Oligopeptide Signaling through TbGPR89 Drives Trypanosome Quorum Sensing.

Trypanosome parasites control their virulence and spread by using quorum sensing (QS) to generate transmissible "stumpy forms" in their host bloodstream. However, the QS signal "stumpy induction factor" (SIF) and its reception mechanism are unknown. Although trypanosomes lack G protein-coupled receptor signaling, we have identified a surface GPR89-family protein that regulates stumpy formation. TbGPR89 is expressed on bloodstream "slender form" trypanosomes, which receive the SIF signal, and when ectopically expressed, TbGPR89 drives stumpy formation in a SIF-pathway-dependent process. Structural modeling of TbGPR89 predicts unexpected similarity to oligopeptide transporters (POT), and when expressed in bacteria, TbGPR89 transports oligopeptides. Conversely, expression of an E. coli POT in trypanosomes drives parasite differentiation, and oligopeptides promote stumpy formation in vitro. Furthermore, the expression of secreted trypanosome oligopeptidases generates a paracrine signal that accelerates stumpy formation in vivo. Peptidase-generated oligopeptide QS signals being received through TbGPR89 provides a mechanism for both trypanosome SIF production and reception.

Genome-wide analysis of copy-number variation in humans with cleft lip and/or cleft palate identifies COBLL1, RIC1, and ARHGEF38 as clefting genes.

Cleft lip with or without cleft palate (CL/P) is a common birth defect with a complex, heterogeneous etiology. It is well established that common and rare sequence variants contribute to the formation of CL/P, but the contribution of copy-number variants (CNVs) to cleft formation remains relatively understudied. To fill this knowledge gap, we conducted a large-scale comparative analysis of genome-wide CNV profiles of 869 individuals from the Philippines and 233 individuals of European ancestry with CL/P with three primary goals: first, to evaluate whether differences in CNV number, amount of genomic content, or amount of coding genomic content existed within clefting subtypes; second, to assess whether CNVs in our cohort overlapped with known Mendelian clefting loci; and third, to identify unestablished Mendelian clefting genes. Significant differences in CNVs across cleft types or in individuals with non-syndromic versus syndromic clefts were not observed; however, several CNVs in our cohort overlapped with known syndromic and non-syndromic Mendelian clefting loci. Moreover, employing a filtering strategy relying on population genetics data that rare variants are on the whole more deleterious than common variants, we identify several CNV-associated gene losses likely driving non-syndromic clefting phenotypes. By prioritizing genes deleted at a rare frequency across multiple individuals with clefts yet enriched in our cohort of individuals with clefts compared to control subjects, we identify COBLL1, RIC1, and ARHGEF38 as clefting genes. CRISPR-Cas9 mutagenesis of these genes in Xenopus laevis and Danio rerio yielded craniofacial dysmorphologies, including clefts analogous to those seen in human clefting disorders.

Maternal Wnt11b regulates cortical rotation during Xenopus axis formation: analysis of maternal-effect wnt11b mutants.

Asymmetric signalling centres in the early embryo are essential for axis formation in vertebrates. These regions (e.g. amphibian dorsal morula, mammalian anterior visceral endoderm) require stabilised nuclear ß-catenin, but the role of localised Wnt ligand signalling activity in their establishment remains unclear. In Xenopus, dorsal ß-catenin is initiated by vegetal microtubule-mediated symmetry breaking in the fertilised egg, known as 'cortical rotation'. Localised wnt11b mRNA and ligand-independent activators of ß-catenin have been implicated in dorsal ß-catenin activation, but the extent to which each contributes to axis formation in this paradigm remains unclear. Here, we describe a CRISPR-mediated maternal-effect mutation in Xenopus laevis wnt11b.L. We find that wnt11b is maternally required for robust dorsal axis formation and for timely gastrulation, and zygotically for left-right asymmetry. Importantly, we show that vegetal microtubule assembly and cortical rotation are reduced in wnt11b mutant eggs. In addition, we show that activated Wnt coreceptor Lrp6 and Dishevelled lack behaviour consistent with roles in early ß-catenin stabilisation, and that neither is regulated by Wnt11b. This work thus implicates Wnt11b in the distribution of putative dorsal determinants rather than in comprising the determinants themselves. This article has an associated 'The people behind the papers' interview.

