Phylogenomic analysis reveals five independently evolved African forage grass clades in the genus Urochloa.

BACKGROUND AND AIMS: The grass genus Urochloa (Brachiaria) sensu lato includes forage crops that are important for beef and dairy industries in tropical and sub-tropical Africa, South America and Oceania/Australia. Economically important species include U. brizantha, U. decumbens, U. humidicola, U. mutica, U. arrecta, U. trichopus, U. mosambicensis and Megathyrsus maximus, all native to the African continent. Perennial growth habits, large, fast growing palatable leaves, intra- and interspecific morphological variability, apomictic reproductive systems and frequent polyploidy are widely shared within the genus. The combination of these traits probably favoured the selection for forage domestication and weediness, but trait emergence across Urochloa cannot be modelled, as a robust phylogenetic assessment of the genus has not been conducted. We aim to produce a phylogeny for Urochloa that includes all important forage species, and identify their closest wild relatives (crop wild relatives). Finally, we will use our phylogeny and available trait data to infer the ancestral states of important forage traits across Urochloa s.l. and model the evolution of forage syndromes across the genus. METHODS: Using a target enrichment sequencing approach (Angiosperm 353), we inferred a species-level phylogeny for Urochloa s.l., encompassing 54 species (~40 % of the genus) and outgroups. Phylogenies were inferred using a multispecies coalescent model and maximum likelihood method. We determined the phylogenetic placement of agriculturally important species and identified their closest wild relatives, or crop wild relatives, based on well-supported monophyly. Further, we mapped key traits associated with Urochloa forage crops to the species tree and estimated ancestral states for forage traits along branch lengths for continuous traits and at ancestral nodes in discrete traits. KEY RESULTS: Agricultural species belong to five independent clades, including U. brizantha and U. decumbens lying in a previously defined species complex. Crop wild relatives were identified for these clades supporting previous sub-generic groupings in Urochloa based on morphology. Using ancestral trait estimation models, we find that five morphological traits that correlate with forage potential (perennial growth habits, culm height, leaf size, a winged rachis and large seeds) independently evolved in forage clades. CONCLUSIONS: Urochloa s.l. is a highly diverse genus that contains numerous species with agricultural potential, including crop wild relatives that are currently underexploited. All forage species and their crop wild relatives naturally occur on the African continent and their conservation across their native distributions is essential. Genomic and phenotypic diversity in forage clade species and their wild relatives need to be better assessed both to develop conservation strategies and to exploit the diversity in the genus for improved sustainability in Urochloa cultivar production.

CDKN2A Gene Mutations: Implications for Hereditary Cancer Syndromes.

Malignant neoplasms, including pancreatic cancer and melanoma, are major global health challenges. This study investigates melanoma pancreatic syndrome, a rare hereditary tumor syndrome associated with CDKN2A gene mutations. CDKN2A mutations contribute to a lifetime risk of melanoma ranging from 28% to 67%. This study reports the clinical features of six individuals with CDKN2A mutations and identifies recurrent alterations such as c.307_308del, c.159G>C and c.71G>C. It highlights the need for CDKN2A mutation testing in suspected cases of familial atypical multiple mole melanoma. Clinically significant variants show associations with melanoma and pancreatic cancer. The challenges of treating individuals with CDKN2A mutations are discussed, and the lack of specific targeted therapies is highlighted. Preclinical studies suggest a potential benefit of CDK4/6 inhibitors, although clinical trials show mixed results. This study underscores the importance of continued research into improved diagnostic and therapeutic strategies to address the complexities of hereditary cancer syndromes.

The CFTR Gene Germline Heterozygous Pathogenic Variants in Russian Patients with Malignant Neoplasms and Healthy Carriers: 11,800 WGS Results.

