The Impact of Polymer Size and Cleavability on the Intravenous Pharmacokinetics of PEG-Based Hyperbranched Polymers in Rats

A better understanding of the impact of molecular size and linkers is important for PEG-based hyperbranched polymers (HBPs) intended as tailored drug delivery vehicles. This study aimed to evaluate the effects of crosslinker chemistry (cleavable disulphide versus non-cleavable ethylene glycol methacrylate (EGDMA) linkers) and molecular weight within the expected size range for efficient renal elimination (22 vs. 48 kDa) on the intravenous pharmacokinetic and biodistribution properties of 89Zr-labelled HBPs in rats. All HBPs showed similar plasma pharmacokinetics over 72 h, despite differences in linker chemistry and size. A larger proportion of HBP with the cleavable linker was eliminated via the urine and faeces compared to a similar-sized HBP with the non-cleavable linker, while size had no impact on the proportion of the dose excreted. The higher molecular weight HBPs accumulated in organs of the mononuclear phagocyte system (liver and spleen) more avidly than the smaller HBP. These results suggest that HBPs within the 22 to 48 kDa size range show no differences in plasma pharmacokinetics, but distinct patterns of organ biodistribution and elimination are evident.

The ITS region provides a reliable DNA barcode for identifying reishi/lingzhi (Ganoderma) from herbal supplements

The dietary supplement industry is rapidly growing yet, a recent study revealed that up to 60% of supplements may have substituted ingredients, some of which can be harmful contaminants or additives. When ingredients cannot be verified morphologically or biochemically, DNA barcoding complemented with a molecular phylogenetic analysis can be a powerful method for species authentication. We employed a molecular phylogenetic analysis for species authentication of the commonly used fungal supplement, reishi (Ganoderma lingzhi), by amplifying and sequencing the nuclear ribosomal internal transcribed spacer regions (ITS) with genus-specific primers. PCR of six powdered samples and one dried sample all sold as G. lucidum representing independent suppliers produced single, strong amplification products in the expected size-range for Ganoderma. Both best-hit BLAST and molecular phylogenetic analyses clearly identified the presence of G. lingzhi DNA in all seven herbal supplements. We detected variation in the ITS sequences among our samples, but all herbal supplement samples fall within a large clade of G. lingzhi ITS sequences. ITS-based phylogenetic analysis is a successful and cost-effective method for DNA-based species authentication that could be used in the herbal supplement industry for this and other fungal and plant species that are otherwise difficult to identify.

The ITS region provides a reliable DNA barcode for identifying reishi/lingzhi (Ganoderma) from herbal supplements

The dietary supplement industry is a growing enterprise, valued at over $100 billion by 2025 yet, a recent study revealed that up to 60% of herbal supplements may have substituted ingredients not listed on their labels, some with harmful contaminants. Substituted ingredients make rigorous quality control testing a necessary aspect in the production of supplements. Traditionally, species have been verified morphologically or biochemically, but this is not possible for all species if the identifying characteristics are lost in the processing of the material. One approach to validating plant and fungal ingredients in herbal supplements is through DNA barcoding complemented with a molecular phylogenetic analysis. This method provides an efficient, objective, rigorous and repeatable method for species identification. We employed a molecular phylogenetic analysis for species authentication of the commonly used fungal supplement, reishi (Ganoderma lingzhi), by amplifying and sequencing the nuclear ribosomal internal transcribed spacer regions (ITS) with genus-specific primers. PCR of six powdered samples and one dried sample sold as G. lucidum representing independent suppliers produced single, strong amplification products in the expected size-range for Ganoderma. Both best-hit BLAST and molecular phylogenetic analyses using a reference panel assembled from Genbank clearly identified the predominant fungal DNA was G. lingzhi in all seven herbal supplements. We detected variation in ITS among our samples, but all samples still fall within a large clade of G. lingzhi. ITS is a successful and cost-effective method for DNA-based species authentication that could be used in the herbal supplement industry for this and other fungal and plant species that are otherwise difficult to identify.

