Optimizing the Vanillin-Acid Sulfuric Method to Total Saponin Content in Leaves of Yerba Mate Clones.

Yerba mate (Ilex paraguariensis) is a forest species consumed in the form of non-alcoholic beverages in South America, with applications in foods, cosmetics, and pharmaceutical industries. The species leaves are globally recognized for their important bioactive compounds, including, saponins. We adjusted the vanillin-acid sulfuric method for determining spectrophotometrically the total saponin in yerba mate leaves. Seeking to maximize the extraction of saponins from leaves, a Doehlert design combined with Response Surface Methodology (RSM) was used, considering ethanol:water ratios and ultrasound times. In addition, the same methodology was used for the analysis of times and temperatures in the vanillin-sulfuric acid reaction heating. The contents of total saponin in mature leaves were compared in four yerba mate clones. The extraction was maximized using 40 % ethanol:60 % water and 60 minutes of ultrasound assisted extraction (UAE) without heating. For the reaction conditions, 70 °C for 10 minutes heating is recommended, and UV/Vis reading from 460 to 680 nm. Using the optimized methodology, total saponin contents ranged from 28.43 to 53.09 mg g-1 in the four yerba mate clones. The significant difference in saponin contents between clones indicate great genetic diversity and potential for clones' selection and extraction of these compounds from yerba mate leaves.

Establishment of an Efficient In Vitro Propagation of Cnidium officinale Makino and Selection of Superior Clones through Flow Cytometric Assessment of DNA Content.

Cnidium officinale is a valuable medicinal plant cultivated in Asia for its rhizomes. This study reports the in vitro regeneration of Cnidium officinale plants and the induction of rhizomes from microshoots. The rhizomatous buds of Cnidium officinale induced multiple shoots on Murashige and Skoog (MS) medium supplemented with 0.5 mg L-1 BA, which led to the regeneration of plants within four weeks of culture. After four weeks of culture, the plants were assessed for fresh weight, the number of leaves, the number of roots, and the length of roots to compare the performance of the different clones. The clones with good growth characteristics were selected with the aid of a flow cytometric analysis of 2C nuclear DNA content. The plants bearing high DNA values showed better growth characteristics. Various factors, namely, sucrose concentration (30, 50, 70, and 90 g L-1), ABA (0, 0.5, 1.0, and 2.0 mg L-1), the synergistic effects of BA (1.0 mg L-1) + NAA (0.5 mg L-1) and BA (1.0 mg L-1) + NAA (0.5 mg L-1) + ABA (1.0 mg L-1) with or without activated charcoal (1 g L-1), and light and dark incubation were tested on rhizome formation from microshoots. The results of the above experiments suggest that MS medium supplemented with 50 g L-1 sucrose, 1.0 mg L-1 ABA, and 1 g L-1 AC is good for the induction of rhizomes from the shoots of Cnidium officinale. Plantlets with rhizomes were successfully transferred to pots, and they showed 100% survival.

Comparative analysis of virus pathogenicity and resistance-breaking between the P- and A-type from the beet necrotic yellow vein virus using infectious cDNA clones.

The A-type of beet necrotic yellow vein virus (BNYVV) is widely distributed in Europe and is one of the major virus types causing rhizomania disease in sugar beet. The closely related P-type is mainly limited to a small region in France (Pithiviers). Both virus types possess four RNAs (RNA1-4), but the P-type harbours an additional fifth RNA species (RNA5). The P-type is associated with stronger disease symptoms and resistance-breaking of Rz1, one of the two resistance genes which are used to control BNYVV infection. These characteristics are presumably due to the presence of RNA5, but experimental evidence for this is lacking. We generated the first infectious cDNA clone of BNYVV P-type to study its pathogenicity in sugar beet in comparison to a previously developed A-type clone. Using this tool, we confirmed the pathogenicity of the P-type clone in the experimental host Nicotiana benthamiana and two Beta species, B. macrocarpa and B. vulgaris. Independent of RNA5, both the A- and the P-type accumulated in lateral roots and reduced the taproot weight of a susceptible sugar beet genotype to a similar extent. In contrast, only the P-type clone was able to accumulate a virus titre in an Rz1-resistant variety whereas the A-type clone failed to infect this variety. The efficiency of the P-type to overcome Rz1 resistance was strongly associated with the presence of RNA5. Only a double resistant variety, harbouring Rz1 and Rz2, prevented an infection with the P-type. Reassortment experiments between the P- and A-type clones demonstrated that both virus types can exchange whole RNA components without losing the ability to replicate and to move systemically in sugar beet. Although our study highlights the close evolutionary relationship between the two virus types, we were able to demonstrate distinct pathogenicity properties that are attributed to the presence of RNA5 in the P-type.

