Whole-Genome Sequencing and Analysis of Tumour-Forming Radish (Raphanus sativus L.) Line.

Spontaneous tumour formation in higher plants can occur in the absence of pathogen invasion, depending on the plant genotype. Spontaneous tumour formation on the taproots is consistently observed in certain inbred lines of radish (Raphanus sativus var. radicula Pers.). In this paper, using Oxford Nanopore and Illumina technologies, we have sequenced the genomes of two closely related radish inbred lines that differ in their ability to spontaneously form tumours. We identified a large number of single nucleotide variants (amino acid substitutions, insertions or deletions, SNVs) that are likely to be associated with the spontaneous tumour formation. Among the genes involved in the trait, we have identified those that regulate the cell cycle, meristem activity, gene expression, and metabolism and signalling of phytohormones. After identifying the SNVs, we performed Sanger sequencing of amplicons corresponding to SNV-containing regions to validate our results. We then checked for the presence of SNVs in other tumour lines of the radish genetic collection and found the ERF118 gene, which had the SNVs in the majority of tumour lines. Furthermore, we performed the identification of the CLAVATA3/ESR (CLE) and WUSCHEL (WOX) genes and, as a result, identified two unique radish CLE genes which probably encode proteins with multiple CLE domains. The results obtained provide a basis for investigating the mechanisms of plant tumour formation and also for future genetic and genomic studies of radish.

Whole-genome sequence of Kosakonia cowanii strain W006, isolated from seeds of Triticum aestivum L.

In this study, we sequence, assemble, and annotate Kosakonia cowanii strain W006, isolated from seeds of Triticum aestivum L. W006 has a single circular chromosome of 4,788,099 bp and 4,466 genes, with a mean G +C content of 56.1%.

Effect of Octamer-Binding Transcription Factor 4 Overexpression on the Neural Induction of Human Dental Pulp Stem Cells.

Stem cell-based therapy is a potential alternative strategy for brain repair, with neural stem cells (NSC) presenting as the most promising candidates. Obtaining sufficient quantities of NSC for clinical applications is challenging, therefore alternative cell types, such as neural crest-derived dental pulp stem cells (DPSC), may be considered. Human DPSC possess neurogenic potential, exerting positive effects in the damaged brain through paracrine effects. However, a method for conversion of DPSC into NSC has yet to be developed. Here, overexpression of octamer-binding transcription factor 4 (OCT4) in combination with neural inductive conditions was used to reprogram human DPSC along the neural lineage. The reprogrammed DPSC demonstrated a neuronal-like phenotype, with increased expression levels of neural markers, limited capacity for sphere formation, and enhanced neuronal but not glial differentiation. Transcriptomic analysis further highlighted the expression of genes associated with neural and neuronal functions. In vivo analysis using a developmental avian model showed that implanted DPSC survived in the developing central nervous system and respond to endogenous signals, displaying neuronal phenotypes. Therefore, OCT4 enhances the neural potential of DPSC, which exhibited characteristics aligning with neuronal progenitors. This method can be used to standardise DPSC neural induction and provide an alternative source of neural cell types.

Synthesis and Luminescent Properties of Barium Molybdate Nanoparticles.

BaMoO4 was obtained via facile mechanochemical synthesis at room temperature and a solid-state reaction. An evaluation of the phase composition and structural and optical properties of BaMoO4 was conducted. The influence of different milling speeds on the preparation of BaMoO4 was explored. A shorter reaction time for the phase formation of BaMoO4 was achieved using a milling speed of 850 rpm. A milling speed of 500 rpm led to partial amorphization of the initial reagents and to prolongation of the synthesis time of up to 3 h of milling time. Solid-state synthesis was performed via heat treatment at 900 °C for 15 h. X-ray diffraction analysis (XRD), infrared (IR) and UV diffuse reflectance (UV-Vis) and photoluminescence (PL) spectroscopy were carried out to characterize the samples. Independently of the method of preparation, the obtained samples had tetragonal symmetry. The average crystallite sizes of all samples, calculated using Scherrer's formula, were in the range of 240 to 1540 Å. IR spectroscopy showed that more distorted structural MoO4 units were formed when the compound was synthesized via a solid-state reaction. The optical band gap energy of the obtained materials was found to decrease from 4.50 to 4.30 eV with increasing crystallite sizes. Green- and blue-light emissions were observed for BaMoO4 phases under excitation wavelengths of 330 and 488 nm. It was established that the intensity of the PL peaks depends on two factors: the symmetry of MoO4 units and the crystallite sizes.