Transferability of Microsatellite Markers across Eleven Species of Magnolia L.

The genus Magnolia (Magnoliaceae) comprises more than 130 species distributed predominantly in temperate and tropical regions in Southeast Asia and is valued worldwide for its ornamental traits as well as for timber and medicinal products, and in trade. Despite their favored status, many species of Magnolia are faced with threats from logging, agricultural land use, development, and collection, and are at risk of extinction. Conservation of these species through habitat preservation and in ex situ collections is needed to prevent extinction. To provide a tool for conservation of Magnolia species, microsatellite markers developed previously for Magnolia ashei were tested in 10 other species of Magnolia to determine their transferability across species. Of the 64 primer pairs tested, 21 amplified alleles in the expected size range in all samples; 11 primer pairs amplified clean products in most, but not all, species; 18 primer pairs consistently amplified a polymerase chain reaction (PCR) product in most species, but had either low peak height or other amplification issues; and 14 primers showed excessive stutter, nonspecific amplification, or no amplification. Cluster analysis using the 129 alleles amplified by these 21 simple sequence repeat (SSR) primer pairs generated groups that corresponded to the known taxonomic relationships in this genus.

Genetic relationships and genotype tracing in date palms (Phoenix dactylifera L.) in Oman, based on microsatellite markers

Microsatellite markers (SSRs) were used to screen and analyse the genetic diversity among clonal genotypes of date palm (Phoenix dactylifera L.) derived by somatic embryogenesis in Oman. Twenty-one palms, representing 14 Omani, five Bahraini, one Iraqi and one Moroccan genotype, were screened with ten microsatellite markers. All primer pairs produced an amplification product in the expected size range and detected high levels of polymorphism among the analysed samples. Correspondence analysis revealed that the genotypes from Bahrain and Iraq showed a close relationship with accessions already grown in Oman. The genotype from Morocco (Medjool) appeared distinct from the rest of the material. Three independent clonal lines derived from a single Khalas Aldahra genotype were found to give identical genetic fingerprints. The value of this work for date palm production and conservation in Oman is discussed.

Development and characterization of polymorphic microsatellite markers in taro (Colocasia esculenta)

Microsatellite-containing sequences were isolated from enriched genomic libraries of taro (Colocasia esculenta (L.) Schott). The sequencing of 269 clones yielded 77 inserts containing repeat motifs. The majority of these (81.7%) were dinucleotide or trinucleotide repeats. The GT/CA repeat motif was the most common, accounting for 42% of all repeat types. From a total of 43 primer pairs designed, 41 produced markers within the expected size range. Sixteen (39%) were polymorphic when screened against a restricted set of taro genotypes from Southeast Asia and Oceania, with an average of 3.2 alleles detected on each locus. These markers represent a useful resource for taro germplasm management, genome mapping, and marker-assisted selection.Key words: Colocasia esculenta, microsatellite-enriched genomic library, simple sequence repeats, germplasm characterization.

063 Detection of Putatively Stress-activated Ty1-copia-like Retrotransposon Sequences in Virus-infected Sweetpotato [Ipomoea batatas (L.) Poir.] Clones