Diversity and population structure of Nordic potato cultivars and breeding clones.

BACKGROUND: The genetic diversity and population structure of breeding germplasm is central knowledge for crop improvement. To gain insight into the genetic potential of the germplasm used for potato breeding in a Nordic breeding program as well as all available accessions from the Nordic genebank (NordGen), 133 potato genotypes were genotyped using the Infinium Illumina 20 K SNP array. After SNP filtering, 11 610 polymorphic SNPs were included in the analysis. In addition, data from three important breeding traits - percent dry matter and uniformity of tuber shape and eye - were scored to measure the variation potato cultivars and breeding clones. RESULTS: The genetic diversity among the genotypes was estimated using principal coordinate analysis based on the genetic distance between individuals, as well as by using the software STRUCTURE. Both methods suggest that the collected breeding material and the germplasm from the gene-bank are closely related, with a low degree of population structure between the groups. The phenotypic distribution among the genotypes revealed significant differences, especially between farmer's cultivars and released cultivars and breeding clones. The percent heterozygosity was similar between the groups, with a mean average of 58-60%. Overall, the breeding germplasm and the accessions from the Nordic genebank seems to be closely related with similar genetic background. CONCLUSION: The genetic potential of available Nordic potato breeding germplasm is low, and for genetic hybridization purposes, genotypes from outside the Nordic region should be employed.

Analysis of genetic diversity and population structure among cultivated potato clones from Korea and global breeding programs.

Characterizing the genetic diversity and population structure of breeding materials is essential for breeding to improve crop plants. The potato is an important non-cereal food crop worldwide, but breeding potatoes remains challenging owing to their auto-tetraploidy and highly heterozygous genome. We evaluated the genetic structure of a 110-line Korean potato germplasm using the SolCAP 8303 single nucleotide polymorphism (SNP) Infinium array and compared it with potato clones from other countries to understand the genetic landscape of cultivated potatoes. Following the tetraploid model, we conducted population structure analysis, revealing three subpopulations represented by two Korean potato groups and one separate foreign potato group within 110 lines. When analyzing 393 global potato clones, country/region-specific genetic patterns were revealed. The Korean potato clones exhibited higher heterozygosity than those from Japan, the United States, and other potato landraces. We also employed integrated extended haplotype homozygosity (iHS) and cross-population extended haplotype homozygosity (XP-EHH) to identify selection signatures spanning candidate genes associated with biotic and abiotic stress tolerance. Based on the informativeness of SNPs for dosage genotyping calls, 10 highly informative SNPs discriminating all 393 potatoes were identified. Our results could help understanding a potato breeding history that reflects regional adaptations and distinct market demands.

Transcriptome and metabolome profiling in different stages of infestation of Eucalyptus urophylla clones by Ralstonia solanacearum.

Eucalyptus urophylla is an economically important tree species that widely planted in tropical and sub-tropical areas around the world, which suffers significant losses due to Ralstonia solanacearum. However, little is known about the molecular mechanism of pathogen-response of Eucalyptus. We collected the vascular tissues of a E. urophylla clone infected by R. solanacearum in the laboratory, and combined transcriptome and metabolome analysis to investigate the defense responses of Eucalyptus. A total of 11 flavonoids that differentially accumulated at the first stage or a later stage after infection. The phenylpropanoid of p-coumaraldehyde, the two alkaloids trigonelline and DL-ephedrine, two types of traditional Chinese medicine with patchouli alcohol and 3-dihydrocadambine, and the amino acid phenylalanine were differentially accumulated after infection, which could be biomarkers indicating a response to R. solanacearum. Differentially expressed genes involved in plant hormone signal transduction, phenylpropanoids, flavonoids, mitogen-activated protein kinase (MAPK) signaling, and amino acid metabolism were activated at the first stage of infection or a later stage, indicating that they may participate in the defense against infection. This study is expected to deliver several insights into the molecular mechanism in response to pathogens in E. urophylla, and the findings have far-reaching implications in the control of E. urophylla pathogens.