Nitrogen-Activated CLV3/ESR-Related 4 (CLE4) Regulates Shoot, Root, and Stolon Growth in Potato.

In potato, high levels of nitrogen (N) can lead to excessive vegetative growth at the expense of tuber development, resulting in lower yield and poor-quality tubers. We found that Solanum tuberosum CLE4 (StCLE4) is expressed most strongly in the roots grown in N-rich media, and it positively regulates potato root growth under N-deficient conditions. We noted that StCLE4 functions as a negative regulator of normal shoot apex development similar to CLV3 in Arabidopsis. Transcriptomic analysis revealed that overexpression of StCLE4 resulted in the repression of the StIT1 gene, a regulator of potato tuber initiation. StCLE4-overexpressing stolons were converted into branches, that were similar to a mild phenotype of the it1 (identity of tuber 1) mutant. We also found that NIN-like proteins, key regulators of nitrate signaling bind to the regulatory sequence of StIT1 in a yeast one-hybrid assay. Taken together, our findings suggest that StCLE4 regulates shoot, root, and stolon growth in potato.

Whole-genome sequence of Paenibacillus amylolyticus strain W018, isolated from Triticum aestivum L. seeds, obtained using nanopore sequencing.

In this study, we performed nanopore sequencing of the genome of Paenibacillus amylolyticus strain W018, isolated from the seeds of winter wheat, cv. Bezostaya 100. The genome size is 7.07 Mb, with a GC content of 45.8%, and contains 8,190 genes.

Sulfonylpiperazine compounds prevent Plasmodium falciparum invasion of red blood cells through interference with actin-1/profilin dynamics.

With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell (RBC) invasion, and have generated analogues with improved potency. Here, we generated resistance to MMV020291 and performed whole genome sequencing of 3 MMV020291-resistant populations. This revealed 3 nonsynonymous single nucleotide polymorphisms in 2 genes; 2 in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wild-type parasites, which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade RBCs. Additionally, the series inhibits the actin-1-dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro cosedimentation experiments using recombinant P. falciparum proteins indicate that potent MMV020291 analogues disrupt the formation of filamentous actin in the presence of profilin. Altogether, this study identifies the first compound series interfering with the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation.

Phytohormone Production by the Endophyte Bacillus safensis TS3 Increases Plant Yield and Alleviates Salt Stress.

Endophytic bacteria can be used to overcome the effect of salinity stress and promote plant growth and nutrient uptake. Bacillus safensis colonizes a wide range of habitats due to survival in extreme environments and unique physiological characteristics, such as a high tolerance for salt, heavy metals, and ultraviolet and gamma radiations. The aim of our study was to examine the salt resistance of the endophytic strain TS3 B. safensis and its ability to produce phytohormones and verify its effect on plant yield in field trials and the alleviation of salt stress in pot experiments. We demonstrate that the strain TS3 is capable of producing enzymes and phytohormones such as IAA, ABA and tZ. In pot experiments with radish and oat plants in salinization, the strain TS3 contributed to the partial removal of the negative effect of salinization. The compensatory effect of the strain TS3 on radish plants during salinization was 46.7%, and for oats, it was 108%. We suppose that such a pronounced effect on the plants grown and the salt stress is connected with its ability to produce phytohormones. Genome analysis of the strain TS3 showed the presence of the necessary genes for the synthesis of compounds responsible for the alleviation of the salt stress. Strain B. safensis TS3 can be considered a promising candidate for developing biofertilizer to alleviate salt stress and increase plant yield.

Targeting malaria parasites with novel derivatives of azithromycin.