More than 275 million people in the world are carriers of a heterozygous mutation of the CFTR gene, associated with cystic fibrosis, the most common autosomal recessive disease among Caucasians. Some recent studies assessed the association between carriers of CFTR variants and some pathologies, including cancer risk. The aim of this study is to analyze the landscape of germline pathogenic heterozygous CFTR variants in patients with diagnosed malignant neoplasms. For the first time in Russia, we evaluated the frequency of CFTR pathogenic variants by whole-genome sequencing in 1800 patients with cancer and compared this with frequencies of CFTR variants in the control group (1825 people) adjusted for age and 10,000 healthy individuals. In the issue, 47 out of 1800 patients (2.6%) were carriers of CFTR pathogenic genetic variants: 0.028 (42/1525) (2.8%) among breast cancer patients, 0.017 (3/181) (1.7%) among colorectal cancer patients and 0.021 (2/94) (2.1%) among ovarian cancer patients. Pathogenic CFTR variants were found in 52/1825 cases (2.85%) in the control group and 221 (2.21%) in 10,000 healthy individuals. Based on the results of the comparison, there was no significant difference in the frequency and distribution of pathogenic variants of the CFTR gene, which is probably due to the study limitations. Obviously, additional studies are needed to assess the clinical significance of the heterozygous carriage of CFTR pathogenic variants in the development of various pathologies in the future, particularly cancer.

Complex polyploid and hybrid species in an apomictic and sexual tropical forage grass group: genomic composition and evolution in Urochloa (Brachiaria) species.

BACKGROUND AND AIMS: Diploid and polyploid Urochloa (including Brachiaria, Panicum and Megathyrsus species) C4 tropical forage grasses originating from Africa are important for food security and the environment, often being planted in marginal lands worldwide. We aimed to characterize the nature of their genomes, the repetitive DNA and the genome composition of polyploids, leading to a model of the evolutionary pathways within the group including many apomictic species. METHODS: Some 362 forage grass accessions from international germplasm collections were studied, and ploidy was determined using an optimized flow cytometry method. Whole-genome survey sequencing and molecular cytogenetic analysis were used to identify chromosomes and genomes in Urochloa accessions belonging to the 'brizantha' and 'humidicola' agamic complexes and U. maxima. KEY RESULTS: Genome structures are complex and variable, with multiple ploidies and genome compositions within the species, and no clear geographical patterns. Sequence analysis of nine diploid and polyploid accessions enabled identification of abundant genome-specific repetitive DNA motifs. In situ hybridization with a combination of repetitive DNA and genomic DNA probes identified evolutionary divergence and allowed us to discriminate the different genomes present in polyploids. CONCLUSIONS: We suggest a new coherent nomenclature for the genomes present. We develop a model of evolution at the whole-genome level in diploid and polyploid accessions showing processes of grass evolution. We support the retention of narrow species concepts for Urochloa brizantha, U. decumbens and U. ruziziensis, and do not consider diploids and polyploids of single species as cytotypes. The results and model will be valuable in making rational choices of parents for new hybrids, assist in use of the germplasm for breeding and selection of Urochloa with improved sustainability and agronomic potential, and assist in measuring and conserving biodiversity in grasslands.

A Unique Observation of a Patient with Vulto-van Silfhout-de Vries Syndrome.

Introduction: Vulto-van Silfhout-de Vries Syndrome (VSVS; OMIM#615828) is a rare hereditary disease associated with impaired intellectual development and speech, delayed psychomotor development, and behavioral anomalies, including autistic behavioral traits and poor eye contact. To date, 27 patients with VSVS have been reported in the literature. Materials and Methods: We describe a 23-year-old male patient with autism spectrum disorder (ASD) who was admitted to the gastroenterological hospital with signs of pseudomembranous colitis. ASD was first noted in the patient at the age of 2.5 years. Later, he developed epileptic seizures and important growth retardation. Prior to the hospitalization, chromosomal aberrations, Fragile X syndrome, and aminoacidopathies/aminoacidurias associated with ASD were excluded. Whole-genome sequencing (WGS) was prescribed to the patient at 23 years old. Results: The patient had a heterozygous carrier of "de novo" variant c.662C > T (p.S221L) in exon 4 of the DEAF1 gene. c.662C > T had not been previously described in genomic databases. According to the ACMG criteria, this missense variant was considered to be pathogenic. VSVS was diagnosed in the patient. Conclusions: The phenotype of the patient is very similar to the data presented in the world literature. However, growth retardation and cachexia, which have not been described previously in the articles, are of interest.