Clone and Function Verification of the OPR gene in Brassica napus Related to Linoleic Acid Synthesis.

BACKGROUND: Fatty acid composition and content affect rapeseed oil quality. Fatty acid synthesis-related genes in rapeseed have been studied globally by researchers. Nevertheless, rapeseed oil is mainly composed of seven different fatty acids (FA), and each fatty acid was regulated by different genes. Furthermore, different FA affect each other, which needs continuous and in-depth research to obtain more clear results in Brassica napus. RESULTS: In this paper, broad-scale miRNA expression profiles were constructed and 21 differentially expressed miRNAs were detected. GO enrichment analysis showed that most up-regulated proteins were involved in transcription factor activity and catalytic activity. KEGG pathway enrichment analysis indicated that 20 pathways involving 36 target genes were enriched, of which the bna00592 pathway may be involved in fatty acid metabolism. The results were verified using a quantitative real-time PCR (RT-qPCR) analysis, we found that the target gene of bna-miR156b > c > g was the OPR (12-oxo-phytodienoic acid reductase). Four copies of OPR gene were found, and the over-expression vectors (pCAMBIA1300-35 s-OPR and pCAMBIA1300-RNAi-OPR) were constructed to verify their functions. In T1 and T2 generation, the content of linoleic acid (LA) increased significantly in OE but deceased in OPRi. CONCLUSIONS: This is the first study to provide four copies of the OPR gene that regulates LA metabolism, can be used for the molecular mechanism of LA and optimizing fatty acid profiles in oilseed for breeding programs.

Impact of Ceftazidime-Avibactam Treatment in the Emergence of Novel KPC Variants in the ST307-Klebsiella pneumoniae High-Risk Clone and Consequences for Their Routine Detection.

The emergence of Klebsiella pneumoniae isolates carrying novel blaKPC variants conferring ceftazidime-avibactam (CAZ/AVI) resistance is being increasingly reported. We evaluated the accuracy of phenotypic methods commonly used in routine clinical laboratories in the detection of novel K. pneumoniae carbapenemase (KPC) enzymes. Additionally, we characterized by whole-genome sequencing (WGS) the KPC-ST307-K. pneumoniae isolates recovered in our hospital before and after CAZ/AVI therapy. Rectal colonization or infection by carbapenem-resistant KPC-3 K. pneumoniae isolates (imipenem MIC, 16 mg/L; meropenem MIC, 8 to >16 mg/L) and CAZ/AVI-susceptible isolates (CAZ/AVI MIC, 1 to 2 mg/L) were first detected in three intensive care unit (ICU) patients admitted between March 2020 and July 2020. KPC K. pneumoniae isolates with increased CAZ/AVI MICs (8 to 32 mg/L) and carbapenem susceptibility (imipenem and meropenem MIC, <1 mg/L) were recovered within 6 to 24 days after CAZ/AVI treatment. WGS confirmed that all KPC K. pneumoniae isolates belonged to the sequence type 307 (ST307) high-risk clone and carried identical antimicrobial resistance genes and virulence factors. The presence of the novel blaKPC-46, blaKPC-66, and blaKPC-92 genes was confirmed in the K. pneumoniae isolates with increased CAZ/AVI MICs and restored carbapenem activity. KPC production was confirmed by immunochromatography, the eazyplex Superbug CRE system, and the Xpert Carba-R assay in all KPC K. pneumoniae isolates, but not in any isolate using chromogenic agar plates for carbapenemase producers (ChromID-CARBA), the KPC/MBL/OXA-48 Confirm kit, and the ß-CARBA test. Nevertheless, all grew in chromogenic agar plates for extended-spectrum ß-lactamase (ESBL) producers (ChromID-ESBL). We report the failure of the most common phenotypic methods used for the detection of novel KPC carbapenemases but not of rapid molecular or immunochromatography assays, thus highlighting their relevance in microbiology laboratories.

Stable Cell Clones Harboring Self-Replicating SARS-CoV-2 RNAs for Drug Screen.