From intuition to AI: evolution of small molecule representations in drug discovery.

Within drug discovery, the goal of AI scientists and cheminformaticians is to help identify molecular starting points that will develop into safe and efficacious drugs while reducing costs, time and failure rates. To achieve this goal, it is crucial to represent molecules in a digital format that makes them machine-readable and facilitates the accurate prediction of properties that drive decision-making. Over the years, molecular representations have evolved from intuitive and human-readable formats to bespoke numerical descriptors and fingerprints, and now to learned representations that capture patterns and salient features across vast chemical spaces. Among these, sequence-based and graph-based representations of small molecules have become highly popular. However, each approach has strengths and weaknesses across dimensions such as generality, computational cost, inversibility for generative applications and interpretability, which can be critical in informing practitioners' decisions. As the drug discovery landscape evolves, opportunities for innovation continue to emerge. These include the creation of molecular representations for high-value, low-data regimes, the distillation of broader biological and chemical knowledge into novel learned representations and the modeling of up-and-coming therapeutic modalities.

AutoDock-SS: AutoDock for Multiconformational Ligand-Based Virtual Screening.

Ligand-based virtual screening (LBVS) can be pivotal for identifying potential drug leads, especially when the target protein's structure is unknown. However, current LBVS methods are limited in their ability to consider the ligand conformational flexibility. This study presents AutoDock-SS (Similarity Searching), which adapts protein-ligand docking for use in LBVS. AutoDock-SS integrates novel ligand-based grid maps and AutoDock-GPU into a novel three-dimensional LBVS workflow. Unlike other approaches based on pregenerated conformer libraries, AutoDock-SS's built-in conformational search optimizes conformations dynamically based on the reference ligand, thus providing a more accurate representation of relevant ligand conformations. AutoDock-SS supports two modes: single and multiple ligand queries, allowing for the seamless consideration of multiple reference ligands. When tested on the Directory of Useful Decoys─Enhanced (DUD-E) data set, AutoDock-SS surpassed alternative 3D LBVS methods, achieving a mean AUROC of 0.775 and an EF1% of 25.72 in single-reference mode. The multireference mode, evaluated on the augmented DUD-E+ data set, demonstrated superior accuracy with a mean AUROC of 0.843 and an EF1% of 34.59. This enhanced performance underscores AutoDock-SS's ability to treat compounds as conformationally flexible while considering the ligand's shape, pharmacophore, and electrostatic potential, expanding the potential of LBVS methods.

Intrinsic lipid binding activity of ATG16L1 supports efficient membrane anchoring and autophagy.

Membrane targeting of autophagy-related complexes is an important step that regulates their activities and prevents their aberrant engagement on non-autophagic membranes. ATG16L1 is a core autophagy protein implicated at distinct phases of autophagosome biogenesis. In this study, we dissected the recruitment of ATG16L1 to the pre-autophagosomal structure (PAS) and showed that it requires sequences within its coiled-coil domain (CCD) dispensable for homodimerisation. Structural and mutational analyses identified conserved residues within the CCD of ATG16L1 that mediate direct binding to phosphoinositides, including phosphatidylinositol 3-phosphate (PI3P). Mutating putative lipid binding residues abrogated the localisation of ATG16L1 to the PAS and inhibited LC3 lipidation. On the other hand, enhancing lipid binding of ATG16L1 by mutating negatively charged residues adjacent to the lipid binding motif also resulted in autophagy inhibition, suggesting that regulated recruitment of ATG16L1 to the PAS is required for its autophagic activity. Overall, our findings indicate that ATG16L1 harbours an intrinsic ability to bind lipids that plays an essential role during LC3 lipidation and autophagosome maturation.

A single KH domain in Bicaudal-C links mRNA binding and translational repression functions to maternal development.