Intracellular Localization and Gene Expression Analysis Provides New Insights on LEA Proteins' Diversity in Anhydrobiotic Cell Line.

Anhydrobiosis, an adaptive ability to withstand complete desiccation, in the nonbiting midge Polypedilum vanderplanki, is associated with the emergence of new multimember gene families, including a group of 27 genes of late embryogenesis abundant (LEA) proteins (PvLea). To obtain new insights into the possible functional specialization of these genes, we investigated the expression and localization of PvLea genes in a P. vanderplanki-derived cell line (Pv11), capable of anhydrobiosis. We confirmed that all but two PvLea genes identified in the genome of P. vanderplanki are expressed in Pv11 cells. Moreover, PvLea genes are induced in Pv11 cells in response to anhydrobiosis-inducing trehalose treatment in a manner highly similar to the larvae of P. vanderplanki during the real induction of anhydrobiosis. Then, we expanded our previous data on PvLEA proteins localization in mammalian cells that were obtained using C-terminal fusions of PvLEA proteins and green fluorescent protein (GFP). We investigated PvLEA localization using N- and C-terminal fusions with GFP in Pv11 cells and the Sf9 insect cell line. We observed an inconsistency of PvLEA localization between different fusion types and different cell cultures, that needs to be taken into account when using PvLEA in the engineering of anhydrobiotic cell lines.

Viral Membrane Fusion Proteins and RNA Sorting Mechanisms for the Molecular Delivery by Exosomes.

The advancement of precision medicine critically depends on the robustness and specificity of the carriers used for the targeted delivery of effector molecules in the human body. Numerous nanocarriers have been explored in vivo, to ensure the precise delivery of molecular cargos via tissue-specific targeting, including the endocrine part of the pancreas, thyroid, and adrenal glands. However, even after reaching the target organ, the cargo-carrying vehicle needs to enter the cell and then escape lysosomal destruction. Most artificial nanocarriers suffer from intrinsic limitations that prevent them from completing the specific delivery of the cargo. In this respect, extracellular vesicles (EVs) seem to be the natural tool for payload delivery due to their versatility and low toxicity. However, EV-mediated delivery is not selective and is usually short-ranged. By inserting the viral membrane fusion proteins into exosomes, it is possible to increase the efficiency of membrane recognition and also ease the process of membrane fusion. This review describes the molecular details of the viral-assisted interaction between the target cell and EVs. We also discuss the question of the usability of viral fusion proteins in developing extracellular vesicle-based nanocarriers with a higher efficacy of payload delivery. Finally, this review specifically highlights the role of Gag and RNA binding proteins in RNA sorting into EVs.

Single-cell transcriptomics of human embryos identifies multiple sympathoblast lineages with potential implications for neuroblastoma origin.

Characterization of the progression of cellular states during human embryogenesis can provide insights into the origin of pediatric diseases. We examined the transcriptional states of neural crest- and mesoderm-derived lineages differentiating into adrenal glands, kidneys, endothelium and hematopoietic tissue between post-conception weeks 6 and 14 of human development. Our results reveal transitions connecting the intermediate mesoderm and progenitors of organ primordia, the hematopoietic system and endothelial subtypes. Unexpectedly, by using a combination of single-cell transcriptomics and lineage tracing, we found that intra-adrenal sympathoblasts at that stage are directly derived from nerve-associated Schwann cell precursors, similarly to local chromaffin cells, whereas the majority of extra-adrenal sympathoblasts arise from the migratory neural crest. In humans, this process persists during several weeks of development within the large intra-adrenal ganglia-like structures, which may also serve as reservoirs of originating cells in neuroblastoma.