The development of antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been hampered by the lack of efficient cell-based replication systems that are amenable to high-throughput screens in biosafety level 2 laboratories. Here we report that stable cell clones harboring autonomously replicating SARS-CoV-2 RNAs without spike (S), membrane (M), and envelope (E) genes can be efficiently derived from the baby hamster kidney (BHK-21) cell line when a pair of mutations were introduced into the non-structural protein 1 (Nsp1) of SARS-CoV-2 to ameliorate cellular toxicity associated with virus replication. In a proof-of-concept experiment we screened a 273-compound library using replicon cells and identified three compounds as novel inhibitors of SARS-CoV-2 replication. Altogether, this work establishes a robust, cell-based system for genetic and functional analyses of SARS-CoV-2 replication and for the development of antiviral drugs. IMPORTANCE SARS-CoV-2 replicon systems that have been reported up to date were unsuccessful in deriving stable cell lines harboring non-cytopathic replicons. The transient expression of viral sgmRNA or a reporter gene makes it impractical for industry-scale screening of large compound libraries using these systems. Here, for the first time, we derived stable cell clones harboring the SARS-CoV-2 replicon. These clones may now be conveniently cultured in a standard BSL-2 laboratory for high throughput screen of compound libraries. Additionally, our stable replicon cells represent a new model system to study SARS-CoV-2 replication.

Pyrrolizidine alkaloids cause cell cycle and DNA damage repair defects as analyzed by transcriptomics in cytochrome P450 3A4-overexpressing HepG2 clone 9 cells.

Pyrrolizidine alkaloids (PAs) are a large group of highly toxic chemical compounds, which are found as cross-contaminants in numerous food products (e.g., honey), dietary supplements, herbal teas, and pharmaceutical herbal medicines. PA contaminations are responsible for serious hepatotoxicity and hepatocarcinogenesis. Health authorities have to set legal limit values to guarantee the safe consumption of plant-based nutritional and medical products without harmful health. Toxicological and chemical analytical methods are conventionally applied to determine legally permitted limit values for PAs. In the present investigation, we applied a highly sensitive transcriptomic approach to investigate the effect of low concentrations of five PAs (lasiocarpine, riddelliine, lycopsamine, echimidine, and monocrotaline) on human cytochrome P450 3A4-overexpressing HepG2 clone 9 hepatocytes. The transcriptomic profiling of deregulated gene expression indicated that the PAs disrupted important signaling pathways related to cell cycle regulation and DNA damage repair in the transfected hepatocytes, which may explain the carcinogenic PA effects. As PAs affected the expression of genes that involved in cell cycle regulation, we applied flow cytometric cell cycle analyses to verify the transcriptomic data. Interestingly, PA treatment led to an arrest in the S phase of the cell cycle, and this effect was more pronounced with more toxic PAs (i.e., lasiocarpine and riddelliine) than with the less toxic monocrotaline. Using immunofluorescence, high fractions of cells were detected with chromosome congression defects upon PA treatment, indicating mitotic failure. In conclusion, the tested PAs revealed threshold concentrations, above which crucial signaling pathways were deregulated resulting in cell damage and carcinogenesis. Cell cycle arrest and DNA damage repair point to the mutagenicity of PAs. The disturbance of chromosome congression is a novel mechanism of Pas, which may also contribute to PA-mediated carcinogenesis. Transcriptomic, cell cycle, and immunofluorescence analyses should supplement the standard techniques in toxicology to unravel the biological effects of PA exposure in liver cells as the primary target during metabolization of PAs.

Biodiversity of South Indian tea clones with detection of plant-based adulterants in tea dust using DNA barcoding.

Tea is by and large a highly penetrated product in south India. Hence the adulteration risk in tea dust gets hiked in the markets. We constructed a standard database using plant plastid markers (rbcL, matK, trnH-psbA, rpoC, rpoB, ycf 1) and nuclear (ITS2) locus from prominent south Indian tea clones representing Assam, China, and Cambod varieties. These barcodes were used as reference algorithm to investigate the authenticity of 10 sampled commercial tea dust by recovering its DNA barcodes using rbcL, matK, and ITS2 loci. PCR amplification success, sequencing efficiency, genetic polymorphisms, BLAST search, and phylogenetic analysis were performed to enhance genotypic information on south tea cultivars and in authenticating the commercial samples of Camellia sinensis. Findings suggest that the chloroplast and nuclear loci can identify tea plant at the genus and varietal level respectively and rbcL as the potential marker for detecting plant-based admixtures coupled with TA cloning after DNA barcoding.