Introduction: The spread of artemisinin resistant Plasmodium falciparum parasites is of global concern and highlights the need to identify new antimalarials for future treatments. Azithromycin, a macrolide antibiotic used clinically against malaria, kills parasites via two mechanisms: 'delayed death' by inhibiting the bacterium-like ribosomes of the apicoplast, and 'quick-killing' that kills rapidly across the entire blood stage development. Methods: Here, 22 azithromycin analogues were explored for delayed death and quick-killing activities against P. falciparum (the most virulent human malaria) and P. knowlesi (a monkey parasite that frequently infects humans). Results: Seventeen analogues showed improved quick-killing against both Plasmodium species, with up to 38 to 20-fold higher potency over azithromycin after less than 48 or 28 hours of treatment for P. falciparum and P. knowlesi, respectively. Quick-killing analogues maintained activity throughout the blood stage lifecycle, including ring stages of P. falciparum parasites (<12 hrs treatment) and were >5-fold more selective against P. falciparum than human cells. Isopentenyl pyrophosphate supplemented parasites that lacked an apicoplast were equally sensitive to quick-killing analogues, confirming that the quick killing activity of these drugs was not directed at the apicoplast. Further, activity against the related apicoplast containing parasite Toxoplasma gondii and the gram-positive bacterium Streptococcus pneumoniae did not show improvement over azithromycin, highlighting the specific improvement in antimalarial quick-killing activity. Metabolomic profiling of parasites subjected to the most potent compound showed a build-up of non-haemoglobin derived peptides that was similar to chloroquine, while also exhibiting accumulation of haemoglobin-derived peptides that was absent for chloroquine treatment. Discussion: The azithromycin analogues characterised in this study expand the structural diversity over previously reported quick-killing compounds and provide new starting points to develop azithromycin analogues with quick-killing antimalarial activity.

Identification and Expression Analysis of the C-TERMINALLY ENCODED PEPTIDE Family in Pisum sativum L.

The C-TERMINALLY ENCODED PEPTIDE(CEP) peptides play crucial roles in plant growth and response to environmental factors. These peptides were characterized as positive regulators of symbiotic nodule development in legume plants. However, little is known about the CEP peptide family in pea. Here, we discovered in pea genome 21 CEP genes (PsCEPs), among which three genes contained additional conserved motifs corresponding to the PIP (PAMP-induced secreted peptides) consensus sequences. We characterized the expression patterns of pea PsCEP genes based on transcriptomic data, and for six PsCEP genes with high expression levels in the root and symbiotic nodules the detailed expression analysis at different stages of symbiosis and in response to nitrate treatment was performed. We suggest that at least three PsCEP genes, PsCEP1, PsCEP7 and PsCEP2, could play a role in symbiotic nodule development, whereas the PsCEP1 and PsCEP13 genes, downregulated by nitrate addition, could be involved in regulation of nitrate-dependent processes in pea. Further functional studies are required to elucidate the functions of these PsCEP genes.

Draft Genome Sequence of Bacillus vallismortis Strain BL01, Isolated from Artemisia lerchiana Web. Roots.

Some strains of Bacillus vallismortis have been reported to be efficient plant-growth-promoting bacteria as well as inducers of systemic resistance. Here, we report the draft genome sequence of Bacillus vallismortis strain BL01, isolated from the roots of Artemisia lerchiana Web.

Draft Genome Sequence of the Tomato Stem Endophyte Bacillus safensis TS3.

Tomato stem endophyte Bacillus safensis TS3 was isolated from surface-sterilized stems of greenhouse tomato plants. Here, we sequenced the complete genome of this strain to understand the molecular mechanisms underlying its beneficial activities.

Transcriptomic Analysis of Radish (Raphanus sativus L.) Roots with CLE41 Overexpression.

The CLE41 peptide, like all other TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF) family CLE peptides, promotes cell division in (pro-)cambium vascular meristem and prevents xylem differentiation. In this work, we analyzed the differential gene expression in the radish primary-growing P35S:RsCLE41-1 roots using the RNA-seq. Our analysis of transcriptomic data revealed a total of 62 differentially expressed genes between transgenic radish roots overexpressing the RsCLE41-1 gene and the glucuronidase (GUS) gene. For genes associated with late embryogenesis, response to abscisic acid and auxin-dependent xylem cell fate determination, an increase in the expression in P35S:RsCLE41-1 roots was found. Among those downregulated, stress-associated genes prevailed. Moreover, several genes involved in xylem specification were also downregulated in the roots with RsCLE41-1 overexpression. Unexpectedly, none of the well-known targets of TDIFs, such as WOX4 and WOX14, were identified as DEGs in our experiment. Herein, we discuss a suggestion that the activation of pathways associated with desiccation resistance, which are more characteristic of late embryogenesis, in roots with RsCLE41-overexpression may be a consequence of water deficiency onset due to impaired vascular specification.

Property activity refinement of 2-anilino 4-amino substituted quinazolines as antimalarials with fast acting asexual parasite activity.