Contrasted histories of organelle and nuclear genomes underlying physiological diversification in a grass species.

C4 photosynthesis evolved multiple times independently in angiosperms, but most origins are relatively old so that the early events linked to photosynthetic diversification are blurred. The grass Alloteropsis semialata is an exception, as this species encompasses C4 and non-C4 populations. Using phylogenomics and population genomics, we infer the history of dispersal and secondary gene flow before, during and after photosynthetic divergence in A. semialata. We further analyse the genome composition of individuals with varied ploidy levels to establish the origins of polyploids in this species. Detailed organelle phylogenies indicate limited seed dispersal within the mountainous region of origin and the emergence of a C4 lineage after dispersal to warmer areas of lower elevation. Nuclear genome analyses highlight repeated secondary gene flow. In particular, the nuclear genome associated with the C4 phenotype was swept into a distantly related maternal lineage probably via unidirectional pollen flow. Multiple intraspecific allopolyploidy events mediated additional secondary genetic exchanges between photosynthetic types. Overall, our results show that limited dispersal and isolation allowed lineage divergence, with photosynthetic innovation happening after migration to new environments, and pollen-mediated gene flow led to the rapid spread of the derived C4 physiology away from its region of origin.

Genetically Modified DR5-Specific TRAIL Variant DR5-B Revealed Dual Antitumor and Protumoral Effect in Colon Cancer Xenografts and an Improved Pharmacokinetic Profile.

Despite the weak clinical efficacy of TRAIL death receptor agonists, a search is under way for new agents that more efficiently activate apoptotic signaling. We previously created a TRAIL DR5-selective variant DR5-B without affinity for the DR4, DcR1, DcR2, and OPG receptors and increased proapoptotic activity in tumor cells. Here we showed that DR5-B significantly inhibited tumor growth in HCT116 and Caco-2 but not in HT-29 xenografts. The antitumor activity of DR5-B was 2.5 times higher in HCT116 xenografts compared to TRAIL. DR5-B at a dose of 2 or 10 mg/kg/d for 10 days inhibited tumor growth in HCT116 xenografts by 26% or 50% respectively, and increased animal survival. Unexpectedly, DR5-B at a higher dose (25 mg/kg/d) inhibited tumor growth only during the first 8 days of drug exposure, while at the end of the monitoring, no effect or even slight stimulation of tumor growth was observed. The pharmacokinetic parameters of DR5-B were comparable to those of TRAIL, except that the half-life was 3.5 times higher. Thus, enhancing TRAIL selectivity to DR5 may increase both antitumor and proliferative activities depending on the concentration and administration regimens.

ddRAD analyses reveal a credible phylogenetic relationship of the four main genera of Bambusa-Dendrocalamus-Gigantochloa complex (Poaceae: Bambusoideae).

The Bambusa-Dendrocalamus-Gigantochloa complex (BDG complex) is the most diversified and phylogenetically recalcitrant group of the paleotropical woody bamboos. Species of this complex occur in tropical and subtropical Asia and most of them are of great economic, cultural and ecological value. The lack of resolution achieved through the analyses of previous molecular datasets has long confounded its phylogenetic estimation and generic delimitation. Here, we adopted a ddRAD-seq strategy to investigate phylogenetic relationships of the four main genera (Bambusa, Dendrocalamus, Gigantochloa, and Melocalamus) in the BDG complex. A total of 102 species were sampled, and SNP data were generated. Both MP and ML analyses of the ddRAD-seq data resulted in a well-resolved topology with Gigantochloa and Melocalamus confirmed as monophyletic, and Melocalamus resolved as sister to the rest of the complex. Bambusa and Dendrocalamus were both resolved as paraphyletic. The phylogenetic relationships were mostly supported by morphological evidence including characters of the branch complement, rachilla, lodicules, filaments and stigma. We also generated and assembled complete plastid genomes of 48 representative species. There were conflicts between the plastome and the ddRAD topologies. Our study demonstrated that RAD-seq can be used to reconstruct evolutionary history of lineages such as the bamboos where ancient hybridization and polyploidy play a significant role. The four genera of the BDG complex have a complex evolutionary history which is likely a product of ancient introgression events.