Bicaudal-C (Bicc1) is a conserved RNA-binding protein that represses the translation of selected mRNAs to control development. In Xenopus embryos, Bicc1 binds and represses specific maternal mRNAs to control anterior-posterior cell fates. However, it is not known how Bicc1 binds its RNA targets or how binding affects Bicc1-dependent embryogenesis. Focusing on the KH domains, we analyzed Bicc1 mutants for their ability to bind RNA substrates in vivo and in vitro Analyses of these Bicc1 mutants demonstrated that a single KH domain, KH2, was crucial for RNA binding in vivo and in vitro, while the KH1 and KH3 domains contributed minimally. The Bicc1 mutants were also assayed for their ability to repress translation, and results mirrored the RNA-binding data, with KH2 being the only domain essential for repression. Finally, maternal knockdown and rescue experiments indicated that the KH domains were essential for the regulation of embryogenesis by Bicc1. These data advance our understanding of how Bicc1 selects target mRNAs and provide the first direct evidence that the RNA binding functions of Bicc1 are essential for both Bicc1-dependent translational repression and maternal vertebrate development.

Comparison of ATP-binding pockets and discovery of homologous recombination inhibitors.

The ATP binding sites of many enzymes are structurally related, which complicates their development as therapeutic targets. In this work, we explore a diverse set of ATPases and compare their ATP binding pockets using different strategies, including direct and indirect structural methods, in search of pockets attractive for drug discovery. We pursue different direct and indirect structural strategies, as well as ligandability assessments to help guide target selection. The analyses indicate human RAD51, an enzyme crucial in homologous recombination, as a promising, tractable target. Inhibition of RAD51 has shown promise in the treatment of certain cancers but more potent inhibitors are needed. Thus, we design compounds computationally against the ATP binding pocket of RAD51 with consideration of multiple criteria, including predicted specificity, drug-likeness, and toxicity. The molecules designed are evaluated experimentally using molecular and cell-based assays. Our results provide two novel hit compounds against RAD51 and illustrate a computational pipeline to design new inhibitors against ATPases.

A gradient of maternal Bicaudal-C controls vertebrate embryogenesis via translational repression of mRNAs encoding cell fate regulators.

Vertebrate Bicaudal-C (Bicc1) has important biological roles in the formation and homeostasis of multiple organs, but direct experiments to address the role of maternal Bicc1 in early vertebrate embryogenesis have not been reported. Here, we use antisense phosphorothioate-modified oligonucleotides and the host-transfer technique to eliminate specifically maternal stores of both bicc1 mRNA and Bicc1 protein from Xenopus laevis eggs. Fertilization of these Bicc1-depleted eggs produced embryos with an excess of dorsal-anterior structures and overexpressed organizer-specific genes, indicating that maternal Bicc1 is crucial for normal embryonic patterning of the vertebrate embryo. Bicc1 is an RNA-binding protein with robust translational repression function. Here, we show that the maternal mRNA encoding the cell-fate regulatory protein Wnt11b is a direct target of Bicc1-mediated repression. It is well established that the Wnt signaling pathway is crucial to vertebrate embryogenesis. Thus, the work presented here links the molecular function of Bicc1 in mRNA target-specific translation repression to its biological role in the maternally controlled stages of vertebrate embryogenesis.

Insights into the Effects of Cancer Associated Mutations at the UPF2 and ATP-Binding Sites of NMD Master Regulator: UPF1.

Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that recognizes post-transcriptionally abnormal transcripts and mediates their degradation. The master regulator of NMD is UPF1, an enzyme with intrinsic ATPase and helicase activities. The cancer genomic sequencing data has identified frequently mutated residues in the CH-domain and ATP-binding site of UPF1. In silico screening of UPF1 stability change as a function over 41 cancer mutations has identified five variants with significant effects: K164R, R253W, T499M, E637K, and E833K. To explore the effects of these mutations on the associated energy landscape of UPF1, molecular dynamics simulations (MDS) were performed. MDS identified stable H-bonds between residues S152, S203, S205, Q230/R703, and UPF2/AMPPNP, and suggest that phosphorylation of Serine residues may control UPF1-UPF2 binding. Moreover, the alleles K164R and R253W in the CH-domain improved UPF1-UPF2 binding. In addition, E637K and E833K alleles exhibited improved UPF1-AMPPNP binding compared to the T499M variant; the lower binding is predicted from hindrance caused by the side-chain of T499M to the docking of the tri-phosphate moiety (AMPPNP) into the substrate site. The dynamics of wild-type/mutant systems highlights the flexible nature of the ATP-binding region in UPF1. These insights can facilitate the development of drug discovery strategies for manipulating NMD signaling in cell systems using chemical tools.