Chromosome-scale reference genome assembly of a diploid potato clone derived from an elite variety.

Potato (Solanum tuberosum L.) is one of the most important crops with a worldwide production of 370 million metric tons. The objectives of this study were (1) to create a high-quality consensus sequence across the two haplotypes of a diploid clone derived from a tetraploid elite variety and assess the sequence divergence from the available potato genome assemblies, as well as among the two haplotypes; (2) to evaluate the new assembly's usefulness for various genomic methods; and (3) to assess the performance of phasing in diploid and tetraploid clones, using linked-read sequencing technology. We used PacBio long reads coupled with 10x Genomics reads and proximity ligation scaffolding to create the dAg1_v1.0 reference genome sequence. With a final assembly size of 812 Mb, where 750 Mb are anchored to 12 chromosomes, our assembly is larger than other available potato reference sequences and high proportions of properly paired reads were observed for clones unrelated by pedigree to dAg1. Comparisons of the new dAg1_v1.0 sequence to other potato genome sequences point out the high divergence between the different potato varieties and illustrate the potential of using dAg1_v1.0 sequence in breeding applications.

The complete mitogenome assemblies of 10 diploid potato clones reveal recombination and overlapping variants.

The potato mitogenome is complex and to understand various biological functions and nuclear-cytoplasmic interactions, it is important to characterize its gene content and structure. In this study, the complete mitogenome sequences of nine diploid potato clones along with a diploid Solanum okadae clone were characterized. Each mitogenome was assembled and annotated from Pacific Biosciences (PacBio) long reads and 10X genomics short reads. The results show that each mitogenome consists of multiple circular molecules with similar structure and gene organization, though two groups (clones 07506-01, DW84-1457, 08675-21 and H412-1 in one group, and clones W5281-2, 12625-02, 12120-03 and 11379-03 in another group) could be distinguished, and two mitogenomes (clone 10908-06 and OKA15) were not consistent with those or with each other. Significant differences in the repeat structure of the 10 mitogenomes were found, as was recombination events leading to multiple sub-genomic circles. Comparison between individual molecules revealed a translocation of ∼774 bp region located between a short repeat of 40 bp in molecule 3 of each mitogenome, and an insertion of the same in molecule 2 of the 10908-06 mitogenome. Finally, phylogenetic analyses revealed a close relationship between the mitogenomes of these clones and previously published potato mitogenomes.

Loss of CD22 expression and expansion of a CD22dim subpopulation in adults with relapsed/refractory B-lymphoblastic leukaemia after treatment with Inotuzumab-Ozogamicin.

Treatment options for relapsed or refractory B-lymphoblastic leukaemia (r/r B-ALL) are limited and the prognosis of these patients remains dismal, but novel immunotherapeutic options such as the anti-CD22 antibody-drug-conjugate Inotuzumab-Ozogamicin (InO) have improved outcomes in these patients. Flow cytometry is essential to assess antigen-expression prior to treatment initiation of antigen-directed immunotherapies. Here, we present flow cytometric and clinical data of three adult patients with r/r B-ALL who failed treatment with InO associated with reduced or lost antigen-expression. In addition, we present comparative data on two different diagnostic CD22-specific antibody clones that exhibit significant differences in staining intensities.

Magnetic Fluid Hyperthermia as Treatment Option for Pancreatic Cancer Cells and Pancreatic Cancer Organoids.

INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is a cancer with a meager prognosis due to its chemotherapy resistance. A new treatment method may be magnetic fluid hyperthermia (MFH). Magnetoliposomes (ML), consisting of superparamagnetic iron oxide nanoparticles (SPION) stabilized with a phospholipid-bilayer, are exposed to an alternating magnetic field (AMF) to generate heat. To optimize this therapy, we investigated the effects of MFH on human PDAC cell lines and 3D organoid cultures. MATERIAL AND METHODS: ML cytotoxicity was tested on Mia PaCa-2 and PANC-1 cells and on PDAC 3D organoid cultures, generated from resected tissue of patients. The MFH was achieved by AMF application with an amplitude of 40-47 kA/m and a frequency of 270 kHz. The MFH effect on the cell viability of the cell lines and the organoid cultures was investigated at two different time points. Clonogenic assays evaluated the impairment of colony formation. Altering ML set-ups addressed differences arising from intra- vs extracellular ML locations. RESULTS: Mia PaCa-2 and PANC-1 cells showed no cytotoxic effects at ML concentrations up to 300 µg(Fe)/mL and 225 µg(Fe)/mL, respectively. ML at a concentration of 225 µg(Fe)/mL were also non-toxic for PDAC organoid cultures. MFH treatment using exclusively extracellular ML presented the highest impact on cell viability. Clonogenic assays demonstrated remarkable impairment as long-term outcome in MFH-treated PDAC cell lines. Additionally, we successfully treated PDAC organoids with extracellular ML-derived MFH, resulting in notably reduced cell viabilities 2h and 24 h post treatment. Still, PDAC organoids seem to partly recover from MFH after 24 h as opposed to conventional 2D-cultures. CONCLUSION: Treatment with MFH strongly diminished pancreatic cancer cell viability in vitro, making it a promising treatment strategy. As organoids resemble the more advanced in vivo conditions better than conventional 2D cell lines, our organoid model holds great potential for further investigations.

Genetic diversity and population structure of advanced clones selected over forty years by a potato breeding program in the USA.

Knowledge regarding genetic diversity and population structure of breeding materials is essential for crop improvement. The Texas A&M University Potato Breeding Program has a collection of advanced clones selected and maintained in-vitro over a 40-year period. Little is known about its genetic makeup and usefulness for the current breeding program. In this study, 214 potato clones were genotyped with the Infinium Illumina 22 K V3 Potato Array. After filtering, a total of 10,106 single nucleotide polymorphic (SNP) markers were used for analysis. Heterozygosity varied by SNP, with an overall average of 0.59. Three groups of tetraploid clones primarily based on potato market classes, were detected using STRUCTURE software and confirmed by discriminant analysis of principal components. The highest coefficient of differentiation observed between the groups was 0.14. Signatures of selection were uncovered in genes controlling potato flesh and skin color, length of plant cycle and tuberization, and carbohydrate metabolism. A core set of 43 clones was obtained using Core Hunter 3 to develop a sub-collection that retains similar genetic diversity as the whole population, minimize redundancies, and facilitates long-term conservation of genetic resources. The comprehensive molecular characterization of our breeding clone bank collection contributes to understanding the genetic diversity of existing potato resources. This analysis could be applied to other breeding programs and assist in the selection of parents, fingerprinting, protection, and management of the breeding collections.

MDR M. tuberculosis outbreak clone in Eswatini missed by Xpert has elevated bedaquiline resistance dated to the pre-treatment era.

BACKGROUND: Multidrug-resistant (MDR) Mycobacterium tuberculosis complex strains not detected by commercial molecular drug susceptibility testing (mDST) assays due to the RpoB I491F resistance mutation are threatening the control of MDR tuberculosis (MDR-TB) in Eswatini. METHODS: We investigate the evolution and spread of MDR strains in Eswatini with a focus on bedaquiline (BDQ) and clofazimine (CFZ) resistance using whole-genome sequencing in two collections ((1) national drug resistance survey, 2009-2010; (2) MDR strains from the Nhlangano region, 2014-2017). RESULTS: MDR strains in collection 1 had a high cluster rate (95%, 117/123 MDR strains) with 55% grouped into the two largest clusters (gCL3, n = 28; gCL10, n = 40). All gCL10 isolates, which likely emerged around 1993 (95% highest posterior density 1987-1998), carried the mutation RpoB I491F that is missed by commercial mDST assays. In addition, 21 (53%) gCL10 isolates shared a Rv0678 M146T mutation that correlated with elevated minimum inhibitory concentrations (MICs) to BDQ and CFZ compared to wild type isolates. gCL10 isolates with the Rv0678 M146T mutation were also detected in collection 2. CONCLUSION: The high clustering rate suggests that transmission has been driving the MDR-TB epidemic in Eswatini for three decades. The presence of MDR strains in Eswatini that are not detected by commercial mDST assays and have elevated MICs to BDQ and CFZ potentially jeopardizes the successful implementation of new MDR-TB treatment guidelines. Measures to limit the spread of these outbreak isolates need to be implemented urgently.

In Silico, In Vitro, and In Vivo Antitumor and Anti-Inflammatory Evaluation of a Standardized Alkaloid-Enriched Fraction Obtained from Boehmeria caudata Sw. Aerial Parts.