Antitumor Activity of Auger Electron Emitter 111In Delivered by Modular Nanotransporter for Treatment of Bladder Cancer With EGFR Overexpression.

Gamma-ray emitting 111In, which is extensively used for imaging, is also a source of short-range Auger electrons (AE). While exhibiting negligible effect outside cells, these AE become highly toxic near DNA within the cell nucleus. Therefore, these radionuclides can be used as a therapeutic anticancer agent if delivered precisely into the nuclei of tumor target cells. Modular nanotransporters (MNTs) designed to provide receptor-targeted delivery of short-range therapeutic cargoes into the nuclei of target cells are perspective candidates for specific intracellular delivery of AE emitters. The objective of this study was to evaluate the in vitro and in vivo efficacy of 111In attached MNTs to kill human bladder cancer cells overexpressing epidermal growth factor receptor (EGFR). The cytotoxicity of 111In delivered by the EGFR-targeted MNT (111In-MNT) was greatly enhanced on EJ-, HT-1376-, and 5637-expressing EGFR bladder cancer cell lines compared with 111In non-targeted control. In vivo microSPECT/CT imaging and antitumor efficacy studies revealed prolonged intratumoral retention of 111In-MNT with t½ = 4.1 ± 0.5 days as well as significant dose-dependent tumor growth delay (up to 90% growth inhibition) after local infusion of 111In-MNT in EJ xenograft-bearing mice.

Preparation, cytotoxicity, and in vivo antitumor efficacy of 111In-labeled modular nanotransporters.

PURPOSE: Modular nanotransporters (MNTs) are a polyfunctional platform designed to achieve receptor-specific delivery of short-range therapeutics into the cell nucleus by receptor-mediated endocytosis, endosome escape, and targeted nuclear transport. This study evaluated the potential utility of the MNT platform in tandem with Auger electron emitting 111In for cancer therapy. METHODS: Three MNTs developed to target either melanocortin receptor-1 (MC1R), folate receptor (FR), or epidermal growth factor receptor (EGFR) that are overexpressed on cancer cells were modified with p-SCN-Bn-NOTA and then labeled with 111In in high specific activity. Cytotoxicity of the 111In-labeled MNTs was evaluated on cancer cell lines bearing the appropriate receptor target (FR: HeLa, SK-OV-3; EGFR: A431, U87MG.wtEGFR; and MC1R: B16-F1). In vivo micro-single-photon emission computed tomography/computed tomography imaging and antitumor efficacy studies were performed with intratumoral injection of MC1R-targeted 111In-labeled MNT in B16-F1 melanoma tumor-bearing mice. RESULTS: The three NOTA-MNT conjugates were labeled with a specific activity of 2.7 GBq/mg with nearly 100% yield, allowing use without subsequent purification. The cytotoxicity of 111In delivered by these MNTs was greatly enhanced on receptor-expressing cancer cells compared with 111In nontargeted control. In mice with B16-F1 tumors, prolonged retention of 111In by serial imaging and significant tumor growth delay (82% growth inhibition) were found. CONCLUSION: The specific in vitro cytotoxicity, prolonged tumor retention, and therapeutic efficacy of MC1R-targeted 111In-NOTA-MNT suggest that this Auger electron emitting conjugate warrants further evaluation as a locally delivered radiotherapeutic, such as for ocular melanoma brachytherapy. Moreover, the high cytotoxicity observed with FR- and EGFR-targeted 111In-NOTA-MNT suggests further applications of the MNT delivery strategy should be explored.

Benchtop isolation and characterization of functional exosomes by sequential filtration.

