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Once considered potential liabilities, the modern era witnesses a renaissance of interest in covalent inhibitors in drug discovery. The available toolbox of electrophilic warheads is limited by constraints on tuning reactivity and selectivity. Following our work on a class of ferroptotic agents termed CETZOLEs, we discovered new tunable heterocyclic electrophiles which are capable of inducing ferroptosis. The biological evaluation demonstrated that thiazoles with an alkyne electrophile at the 2-position selectively induce ferroptosis with high potency. Density functional theory calculations and NMR kinetic studies demonstrated the ability of our heterocycles to undergo thiol addition, an apparent prerequisite for cytotoxicity. Chemoproteomic analysis indicated several potential targets, the most prominent among them being GPX4 protein. These results were further validated by western blot analysis and the cellular thermal shift assay. Incorporation of these heterocycles into appropriate pharmacophores generated highly cytotoxic agents such as the analogue

Reactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) μ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol μ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol μ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability., The authors previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP). Here they reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during double strand break repair by pol μ.

Across all walks of life, certain transfer RNA (tRNA) transcripts contain introns. Pre-tRNAs with introns require splicing to form the mature anticodon stem loop (ASL). In eukaryotes, tRNA splicing is initiated by the heterotetrameric tRNA splicing endonuclease (TSEN) complex. All TSEN subunits are essential and mutations within the complex are associated with a family of neurodevelopmental disorders known as pontocerebellar hypoplasia (PCH). The pathogenesis of PCH is poorly understood. Moreover, a lack of structures for any eukaryotic TSEN complex has hindered our understanding of tRNA recognition and processing. Here, we report Cryo-Electron Microscopy (cryo-EM) structures of the human TSEN•pre-tRNA complex, trapped in the pre-cleavage state, at near atomic resolution. These structures reveal the overall architecture of the complex, along with extensive tRNA binding interfaces within the complex. Although it shares structural homology with archaeal TSENs, the human TSEN complex contains additional features important for recognizing the acceptor stem and D-arm of the pre-tRNA. Our findings also establish the TSEN54 subunit as more than a simple molecular ruler; it functions as a pivotal scaffold for the pre-tRNA and the two endonuclease subunits, TSEN2 and TSEN34. Finally, the human TSEN structures enable detailed visualization of the molecular environments of PCH-causing missense mutations, providing crucial insight into the mechanism of eukaryotic pre-tRNA splicing and neurodevelopmental disease.

Graphical abstract, Highlights • The SARS-CoV-2 main protease is a prime drug target. • Coronavirus main proteases share a structurally conserved substrate-binding region. • The structure and sequence similarity of SARS-CoV-2 to SARS-CoV readily allows testing of inhibitors designed for the latter. • Both covalent and non-covalent inhibitors of the SARS-CoV-2 main protease have been designed., The coronavirus disease 2019 (COVID-19) pandemic has prompted an urgent need for new treatment strategies. No target-specific drugs are currently available for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but new drug candidates targeting the viral replication cycle are being explored. A prime target of drug-discovery efforts is the SARS-CoV-2 main protease (Mpro). The main proteases of different coronaviruses, including SARS-CoV-2, SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), share a structurally conserved substrate-binding region that can be exploited to design new protease inhibitors. With the recent reporting of the X-ray crystal structure of the SARS-CoV-2 Mpro, studies to discover Mpro inhibitors using both virtual and in vitro screening are progressing rapidly. This review focusses on the recent developments in the search for small-molecule inhibitors targeting the SARS-CoV-2 Mpro.

Covalent inhibitors have historically been considered potential liabilities in medicinal chemistry. The modern era witnesses a renaissance of interest in irreversible binders. The available toolbox of electrophilic warheads is limited with constraints on tuning reactivity and selectivity. Following our initial work on a new class of ferroptotic agents termed CETZOLEs, we discovered new tunable heterocyclic electrophiles, which are capable of inducing ferroptosis. Extensive biological evaluation demonstrated that thiazoles with an alkyne electrophile at the 2-position selectively induce ferroptosis with high potency. Density functional theory (DFT) calculations and NMR kinetic studies demonstrated the ability of our heterocycles to undergo thiol addition, which appears to be a prerequisite for cytotoxicity. Chemoproteomic analysis by in-gel fluorescence and pulldown experiments indicated several potential targets, the most prominent among them being GPX4 protein. The results from the proteomic data were further validated by western blot analysis and CETSA. Incorporation of our heterocycles into appropriate pharmacophores generates highly cytotoxic agents such as the analog BCP-T. A, which demonstrated low nM IC50 values in a series of ferroptosis-sensitive cell lines.