In the context of the cancer-inflammation relationship and the use of natural products as potential antitumor and anti-inflammatory agents, the alkaloid-enriched fraction of Boehmeriacaudata (BcAEF) aerial parts was evaluated. In vitro antiproliferative studies with human tumor cell lines showed high activity at low concentrations. Further investigation on NCI-H460 cells showed an irreversible effect on cell proliferation, with cell cycle arrest at G2/M phase and programmed cell death induction. Molecular docking studies of four alkaloids identified in BcAEF with colchicine's binding site on ß-tubulin were performed, suggesting (-)-C (15R)-hydroxycryptopleurine as the main inductor of the observed mitotic death. In vivo studies showed that BcAEF was able to reduce Ehrlich tumor volume progression by 30 to 40%. Checking myeloperoxidase activity, BcAEF reduced neutrophils migration towards the tumor. The in vivo anti-inflammatory activity was evaluated by chemically induced edema models. In croton oil-induced ear edema and carrageenan (CG)-induced paw edema models, BcAEF reduced edema around 70 to 80% together with inhibition of activation and/or migration of neutrophils to the inflammatory area. All together the results presented herein show BcAEF as a potent antitumor agent combining antiproliferative and anti-inflammatory properties, which could be further explored in (pre)clinical studies.

Hand in hand: intrinsic and extrinsic drivers of aging and clonal hematopoiesis.

Over the past 25 years, the importance of hematopoietic stem cell (HSC) aging in overall hematopoietic and immune system health span has been appreciated. Much work has been done in model organisms to understand the intrinsic dysregulation that occurs in HSCs during aging, with the goal of identifying modifiable mechanisms that represent the proverbial "fountain of youth." Much more recently, the discovery of somatic mutations that are found to provide a selective advantage to HSCs and accumulate in the hematopoietic system during aging, termed clonal hematopoiesis (CH), inspires revisiting many of these previously defined drivers of HSC aging in the context of these somatic mutations. To truly understand these processes and develop a holistic picture of HSC aging, ongoing and future studies must include investigation of the critical changes that occur in the HSC niche or bone marrow microenvironment with aging, as increasing evidence supports that these HSC-extrinsic alterations provide necessary inflammation, signaling pathway activation or repression, and other selective pressures to favor HSC aging-associated phenotypes and CH. Here, we provide our perspectives based on the past 8 years of our own laboratory's investigations into these mechanisms and chart a path for integrative studies that, in our opinion, will provide an ideal opportunity to discover HSC and hematopoietic health span-extending interventions. This path includes examining when and how aging-associated HSC-intrinsic and HSC-extrinsic changes accumulate over time in different individuals and developing new models to track and test relevant HSC-extrinsic changes, complementary to innovative HSC lineage tracing systems that have recently been developed.

Panax quinquefolium L. Ginsenosides from Hairy Root Cultures and Their Clones Exert Cytotoxic, Genotoxic and Pro-Apoptotic Activity towards Human Colon Adenocarcinoma Cell Line Caco-2.

American ginseng, Panax quinquefolium (L.), is traditionally used in folk medicine. It exhibits a range of anti-inflammatory, hepatoprotective, anti-diabetic, anti-obesity, anti-hyperlipidemic and anti-carcinogenic effects. Its main components are ginsenosides, also known as panaxosides or triterpene saponins. In order to obtain high yields of ginsenosides, different methods of controlled production are involved, i.e., with hairy root cultures. However, they are still employed under in vitro conditions. Our studies revealed that hairy root cultures subjected to an elicitation process can be considered as a potent source of ginsenosides. The present study examines the biological activity of ginseng hairy root cultures against the Caco-2 human adenocarcinoma cell line. Among our six different clones of P. quinquefolium hairy roots, extracts B and Be (treated with elicitor) were the strongest inhibitors of the cellular metabolic activity. While all extracts induced DNA damage, B and Be also generated reactive oxygen species (ROS) in a concentration-dependent manner, which was correlated with the depletion of the mitochondrial membrane potential and induction of apoptosis. These findings indicate that further research concerning P. quinquefolium hairy root cultures should focus on the activity of rare ginsenosides and other biologically active compound profiles (i.e., phenolic compounds).