Role of maternal Xenopus syntabulin in germ plasm aggregation and primordial germ cell specification.

The localization and organization of mitochondria- and ribonucleoprotein granule-rich germ plasm is essential for many aspects of germ cell development. In Xenopus, germ plasm is maternally inherited and is required for the specification of primordial germ cells (PGCs). Germ plasm is aggregated into larger patches during egg activation and cleavage and is ultimately translocated perinuclearly during gastrulation. Although microtubule dynamics and a kinesin (Kif4a) have been implicated in Xenopus germ plasm localization, little is known about how germ plasm distribution is regulated. Here, we identify a role for maternal Xenopus Syntabulin in the aggregation of germ plasm following fertilization. We show that depletion of sybu mRNA using antisense oligonucleotides injected into oocytes results in defects in the aggregation and perinuclear transport of germ plasm and subsequently in reduced PGC numbers. Using live imaging analysis, we also characterize a novel role for Sybu in the collection of germ plasm in vegetal cleavage furrows by surface contraction waves. Additionally, we show that a localized kinesin-like protein, Kif3b, is also required for germ plasm aggregation and that Sybu functionally interacts with Kif3b and Kif4a in germ plasm aggregation. Overall, these data suggest multiple coordinate roles for kinesins and adaptor proteins in controlling the localization and distribution of a cytoplasmic determinant in early development.

Mono-Substituted Hydrocarbon Diastereomer Combinations Reveal Stapled Peptides with High Structural Fidelity.

Modified peptides, such as stapled peptides, which replicate the structure of α-helical protein segments, represent a potential therapeutic advance. However, the 3D solution structure of these stapled peptides is rarely explored beyond the acquisition of circular dichroism (CD) data to quantify bulk peptide helicity; the detailed backbone structure, which underlies this, is typically undefined. Diastereomeric stapled peptides based on helical sections of three proteins (αSyn, Cks1 and CK1α) were generated; their overall helicity was quantified by CD; and the most helical peptide from each series was selected for structural analysis. Solution-phase models for the optimised peptides were generated using NMR-derived restraints and a modified CHARMM22 force field. Comparing these models with PDB structures allowed deviation between the stapled peptides and critical helical regions to be evaluated. These studies demonstrate that CD alone is not sufficient to assess the structural fidelity of a stapled peptide.

Vertebrate Axial Patterning: From Egg to Asymmetry.

The emergence of the bilateral embryonic body axis from a symmetrical egg has been a long-standing question in developmental biology. Historical and modern experiments point to an initial symmetry-breaking event leading to localized Wnt and Nodal growth factor signaling and subsequent induction and formation of a self-regulating dorsal "organizer." This organizer forms at the site of notochord cell internalization and expresses primarily Bone Morphogenetic Protein (BMP) growth factor antagonists that establish a spatiotemporal gradient of BMP signaling across the embryo, directing initial cell differentiation and morphogenesis. Although the basics of this model have been known for some time, many of the molecular and cellular details have only recently been elucidated and the extent that these events remain conserved throughout vertebrate evolution remains unclear. This chapter summarizes historical perspectives as well as recent molecular and genetic advances regarding: (1) the mechanisms that regulate symmetry-breaking in the vertebrate egg and early embryo, (2) the pathways that are activated by these events, in particular the Wnt pathway, and the role of these pathways in the formation and function of the organizer, and (3) how these pathways also mediate anteroposterior patterning and axial morphogenesis. Emphasis is placed on comparative aspects of the egg-to-embryo transition across vertebrates and their evolution. The future prospects for work regarding self-organization and gene regulatory networks in the context of early axis formation are also discussed.

The dynamics of plus end polarization and microtubule assembly during Xenopus cortical rotation.