Weligama Coconut Leaf Wilt Disease (WCLWD) is a non-lethal, but debilitating phytoplasma disease found in coconut palms in Sri Lanka which is confined to the Southern Province of the country, well-away from the major coconut growing area. If it spreads to the major coconut growing area, it might severely damage the coconut industry in Sri Lanka. Government commenced a disease control program to eradicate the disease and, more importantly to prevent spreading of the disease to major coconut growing areas. The major constraint in this program is the lack of an accurate and reliable method for identifying affected palms. Visual symptoms are used to identify the affected palms for removal, yet growers are not always convinced of the method of resisting palm removal. This poses a serious threat to the implementation of the disease control program. Although a Nested-PCR-based disease diagnosis was established earlier, the detection rate and reliability need further improvements. Therefore, an urgent necessity for a more reliable disease detection method has arisen. In the current study, a Real-Time Polymerase Chain Reaction (qPCR) powered by a pair of primers and a probe designed from the published partial sequences of the WCLWD phytoplasma was validated with 202 coconut samples and a detection rate of above 95% was achieved. This newly established detection system was highly reliable and a way forward for controlling the WCLWD disease in Sri Lanka.

DNA replication and repair reactions involve the addition of a deoxynucleoside monophosphate onto a growing DNA strand with the loss of pyrophosphate. This chemical reaction is also reversible; the addition of pyrophosphate generates a deoxynucleoside triphosphate, thereby shortening the DNA by one nucleotide. The forward DNA synthesis and reverse pyrophosphorolysis reactions strictly require the presence of divalent metals, usually magnesium, at the reactive center as cofactors. The overall equilibrium enzymatic reaction strongly favors DNA synthesis over pyrophosphorolysis with natural substrates. The DNA polymerase β chemical reaction has been structurally and kinetically characterized, employing natural and chemically modified substrates. Substituting an imido-moiety (NH) for the bridging oxygen between Pβ and Pγ of dGTP dramatically decreased the overall enzymatic activity and resulted in a chemical equilibrium that strongly favors the reverse reaction (i.e., K ≪ 1). Using QM/MM calculations in conjunction with the utilization of parameters such as quantum mechanically derived atomic charges, we have examined the chemical foundation for the altered equilibrium with this central biological reaction. The calculations indicate that the rapid reverse reaction is likely due, in part, to the increased nucleophilicity of the reactive oxygen on the tautomeric form of imidodiphosphate.

We report the properties of two mutations in the exonuclease domain of the Saccharomyces cerevisiae DNA polymerase ϵ. One, pol2-Y473F, increases the mutation rate by about 20-fold, similar to the catalytically dead pol2-D290A/E290A mutant. The other, pol2-N378K, is a stronger mutator. Both retain the ability to excise a nucleotide from double-stranded DNA, but with impaired activity. pol2-Y473F degrades DNA poorly, while pol2-N378K degrades single-stranded DNA at an elevated rate relative to double-stranded DNA. These data suggest that pol2-Y473F reduces the capacity of the enzyme to perform catalysis in the exonuclease active site, while pol2-N378K impairs partitioning to the exonuclease active site. Relative to wild-type Pol ϵ, both variants decrease the dNTP concentration required to elicit a switch between proofreading and polymerization by more than an order of magnitude. While neither mutation appears to alter the sequence specificity of polymerization, the N378K mutation stimulates polymerase activity, increasing the probability of incorporation and extension of a mismatch. Considered together, these data indicate that impairing the primer strand transfer pathway required for proofreading increases the probability of common mutations by Pol ϵ, elucidating the association of homologous mutations in human DNA polymerase ϵ with cancer.

Post-transcriptional processes mediated by mRNA binding proteins represent important control points in gene expression. In eukaryotes, mRNAs containing specific AU-rich motifs are regulated by binding of tristetraprolin (TTP) family tandem zinc finger proteins, which promote mRNA deadenylation and decay, partly through interaction of a conserved C-terminal CNOT1 binding (CNB) domain with CCR4–NOT protein complexes. The social ameba Dictyostelium discoideum shared a common ancestor with humans more than a billion years ago, and expresses only one TTP family protein, TtpA, in contrast to three members expressed in humans. Evaluation of ttpA null-mutants identified six transcripts that were consistently upregulated compared to WT during growth and early development. The 3′-untranslated regions (3′-UTRs) of all six ‘TtpA-target’ mRNAs contained multiple TTP binding motifs (UUAUUUAUU), and one 3′-UTR conferred TtpA post-transcriptional stability regulation to a heterologous mRNA that was abrogated by mutations in the core TTP-binding motifs. All six target transcripts were upregulated to similar extents in a C-terminal truncation mutant, in contrast to less severe effects of analogous mutants in mice. All six target transcripts encoded probable membrane proteins. In Dictyostelium, TtpA may control an ‘RNA regulon’, where a single RNA binding protein, TtpA, post-transcriptionally co-regulates expression of several functionally related proteins.

Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants. ONE-SENTENCE SUMMARY: A cost-effective, high-throughput, adaptable pipeline capable of identifying effective humanized nanobodies against SARS-CoV-2.

Structural snapshots of protein/ligand complexes are a prerequisite for gaining atomic level insight into enzymatic reaction mechanisms. An important group of enzymes has been deprived of this analytical privilege: members of the protein tyrosine phosphatase (PTP) superfamily with catalytic WPD-loops lacking the indispensable general-acid/base within a tryptophan-proline-aspartate/glutamate context. Here, we provide the ligand/enzyme crystal complexes for one such PTP outlier: Arabidopsis thaliana Plant and Fungi Atypical Dual Specificity Phosphatase 1 (AtPFA-DSP1), herein unveiled as a regioselective and efficient phosphatase towards inositol pyrophosphate (PP-InsP) signaling molecules. Although the WPD loop is missing its canonical tripeptide motif, this structural element contributes to catalysis by assisting PP-InsP delivery into the catalytic pocket, for a choreographed exchange with phosphate reaction product. Subsequently, an intramolecular proton donation by PP-InsP substrate is posited to substitute functionally for the absent aspartate/glutamate general-acid. Overall, we expand mechanistic insight into adaptability of the conserved PTP structural elements.

Despite the advances in treatment strategies, cancer is still the second leading cause of death in the USA. A majority of the currently used cancer drugs have limitations in their clinical use due to poor selectivity, toxic side effects and multiple drug resistance, warranting the development of new anticancer drugs of different mechanisms of action. Here we describe the design, synthesis and initial biological evaluation of a new class of antimitotic agents that modulate tubulin polymerization. Structurally, these compounds are chalcone mimics containing a 1-(1H-imidazol-2-yl)ethan-1-one moiety, which was initially introduced to act as a metal-binding group and inhibit histone deacetylase enzymes. Although several analogues selectively inhibited purified HDAC8 with IC(50) values in low micromolar range, tissue culture studies suggest that HDAC inhibition is not a major mechanism responsible for cytotoxicity. The compounds demonstrated cell growth inhibition with GI(50) values of upper nanomolar to low micromolar potency with significant selectively for cancer over normal cells. Interestingly, several compounds arrested HeLaM cells in mitosis and seem to target tubulin to cause mitotic arrest. For example, when combined with inhibitors of Aurora B kinase, they led to dramatic disassembly of the mitotic spindle. In-vitro tubulin polymerization studies showed that the compounds reduced the rate of polymerization of microtubules during the elongation phase and lowered the amount of polymerized tubulin during the plateau phase. Finally, in silico docking studies identified binding of IPE-7 to the colchicine site with similar affinity as the test compound D64131. These compounds represent a new antimitotic pharmacophore with limited HDAC inhibitory activity.

The cockroach allergen Bla g 1 forms a novel fold consisting of 12 amphipathic alpha-helices enclosing an exceptionally large hydrophobic cavity which was previously demonstrated to bind a variety of lipids. Since lipid-dependent immunoactivity is observed in numerous allergens, understanding the structural basis of this interaction could yield insights into the molecular determinants of allergenicity. Here, we report atomic modelling of Bla g 1 bound to both fatty-acid and phospholipids ligands, with 8 acyl chains suggested to represent full stoichiometric binding. This unusually high occupancy was verified experimentally, though both modelling and circular dichroism indicate that the general alpha-helical structure is maintained regardless of cargo loading. Fatty-acid cargoes significantly enhanced thermostability while inhibiting cleavage by cathepsin S, an endosomal protease essential for antigen processing and presentation; the latter of which was found to correlate to a decreased production of known T-cell epitopes. Both effects were strongly dependent on acyl chain length, with 18–20 carbons providing the maximal increase in melting temperature (~20 °C) while completely abolishing proteolysis. Diacyl chain cargoes provided similar enhancements to thermostability, but yielded reduced levels of proteolytic resistance. This study describes how the biophysical properties of Bla g 1 ligand binding and digestion may relate to antigen processing, with potential downstream implications for immunogenicity.