Early and minimally invasive detection of malignant events or other pathologies is of utmost importance in the pursuit of improved patient care and outcomes. Recent evidence indicates that exosomes and extracellular vesicles in serum and body fluids can contain nucleic acid, protein, and other biomarkers. Accordingly, there is great interest in applying these clinically as prognostic, predictive, pharmacodynamic, and early detection indicators. Nevertheless, existing exosome isolation methods can be time-consuming, require specialized equipment, and/or present other inefficiencies regarding purity, reproducibility and assay cost. We have developed a straightforward, three-step protocol for exosome isolation of cell culture supernatants or large volumes of biofluid based on sequential steps of dead-end pre-filtration, tangential flow filtration (TFF), and low-pressure track-etched membrane filtration that we introduce here. Our approach yields exosome preparations of high purity and defined size distribution and facilitates depletion of free protein and other low-molecular-weight species, extracellular vesicles larger than 100nm, and cell debris. Samples of exosomes prepared using the approach were verified morphologically by nanoparticle tracking analysis and electron microscopy, and mass spectrometry analyses confirmed the presence of previously reported exosome-associated proteins. In addition to being easy-to-implement, sequential filtration yields exosomes of high purity and, importantly, functional integrity as a result of the relatively low-magnitude manipulation forces employed during isolation. This answers an unmet need for preparation of minimally manipulated exosomes for investigations into exosome function and basic biology. Further, the strategy is amenable to translation for clinical exosome isolations because of its speed, automatability, scalability, and specificity for isolating exosomes from complex biological samples.

Molecular phylogenetics of tribe Poranthereae (Phyllanthaceae; Euphorbiaceae sensu lato).

Novel insights into the evolutionary history of a taxonomically complex tropical plant group were gained in this study using DNA sequence data. A molecular phylogenetic analysis of the newly circumscribed and expanded tribe Poranthereae (Phyllanthaceae) is presented. Sampling included 97 accessions for 63 of c. 120 species. Largely congruent results have been obtained from nuclear ribosomal ITS and plastid matK sequences. These analyses support the recognition of Andrachne and Leptopus as distinct genera. The deceptively similar Andrachne section Phyllanthopsis, Andrachne ovalis, and Leptopus decaisnei are separate lineages to be segregated. Zimmermannia and Zimmermanniopsis are embedded in Meineckia; Oreoporanthera is embedded in Poranthera; and Archileptopus is embedded in Leptopus. Andrachne section Pseudophyllanthus is polyphyletic, the two Madagascan endemics emerging as a sister clade to Meineckia. The noncontiguous distributions of Andrachne sensu lato and Leptopus sensu lato were found to be the result of separate evolutionary histories of morphologically similar clades, whereas Andrachne sensu stricto and Meineckia remain geographically disjunct. Actephila and Leptopus are sisters with a sympatric distribution in humid Asia. Presence of petals appears to be plesiomorphic for the tribe. Petals are reduced in Actephila and Oreoporanthera and lost in the Meineckia clade.

Effect of 5-azacytidine and trichostatin A on somatic centromere association in wheat.

Both homologous and non-homologous chromosomes in wheat associate via their centromeric hetero chromatin in the developing xylem vessel cells of the root. The antimetabolite 5-azacytidine (which reduces DNA methylation) decreases the overall level of centromere association. Treatment with 5-azacytidine caused a more marked reduction in the level of homologous chromosome association observed in a wheat line carrying a pair of marked chromosomes. On the other hand, treatment of wheat seedlings with trichostatin A (which increases histone acetylation) raises the overall level of centromere association. The Ph1 locus controls the specificity of both somatic and meiotic pairing of homologous centromeres in wheat. The level of non-homologously associated centromeres is, however, reduced in the presence of Ph1 compared with its absence, even after treatment with either drug. Thus these two drugs, which have been shown to affect chromatin structure, do affect chromosome association, but Ph1 must act at least in part by a different mechanism.