The self-organization of dorsally-directed microtubules during cortical rotation in the Xenopus egg is essential for dorsal axis formation. The mechanisms controlling this process have been problematic to analyze, owing to difficulties in visualizing microtubules in living egg. Also, the order of events occurring at the onset of cortical rotation have not been satisfactorily visualized in vivo and have been inferred from staged fixed samples. To address these issues, we have characterized the dynamics of total microtubule and plus end behavior continuously throughout cortical rotation, as well as in oocytes and unfertilized eggs. Here, we show that the nascent microtubule network forms in the cortex but associates with the deep cytoplasm at the start of rotation. Importantly, plus ends remain cortical and become increasingly more numerous and active prior to rotation, with dorsal polarization occurring rapidly after the onset of rotation. Additionally, we show that vegetally localized Trim36 is required to attenuate dynamic plus end growth, suggesting that vegetal factors are needed to locally coordinate growth in the cortex.

Structural Basis for the Inverted Repeat Preferences of mariner Transposases.

The inverted repeat (IR) sequences delimiting the left and right ends of many naturally active mariner DNA transposons are non-identical and have different affinities for their transposase. We have compared the preferences of two active mariner transposases, Mos1 and Mboumar-9, for their imperfect transposon IRs in each step of transposition: DNA binding, DNA cleavage, and DNA strand transfer. A 3.1 Å resolution crystal structure of the Mos1 paired-end complex containing the pre-cleaved left IR sequences reveals the molecular basis for the reduced affinity of the Mos1 transposase DNA-binding domain for the left IR as compared with the right IR. For both Mos1 and Mboumar-9, in vitro DNA transposition is most efficient when the preferred IR sequence is present at both transposon ends. We find that this is due to the higher efficiency of cleavage and strand transfer of the preferred transposon end. We show that the efficiency of Mboumar-9 transposition is improved almost 4-fold by changing the 3' base of the preferred Mboumar-9 IR from guanine to adenine. This preference for adenine at the reactive 3' end for both Mos1 and Mboumar-9 may be a general feature of mariner transposition.

Hetero-trans-ß-glucanase, an enzyme unique to Equisetum plants, functionalizes cellulose.

Cell walls are metabolically active components of plant cells. They contain diverse enzymes, including transglycanases (endotransglycosylases), enzymes that 'cut and paste' certain structural polysaccharide molecules and thus potentially remodel the wall during growth and development. Known transglycanase activities modify several cell-wall polysaccharides (xyloglucan, mannans, mixed-linkage ß-glucan and xylans); however, no transglycanases were known to act on cellulose, the principal polysaccharide of biomass. We now report the discovery and characterization of hetero-trans-ß-glucanase (HTG), a transglycanase that targets cellulose, in horsetails (Equisetum spp., an early-diverging genus of monilophytes). HTG is also remarkable in predominantly catalysing hetero-transglycosylation: its preferred donor substrates (cellulose or mixed-linkage ß-glucan) differ qualitatively from its acceptor substrate (xyloglucan). HTG thus generates stable cellulose-xyloglucan and mixed-linkage ß-glucan-xyloglucan covalent bonds, and may therefore strengthen ageing Equisetum tissues by inter-linking different structural polysaccharides of the cell wall. 3D modelling suggests that only three key amino acid substitutions (Trp → Pro, Gly → Ser and Arg → Leu) are responsible for the evolution of HTG's unique specificity from the better-known xyloglucan-acting homo-transglycanases (xyloglucan endotransglucosylase/hydrolases; XTH). Among land plants, HTG appears to be confined to Equisetum, but its target polysaccharides are widespread, potentially offering opportunities for enhancing crop mechanical properties, such as wind resistance. In addition, by linking cellulose to xyloglucan fragments previously tagged with compounds such as dyes or indicators, HTG may be useful biotechnologically for manufacturing stably functionalized celluloses, thereby potentially offering a commercially valuable 'green' technology for industrially manipulating biomass.

Maternal Dead-End1 is required for vegetal cortical microtubule assembly during Xenopus axis specification.