The androgen receptor (AR) regulates male sexual development. We have now investigated AR homodimerization, hormone-dependent monomerization and nuclear translocation in PC-3 and COS-1 cells, by utilizing mutations associated with the androgen insensitivity syndrome: Pro767Ala, Phe765Leu, Met743Val and Trp742Arg. AR wild type (WT) was expressed as a homodimer in the cytoplasm, while none of these mutants formed homodimers. Unlike AR WT which responded to 1 nM dihydrotestosterone (DHT) to dissociate and translocate into the nucleus, AR Pro767Ala and Phe765Leu mutants remain as the monomer in the cytoplasm. In the crystal structure of the AR LBD homodimer, Pro767 and Phe765 reside closely on a loop that constitutes the dimer interface; their sidechains interact with the Pro767 of the other monomer and with the DHT molecule in the ligand-binding pocket. These observations place Phe765 at a position to facilitate DHT binding to Pro767 and lead to dissociation of the AR homodimer in the cytoplasm. This Pro-Phe Met relay may constitute a structural switch that mediates androgen signaling and is conserved in other steroid hormone receptors.

Several mutations in the gene for the mitochondrial single stranded DNA binding protein (SSBP1) have recently been implicated in human disease, but initial reports are insufficient to explain the molecular mechanism of disease, including the possible role of SSBP1 heterotetramers in heterozygous patients. Here we employed molecular simulations to model the dynamics of wild type and 31 variant SSBP1 tetramer systems, including 7 variant homotetramer and 24 representative heterotetramer systems. Our simulations indicate that all variants are stable and most have stronger intermonomer interactions, reduced solvent accessible surface areas, and a net loss of positive surface charge. We then used structural alignments and phosphate binding simulations to predict DNA binding surfaces on SSBP1. Our models suggest that nearly the entire surface of SSBP1, excluding flexible loops and protruding helices, is available for DNA binding, and we observed several potential DNA binding hotspots. Changes to the protein surface in variant SSBP1 tetramers potentially alter anchor points or wrapping paths, rather than abolishing binding altogether. Overall, our findings disqualify tetramer destabilization or gross disruption of DNA binding as mechanisms of disease. Instead, they are consistent with subtle changes to DNA binding, wrapping, or release that cause rare but consequential failures of mtDNA maintenance, which, in turn, are consistent with the late onset of disease in most of the reported SSBP1 cases.

Nsp15 is a uridine specific endoribonuclease that coronaviruses employ to cleave viral RNA and evade host immune defense systems. Previous structures of Nsp15 from across Coronaviridae revealed that Nsp15 assembles into a homo-hexamer and has a conserved active site similar to RNase A. Beyond a preference for cleaving RNA 3’ of uridines, it is unknown if Nsp15 has any additional substrate preferences. Here we used cryo-EM to capture structures of Nsp15 bound to RNA in pre- and post-cleavage states. The structures along with molecular dynamics and biochemical assays revealed critical residues involved in substrate specificity, nuclease activity, and oligomerization. Moreover, we determined how the sequence of the RNA substrate dictates cleavage and found that outside of polyU tracts, Nsp15 has a strong preference for purines 3’ of the cleaved uridine. This work advances our understanding of how Nsp15 recognizes and processes viral RNA and will aid in the development of new anti-viral therapeutics.

The mosquito protein AEG12 is up-regulated in response to blood meals and flavivirus infection though its function remained elusive. Here, we determine the three-dimensional structure of AEG12 and describe the binding specificity of acyl-chain ligands within its large central hydrophobic cavity. We show that AEG12 displays hemolytic and cytolytic activity by selectively delivering unsaturated fatty acid cargoes into phosphatidylcholine-rich lipid bilayers. This property of AEG12 also enables it to inhibit replication of enveloped viruses such as Dengue and Zika viruses at low micromolar concentrations. Weaker inhibition was observed against more distantly related coronaviruses and lentivirus, while no inhibition was observed against the nonenveloped virus adeno-associated virus. Together, our results uncover the mechanistic understanding of AEG12 function and provide the necessary implications for its use as a broad-spectrum therapeutic against cellular and viral targets.