Malaria is a devastating disease caused by Plasmodium parasites. Emerging resistance against current antimalarial therapeutics has engendered the need to develop antimalarials with novel structural classes. We recently described the identification and initial optimization of the 2-anilino quinazoline antimalarial class. Here, we refine the physicochemical properties of this antimalarial class with the aim to improve aqueous solubility and metabolism and to reduce adverse promiscuity. We show the physicochemical properties of this class are intricately balanced with asexual parasite activity and human cell cytotoxicity. Structural modifications we have implemented improved LipE, aqueous solubility and in vitro metabolism while preserving fast acting P. falciparum asexual stage activity. The lead compounds demonstrated equipotent activity against P. knowlesi parasites and were not predisposed to resistance mechanisms of clinically used antimalarials. The optimized compounds exhibited modest activity against early-stage gametocytes, but no activity against pre-erythrocytic liver parasites. Confoundingly, the refined physicochemical properties installed in the compounds did not engender improved oral efficacy in a P. berghei mouse model of malaria compared to earlier studies on the 2-anilino quinazoline class. This study provides the framework for further development of this antimalarial class.

Optimisation of 2-(N-phenyl carboxamide) triazolopyrimidine antimalarials with moderate to slow acting erythrocytic stage activity.

Malaria is a devastating parasitic disease caused by parasites from the genus Plasmodium. Therapeutic resistance has been reported against all clinically available antimalarials, threatening our ability to control the disease and therefore there is an ongoing need for the development of novel antimalarials. Towards this goal, we identified the 2-(N-phenyl carboxamide) triazolopyrimidine class from a high throughput screen of the Janssen Jumpstarter library against the asexual stages of the P. falciparum parasite. Here we describe the structure activity relationship of the identified class and the optimisation of asexual stage activity while maintaining selectivity against the human HepG2 cell line. The most potent analogues from this study were shown to exhibit equipotent activity against P. falciparum multidrug resistant strains and P. knowlesi asexual parasites. Asexual stage phenotyping studies determined the triazolopyrimidine class arrests parasites at the trophozoite stage, but it is likely these parasites are still metabolically active until the second asexual cycle, and thus have a moderate to slow onset of action. Non-NADPH dependent degradation of the central carboxamide and low aqueous solubility was observed in in vitro ADME profiling. A significant challenge remains to correct these liabilities for further advancement of the 2-(N-phenyl carboxamide) triazolopyrimidine scaffold as a potential moderate to slow acting partner in a curative or prophylactic antimalarial treatment.

Structure activity refinement of phenylsulfonyl piperazines as antimalarials that block erythrocytic invasion.

The emerging resistance to combination therapies comprised of artemisinin derivatives has driven a need to identify new antimalarials with novel mechanisms of action. Central to the survival and proliferation of the malaria parasite is the invasion of red blood cells by Plasmodium merozoites, providing an attractive target for novel therapeutics. A screen of the Medicines for Malaria Venture Pathogen Box employing transgenic P. falciparum parasites expressing the nanoluciferase bioluminescent reporter identified the phenylsulfonyl piperazine class as a specific inhibitor of erythrocyte invasion. Here, we describe the optimization and further characterization of the phenylsulfonyl piperazine class. During the optimization process we defined the functionality required for P. falciparum asexual stage activity and determined the alpha-carbonyl S-methyl isomer was important for antimalarial potency. The optimized compounds also possessed comparable activity against multidrug resistant strains of P. falciparum and displayed weak activity against sexual stage gametocytes. We determined that the optimized compounds blocked erythrocyte invasion consistent with the asexual activity observed and therefore the phenylsulfonyl piperazine analogues described could serve as useful tools for studying Plasmodium erythrocyte invasion.

Plant tumors: a hundred years of study.

MAIN CONCLUSION: The review provides information on the mechanisms underlying the development of spontaneous and pathogen-induced tumors in higher plants. The activation of meristem-specific regulators in plant tumors of various origins suggests the meristem-like nature of abnormal plant hyperplasia. Plant tumor formation has more than a century of research history. The study of this phenomenon has led to a number of important discoveries, including the development of the Agrobacterium-mediated transformation technique and the discovery of horizontal gene transfer from bacteria to plants. There are two main groups of plant tumors: pathogen-induced tumors (e.g., tumors induced by bacteria, viruses, fungi, insects, etc.), and spontaneous ones, which are formed in the absence of any pathogen in plants with certain genotypes (e.g., interspecific hybrids, inbred lines, and mutants). The causes of the transition of plant cells to tumor growth are different from those in animals, and they include the disturbance of phytohormonal balance and the acquisition of meristematic characteristics by differentiated cells. The aim of this review is to discuss the mechanisms underlying the development of most known examples of plant tumors.