Previous work by our group has detected the presence of a heterogeneous population of Ty1-copia-like reverse transcriptase retrotransposon sequences in the sweetpotato genome. Recently, we detected the presence of putatively active Ty1-copia-like reverse transcriptase sequences from a virus-infected `Beauregard' sweetpotato clone. In the current study, we report the differential detection of putatively stress-activated sequences in clones from seedling 91-189. The clones were infected with different combinations of virus isolates followed by extraction of leaf RNA samples at three sampling dates (weeks 2, 4, and 6) after inoculation. After repeated DNAse treatments to eliminate contaminating DNA, the RNA samples were subjected to first strand cDNA synthesis using random decamer primers followed by PCR analysis utilizing Ty1-copia reverse transcriptase-specific primers. Through this approach, we detected amplified fragments within the expected size range (280-300 bp) from clones infected with isolates of sweetpotato leaf curl (SPLC) and feathery mottle viruses (FMV) (week 2 and 6) and FMV (week 4). We were unable to detect PCR products from the noninfected clones or the other infected samples. The data suggests that specific viruses may be involved in the expression of these Ty1-copia-related reverse transcriptase sequences. It also appears that sampling at various dates is necessary to detect putative activity over time. This preliminary information is essential before proceeding to the construction and screening of cDNA libraries to isolate and fully characterize the putatively active sweetpotato Ty1-copia-like retrotransposon sequences. Through the partial or complete characterization of sweetpotato Ty1-copia elements, sequences that correspond to cis-regulatory element(s) can be identified and further studied for their roles in responding to specific stress factors.

470 million years of conservation of microsatellite loci among fish species

Primers for 18 microsatellite loci originally isolated from whiting (6), stickleback ( Gasterosteus aculeatus, n — 5) and cod ( Gadus morhua, n = 7) were tested across a panel of diverse fish species, representing the three principal superclasses and most principal superorders of fish, to examine conservation of microsatellite regions across distantly related taxa. Three methods were used. First, amplified fragments were analysed by Southern blotting using the relevant microsatellite motif probes. A total of 17 of the tested primer pairs gave a product in the expected size range in at least four of 11 tested species. Second, for two study loci the amplified polymerase chain reaction products were cloned and sequenced in five fish species to reveal a high level of conservation of the flanking and microsatellite sequences. Finally, the 17 loci successfully amplified in non-source species were tested for polymorphism in groups of unrelated individuals from nine species, in several cases revealing extensive polymorphism. Levels of polymorphism were generally high in species from which the loci were derived or among closely related species. The conservation of flanking sequences for particular microsatellite motifs over the span of fish evolution represented in the test species (470 million years) far exceeds that hitherto reported and lends support to the suggestion (derived from studies of whales and marine turtles) that the rate of base substitution in nuclear and mitochondrial sequences is lower in aquatic than terrestrial organisms. A further explanation could be that these sequences, although generally considered neutral, may play an important role in eukaryotic genomes, and may be under strong selective constraints. The study suggests that heterologous primers will be a ready source of polymorphic markers among fish species, but also indicates that caution should be used in cross-species comparisons of variability.

Frequencies and sequence characteristics of di-, tri-, and tetra-nucleotide microsatellites in wheat

Microsatellites have emerged as an important source of genetic markers for eukaryotic genomes. In this report, two wheat (Triticum aestivum L.) genomic libraries were screened for several di-, tri-, and tetra-nucleotide tandem repeats. Clones containing (AC)n, (AG)n, (TCT)n, and (TTG)n repeats were isolated and sequenced. On average, there was one (AC)n microsatellite every 292 kbp and one (AG)n microsatellite every 212 kbp. The trinucleotide tandem repeats (TCT)n and (TTG)n were about 10 times less common than the two dinucleotide tandem repeats tested and tetranucleotide tandem repeats were rare. Many of the microsatellites had more than 10 repeats. The maximum repeat number found for (AC)n was 36 and for (TCT)n was more than 50. The prevailing category of (AG)n microsatellites from (AG)n isolates was perfect repeats. About half of the (AC)n microsatellites were compound repeats, while most of the (TCT)n microsatellites were imperfect repeats. In a small sample, (TTG)n microsatellites consisted mainly of compound repeats. The most frequently associated repeats were (AC)n with (AG)n, (TCT)n with (TCC)n, and (TTG)n with (TGG)n. Among 32 pairs of microsatellite primers surveyed, seven produced polymorphic products in the expected size range and these loci were mapped using a hexaploid wheat mapping population or aneuploid stocks. Key words : wheat, Triticum aestivum L., microsatellites, polymorphism, sequence characteristics.