Nsp15, a uridine specific endoribonuclease conserved across coronaviruses, processes viral RNA to evade detection by host defense systems. Crystal structures of Nsp15 from different coronaviruses have shown a common hexameric assembly, yet how the enzyme recognizes and processes RNA remains poorly understood. Here we report a series of cryo-EM reconstructions of SARS-CoV-2 Nsp15, in both apo and UTP-bound states. The cryo-EM reconstructions, combined with biochemistry, mass spectrometry, and molecular dynamics, expose molecular details of how critical active site residues recognize uridine and facilitate catalysis of the phosphodiester bond. Mass spectrometry revealed the accumulation of cyclic phosphate cleavage products, while analysis of the apo and UTP-bound datasets revealed conformational dynamics not observed by crystal structures that are likely important to facilitate substrate recognition and regulate nuclease activity. Collectively, these findings advance understanding of how Nsp15 processes viral RNA and provide a structural framework for the development of new therapeutics.

The coconut palm (Cocos nucifera L.) is grown throughout the tropical world and virtually every part of the palm possesses some human use. It is a monotypic species in the genus Cocos and belongs to the Arecaceae family. Genetic diversity in coconut is mainly compartmentalized between the tall and dwarf palm types. The coconut crop is spread across the globe; hence, different agro-climatic regions contribute to its rich germplasm diversity. Besides morphological descriptors, molecular markers, namely RAPD, ISSR, ISTR, SSR, AFLP, etc. have been widely used to characterize the genetic diversity of coconut originating from different regions. With the expansion of the knowledge of the coconut genomic resources including its genome sequences, DNA sequence-based markers are also being widely utilized presently to characterize the genetic diversity and population structure. Further, genomics and transcriptomics approaches are being utilized to unravel the gene diversity and gene-function relationships. This chapter elaborates the principles and applications of diversity analysis of coconut using various molecular markers and other genomic approaches of coconut that are in vogue.

The enormous potential of DNA-based molecular markers has greatly assisted crop improvement programs of various crops, including non-model species like plantation crops. Despite the perennial nature, analysis of genomic loci governing quantitative traits of economic importance in coconut has gained impetus due to rapid strides in utilization of DNA markers and development of linkage maps. Although various molecular markers have been used to develop linkage maps and to delineate trait-marker association studies, genome-wide association analysis or linkage disequilibrium analysis-based mapping is less explored in coconut. The advent of next-generation sequencing technologies and the availability of whole-genome sequences of coconut genotypes would spur the genome-wide trait-marker association analysis and fine-mapping studies. This chapter discusses the perspective of QTL mapping investigations in coconut and proposes a way forward in the context of developments in genomics-assisted breeding.

We recently reported a new class of imidazole-based chalcones as potential antimitotic agents. In view of their promising cytotoxic activity, a comprehensive structure-activity relationship (SAR) of these compounds was undertaken focusing on four major structural variations: the length of the molecule, the Michael acceptor character, the nature and substitution pattern of ring B, and the nature of the amide functionality tethering ring B. These second-generation analogs (IBCs) demonstrated a superior bioactivity profile than the previously reported imidazole chalcones (referred to as IPEs). The analog IBC-2 with one less methylene group (nor series) and para-fluoro substituted ring B demonstrated the best cytotoxicity profile among the library of compounds. A computational analysis of the NCI-60 data associated both IBCs and the previously reported IPEs with the privileged pharmacological pharmacophore of chalcones. Interestingly, biological studies suggest that the imidazole ring is essential for cytotoxic activity of the elongated chalcone analogues. Immunofluorescence studies revealed that IBC-2, unlike IPEs, has the ability to induce microtubule catastrophe independently of Aurora-B inhibition. The effects of IBC-2 on microtubule dynamics are similar to those of Nocodazole, but the cell cycle effects appear to be different. In-silico studies demonstrate that the members of the new series have the ability to bind to the colchicine binding site of β-tubulin with binding scores similar to those of IPEs, corresponding chalcones and Nocodazole. Although tubulin binding can partially explain the biological effects of IBC-2, on-going target identification studies are aimed at further investigation of its biological targets.