Using Dental Pulp Stem Cells for Stroke Therapy.

Stroke is a leading cause of permanent disability world-wide, but aside from rehabilitation, there is currently no clinically-proven pharmaceutical or biological agent to improve neurological disability. Cell-based therapies using stem cells, such as dental pulp stem cells, are a promising alternative for treatment of neurological diseases, including stroke. The ischaemic environment in stroke affects multiple cell populations, thus stem cells, which act through cellular and molecular mechanisms, are promising candidates. The most common stem cell population studied in the neurological setting has been mesenchymal stem cells due to their accessibility. However, it is believed that neural stem cells, the resident stem cell of the adult brain, would be most appropriate for brain repair. Using reprogramming strategies, alternative sources of neural stem and progenitor cells have been explored. We postulate that a cell of closer origin to the neural lineage would be a promising candidate for reprogramming and modification towards a neural stem or progenitor cell. One such candidate population is dental pulp stem cells, which reside in the root canal of teeth. This review will focus on the neural potential of dental pulp stem cells and their investigations in the stroke setting to date, and include an overview on the use of different sources of neural stem cells in preclinical studies and clinical trials of stroke.

Identification, expression, and functional analysis of CLE genes in radish (Raphanus sativus L.) storage root.

BACKGROUND: Radish (Raphanus sativus L.) is a widespread agricultural plant forming storage root due to extensive secondary growth which involves cambium proliferation and differentiation of secondary conductive tissues. Closely related to the model object Arabidopsis thaliana, radish is a suitable model for studying processes of secondary growth and storage root development. CLE peptides are a group of peptide phytohormones which play important role in the regulation of primary meristems such as SAM, RAM, and procambium, as well as secondary meristems. However, the role of CLE peptides in lateral growth of root during storage root formation has not been studied to date. RESULTS: In present work we studied the role of CLE peptides in the development of storage root in radish. We have identified 18 CLE genes of radish (RsCLEs) and measured their expression in various plant organs and also at different stages of root development in R. sativus and Raphanus raphanistrum-its close relative which does not form storage root. We observed significant decline of expression levels for genes RsCLE1, 2, 11, 13, and 16, and also multifold increase of expression levels for genes RsCLE19, and 41 during secondary root growth in R. sativus but not in R. raphanistrum. Expression of RsCLE 2, 19, and 41 in R. sativus root was confined to certain types of tissues while RsCLE1, 11, 13, and 16 expressed throughout the root. Experiments on overexpression of RsCLE2, 19 and 41 or treatment of radish plants with synthetic CLE peptides revealed that CLE19 and CLE2 increase the number of xylem elements, and CLE41 induces the formation of extra cambium foci in secondary xylem. Expression levels of RsCLE2 and 19 strongly decrease in response to exogenous cytokinin, while auxin causes dramatic increase of RsCLE19 expression level and decrease of RsCLE41 expression. CONCLUSIONS: Our data allow us to hypothesize about the role of RsCLE2, 19 and 41 genes in the development of storage root of Raphanus sativus, e.g. RsCLE19 may play a role in auxin-dependent processes of xylem differentiation and RsCLE41 stimulates cambium activity.

Initiation of spontaneous tumors in radish (Raphanus sativus): Cellular, molecular and physiological events.

In plant meristems, the balance of cell proliferation and differentiation is maintained by phytohormones, specifically auxin and cytokinin, as well as transcription factors. Changing of the cytokinin/auxin balance in plants may lead to developmental abnormalities, and in particular, to the formation of tumors. The examples of spontaneous tumor formation in plants include tumors formed on the roots of radish (Raphanus sativus) inbred lines. Previously, it was found that the cytokinin/auxin ratio is altered in radish tumors. In this study, a detailed histological analysis of spontaneous radish tumors was performed, revealing a possible mechanism of tumor formation, namely abnormal cambial activity. The analysis of cell proliferation patterns revealed meristematic foci in radish tumors. By using a fusion of an auxin-responsive promoter (DR5) and a reporter gene, the involvement of auxin in developmental processes in tumors was shown. In addition, the expression of the root meristem-specific WUSCHEL-related homeobox 5 (WOX5) gene was observed in cells adjacent to meristematic foci. Taken together, the results of the present study show that tumor tissues share some characteristics with root apical meristems, including the presence of auxin-response maxima in meristematic foci with adjacent cells expressing WOX5.