Vertebrate axis specification is an evolutionarily conserved developmental process that relies on asymmetric activation of Wnt signaling and subsequent organizer formation on the future dorsal side of the embryo. Although roles of Wnt signaling during organizer formation have been studied extensively, it is unclear how the Wnt pathway is asymmetrically activated. In Xenopus and zebrafish, the Wnt pathway is triggered by dorsal determinants, which are translocated from the vegetal pole to the future dorsal side of the embryo shortly after fertilization. The transport of dorsal determinants requires a unique microtubule network formed in the vegetal cortex shortly after fertilization. However, molecular mechanisms governing the formation of vegetal cortical microtubule arrays are not fully understood. Here we report that Dead-End 1 (Dnd1), an RNA-binding protein required for primordial germ cell development during later stages of embryogenesis, is essential for Xenopus axis specification. We show that knockdown of maternal Dnd1 specifically interferes with the formation of vegetal cortical microtubules. This, in turn, impairs translocation of dorsal determinants, the initiation of Wnt signaling, organizer formation, and ultimately results in ventralized embryos. Furthermore, we found that Dnd1 binds to a uridine-rich sequence in the 3'-UTR of trim36, a vegetally localized maternal RNA essential for vegetal cortical microtubule assembly. Dnd1 anchors trim36 to the vegetal cortex in the egg, promoting high concentrations of Trim36 protein there. Our work thus demonstrates a novel and surprising function for Dnd1 during early development and provides an important link between Dnd1, mRNA localization, the microtubule cytoskeleton and axis specification.

Novel triazolothiadiazines act as potent anticancer agents in liver cancer cells through Akt and ASK-1 proteins.

Newly designed triazolothiadiazines incorporating with structural motifs of nonsteroidal analgesic anti-inflammatory drugs were synthesized and screened for their bioactivity against epithelial cancer cells. Compounds with bioactivities less then ∼5µM (IC50) were further analyzed and showed to induce apoptotic cell death and SubG1 cell cycle arrest in liver cancer cells. Among this group, two compounds (1g and 1h) were then studied to identify the mechanism of action. These molecules triggered oxidative stress induced apoptosis through ASK-1 protein activation and Akt protein inhibition as demonstrated by downstream targets such as GSK3ß, ß-catenin and cyclin D1. QSAR and molecular docking models provide insight into the mechanism of inhibition and indicate the optimal direction of future synthetic efforts. Furthermore, molecular docking results were confirmed with in vitro COX bioactivity studies. This study demonstrates that the novel triazolothiadiazine derivatives are promising drug candidates for epithelial cancers, especially liver cancer.

Safe Routes to Play? Pedestrian and Bicyclist Crashes Near Parks in Los Angeles.

BACKGROUND: Areas near parks may present active travelers with higher risks than in other areas due to the confluence of more pedestrians and bicyclists, younger travelers, and the potential for increased traffic volumes. These risks may be amplified in low-income and minority neighborhoods due to generally higher rates of active travel or lack of safety infrastructure. This paper examines active travel crashes near parks and builds on existing research around disparities in park access and extends research from the Safe Routes to School and Safe Routes to Transit movements to parks. METHODS: We utilized the Green Visions Parks coverage, encompassing Los Angeles County and several other cities in the LA Metropolitan area. We used negative bionomial regression modeling techniques and ten years of geolocated pedestrian and bicyclist crash data to assess the number of active travel injuries within a quarter mile (~400m) buffer around parks. We controlled for differential exposures to active travel using travel survey data and Bayesian smoothing models. RESULTS: Of 1,311,736 parties involved in 608,530 crashes, there were 896,359 injuries and 7317 fatalities. The number of active travel crash injuries is higher within a quarter-mile of a park, with a ratio of 1.52 per 100,000 residents, compared to areas outside that buffer. This higher rate near parks is amplified in neighborhoods with high proportions of minority and low-income residents. Higher traffic levels are highly predictive of active travel crash injuries. CONCLUSIONS: Planners should consider the higher risks of active travel near parks and the socioeconomic modification of these risks. Additional traffic calming and safety infrastructure may be needed to provide safe routes to parks.