Nsp15, a uridine specific endoribonuclease conserved across coronaviruses, processes viral RNA to evade detection by host defense systems. Crystal structures of Nsp15 from different coronaviruses have shown a common hexameric assembly, yet how the enzyme recognizes and processes RNA remains poorly understood. Here we report a series of cryo-EM reconstructions of SARS-CoV-2 Nsp15, in both apo and UTP-bound states. The cryo-EM reconstructions, combined with biochemistry, mass spectrometry, and molecular dynamics, expose molecular details of how critical active site residues recognize uridine and facilitate catalysis of the phosphodiester bond. Mass spectrometry revealed the accumulation of cyclic phosphate cleavage products, while analysis of the apo and UTP-bound datasets revealed conformational dynamics not observed by crystal structures that are likely important to facilitate substrate recognition and regulate nuclease activity. Collectively, these findings advance understanding of how Nsp15 processes viral RNA and provide a structural framework for the development of new therapeutics., Nsp15 is a uridine specific endoribonuclease present in all coronaviruses. Here, the authors determine the cryo-EM structures of SARS-CoV-2 Nsp15 in the apo and UTP-bound states, which together with biochemical experiments, mass spectrometry and molecular dynamics simulations provide insights into the catalytic mechanism of Nsp15 and its conformational dynamics.

Estrogen insensitivity syndrome (EIS) arises from rare mutations in estrogen receptor-α (ERα, encoded by ESR1 gene) resulting in the inability of estrogen to exert its biological effects. Due to its rarity, mutations in ESR1 gene and the underlying molecular mechanisms of EIS have not been thoroughly studied. Here, we investigate known ESR1 mutants, Q375H and R394H, associated with EIS patients using in vitro and in vivo systems. Comparison of the transcriptome and deoxyribonucleic acid methylome from stable cell lines of both Q375H and R394H clinical mutants shows a differential profile compared with wild-type ERα, resulting in loss of estrogen responsiveness. Molecular dynamic simulation shows that both ESR1 mutations change the ERα conformation of the ligand-receptor complexes. Furthermore, we generated a mouse model Esr1-Q harboring the human mutation using CRISPR/Cas9 genome editing. Female and male Esr1-Q mice are infertile and have similar phenotypes to αERKO mice. Overall phenotypes of the Esr1-Q mice correspond to those observed in the patient with Q375H. Finally, we explore the effects of a synthetic progestogen and a gonadotropin-releasing hormone inhibitor in the Esr1-Q mice for potentially reversing the impaired female reproductive tract function. These findings provide an important basis for understanding the molecular mechanistic consequences associated with EIS.

In situ conservation of coconut germplasm refers to the maintenance of coconut genetic diversity in the agroecosystems where it has evolved. Ex situ conservation comprises all coconut germplasm currently maintained as living trees in field genebanks, screen houses, and elsewhere beyond their host agroecosystems. Ex situ conservation includes in vitro conservation, as well as cryopreservation of pollen, embryos, and embryogenic callus. This chapter presents the variety of existing approaches to conserve coconut germplasm, together with a prospective approach assessing how farmers could better benefit from the advances recently achieved in coconut biotechnology for the sustainable conservation of coconut diversity. This chapter also emphasizes biological and sociological constraints jeopardizing conservation. Ex situ coconut conservation is facing a crisis. Among the 24 registered coconut genebanks, at least 16, including 3 out of the 5 international genebanks, do not have enough capability, laboratory space, equipment, staff, and/or a budget needed to make reliable controlled pollinations for the rejuvenating or maintenance of the germplasm. The most efficient way to conserve coconut diversity should include a multifunctional approach, involving the conservation of additional appropriate crops or species by the same team, at the same site or in the same laboratory. Such multifunctional approaches that integrate “networked” and multifunctional genebanks and harness the latest developments in tissue culture and cryopreservation will help address the unique constraints facing coconut conservation and could help strengthen the networks and communications needed to ensure effective conservation.

A new resveratrol trimer, vateriferol (1), having four cis-oriented methine protons and constituting four contiguous stereocenters, was isolated from the bark extract of Vateria copallifera by bioassay-guided fractionation using a combination of normal, reversed phase, and size exclusion column chromatography. The structure was established based on its spectroscopic data. Vateriferol (1) was evaluated in vitro for its antioxidant capacity, enzyme inhibitory activity, growth inhibitory activity on a number of cancer cell lines, neuroprotective activity, and anti-inflammatory activity. Vateriferol (1) exhibited AChE inhibitory activity (IC(50) 8.4 ± 0.2 μM), ORAC activity (2079 ± 0.20 TE/g), and neuroprotective activity at 1.5 μM using PC12 cells deprived of oxygen and glucose and lowered NO levels in lipopolysaccharide-stimulated SIM-A9 microglial cells at 14.7 and 73.6 μM. Vateriferol (1) exhibited weak cytotoxic potency (