aryltetralin lignan, is the source of important anticancer drugs etoposide, teniposide, and etopophos. Roots/rhizome of Podophyllum hexandrum form one of the most important sources of podophyllotoxin. In order to understand genes involved in podophyllotoxin biosynthesis, two suppression subtractive hybridization libraries were synthesized, one each from root/rhizome and leaves using high and low podophyllotoxin-producing plants of P. hexandrum. Sequencing of clones identified a total of 1,141 Expressed Sequence Tags (ESTs) resulting in 354 unique ESTs. Several unique ESTs showed sequence similarity to the genes involved in metabolism, stress/defense responses, and signalling pathways. A few ESTs also showed high sequence similarity with genes which were shown to be involved in podophyllotoxin biosynthesis in other plant species such as pinoresinol/lariciresinol reductase. A full length coding sequence of pinoresinol/lariciresinol reductase (PLR) has been cloned from P. hexandrum which was found to encode protein with 311 amino acids and show sequence similarity with PLR from Forsythia intermedia and Linum spp. Spatial and stress-inducible expression pattern of PhPLR and other known genes of podophyllotoxin biosynthesis, secoisolariciresinol dehydrogenase (PhSDH), and dirigent protein oxidase (PhDPO) have been studied. All the three genes showed wounding and methyl jasmonate-inducible expression pattern. The present work would form a basis for further studies to understand genomics of podophyllotoxin biosynthesis in P. hexandrum.

Genetic structure of Indian valerian (Valeriana jatamansi) populations in western Himalaya revealed by AFLP.

Valeriana jatamansi Jones is a natural tetraploid species indigenous to the Indian Himalaya. To assess its genetic diversity and population structure, we analyzed six natural populations from the western Himalayan region using amplified fragment length polymorphism. An analysis of molecular variance found that 93% of the genetic variation of V. jatamansi was within populations and 7% among populations. The correlation between genetic and geographic distances (r = 0.14) was not significant. Though the populations are well separated, the lack of distinct genetic variation between populations may be due to either recent rapid fragmentation from a wide and continuous area resulting in genetically similar populations or wide dispersal of seed by wind, since the follicles are feathery. Polyploidy may be the reason for the lack of genetic impoverishment due to fragmentation.