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Journal of Chromatography. A Jun 2024In recent years, many nucleic acid-based pharmaceuticals have been approved and entered the market, and even a larger number are in late stage clinical trials....
In recent years, many nucleic acid-based pharmaceuticals have been approved and entered the market, and even a larger number are in late stage clinical trials. Conventional oligonucleotides are facing issues in vivo like fast renal clearance and nuclease degradation. Therefore, to increase their stability, phosphorothioation is a frequent modification of therapeutic oligonucleotides (ONs) which also leads to improved binding affinity facilitating cell internalization and intracellular distribution. At the same time, by replacing a phosphodiester linkage with a phosphorothioate group, a phosphorous stereogenic center is generated which causes the formation of R- and S-diastereomers. It increases the structural diversity. For example, with 15 of those phosphorothioate (PS) linkages, 32,768 different diastereomers are expected. Since the phosphorothioate is introduced non-stereoselectively, the molecular complexity of the resultant phosphorothioate ON products is tremendously increased impeding the chromatographic separation in the course of quality control. Since distinct phosphorothioate diastereomers have different bioactivities and pharmacological properties, there is increasing interest in implications of stereoisomerism of phosphorothiate oligonucleotides. From a quality and regulatory viewpoint, batch-to-batch reproducibility of the diastereomer profile may be of significant concern. In order to address this issue, this study investigates the stereoselectivity of LC methods for two phosphorothioate oligonucleotide (PSO) compounds differing in their molecular size and numbers of PS linkages. Diastereoselectivity of ion-pairing reversed-phase liquid chromatography (IP-RPLC), RPLC without ion-pairing agents and LC with chiral polysaccharide-based column were evaluated for model PSOs and an active pharmaceutical ingredient (API) of PSO with trivalent N-acetylgalactosamine (GalNAc) conjugate. Due to the structural complexity of PSOs, the separation power for the diastereomer mixture was increased by using sequential selective comprehensive two-dimensional chromatography with an amylose tris(α-methylbenzylcarbamate)-immobilized chiral stationary phase (CSP) in the first dimension and ion-pair RPLC with ethylammonium acetate in the second dimension. Improved diastereomer selectivity was obtained and a larger number of peaks could be separated.
PubMed: 38879975
DOI: 10.1016/j.chroma.2024.465076 -
Rice (New York, N.Y.) Jun 2024Improving rice quality remains a crucial breeding objective, second only to enhancing yield, yet progress in quality improvement lags behind yield. The high temperature...
Improving rice quality remains a crucial breeding objective, second only to enhancing yield, yet progress in quality improvement lags behind yield. The high temperature and ripening conditions in Southern China often result in poor rice quality, impacting hybrid rice production and utilization. Therefore, to address this challenge, analyzing the molecular basis of high-quality traits is essential for molecular design breeding of high-quality hybrid rice varieties. In this study, we investigated the molecular basis of grain shape, amylose content, gel consistency, gelatinization temperature, and aroma, which influence rice quality. We discovered that quality related alleles gs3, GW7, gw8, chalk5, Wx, ALK, and fgr can enhance rice quality when applied in breeding programs. Polymerization of gs3, GW7, gw8, and chalk5 genes improves rice appearance quality. The gs3 and GW7 allele polymerization increasing the grain's length-width ratio, adding the aggregation of gw8 allele can further reducing grain width. The chalk5 gene regulates low chalkiness, but low correlation to chalkiness was exhibited with grain widths below 2.0 mm, with minimal differences between Chalk5 and chalk5 alleles. Enhancing rice cooking and eating quality is achieved through Wx and ALK gene polymerization, while introducing the fgr(E7) gene significantly improved rice aroma. Using molecular marker-assisted technology, we aggregated these genes to develop a batch of indica hybrid rice parents with improved rice quality are obtained. Cross-combining these enhanced parents can generate new, high-quality hybrid rice varieties suitable for cultivation in Southern China. Therefore, our findings contribute to a molecular breeding model for grain quality improvement in high-quality indica hybrid rice. This study, along with others, highlights the potential of molecular design breeding for enhancing complex traits, particularly rice grain quality.
PubMed: 38874692
DOI: 10.1186/s12284-024-00719-7 -
PloS One 2024BRRI31R is one of the Bangladesh's most promising restorer lines due to its abundant pollen producing capacity, strong restoring ability, good combining ability, high...
Pyramiding of bacterial blight resistance genes into promising restorer BRRI31R line through marker-assisted backcross breeding and evaluation of agro-morphological and physiochemical characteristics of developed resistant restorer lines.
BRRI31R is one of the Bangladesh's most promising restorer lines due to its abundant pollen producing capacity, strong restoring ability, good combining ability, high outcrossing rate and genetically diverse from cytoplasmic male sterile (CMS) line. But the drawback of this line is that it is highly susceptible to bacterial blight (BB) disease of rice caused by Xanthomonas oryzae pv. oryzae. The present study highlighted the pyramiding of effective BB resistance genes (xa5, xa13 and Xa21) into the background of BRRI31R, through marker-assisted backcrossing (MABC). Backcross progenies were confirmed and advanced based on the foreground selection of target genes. Pyramided lines were used for pathogenicity test against five Bangladeshi Xanthomonas oryzae (BXo) races (BXo93, BXo220, BXo822, BXo826, BXo887) and confirmed the dominant fertility restore genes, Rf3 and Rf4 and further validated against SNP markers for more confirmation of target resistance genes. All pyramided restorer lines consisted of Xa4 (in built), xa5, xa13, Xa21, and Chalk5 with two fertility restorer genes, Rf3, Rf4. and these restorer lines showed intermediate amylose content (<25%). Restorer lines BRRI31R-MASP3 and BRRI31R-MASP4 showed high levels of resistance against five virulent BXo races and SNP genotyping revealed that these lines also contained a blast resistance gene Pita races. Gene pyramided restorer lines, BRRI31R-MASP3 and BRRI31R-MASP4 can directly be used as a male parent for the development of new BB resistant hybrid rice variety or could be used as a replacement of restorer line of BRRI hybrid dhan5 and 7 to enhance the quality of hybrid seeds as well as rice production in Bangladesh.
Topics: Disease Resistance; Plant Diseases; Xanthomonas; Oryza; Plant Breeding; Genes, Plant; Genetic Markers; Crosses, Genetic
PubMed: 38865348
DOI: 10.1371/journal.pone.0301342 -
Current Research in Food Science 2024The objective of this work was to completely replace margarine with peanut diacylglycerol oil/ethyl cellulose-glycerol monostearate oleogel (DEC/GMS) oleogel, and...
The objective of this work was to completely replace margarine with peanut diacylglycerol oil/ethyl cellulose-glycerol monostearate oleogel (DEC/GMS) oleogel, and evaluate its effect on starch digestibility of cakes. The in vitro digestibility analysis demonstrated that the DEC/GMS-6 cake exhibited a 26.36% increase in slowly digestible starch (SDS) and resistant starch (RS) contents, compared to cakes formulated with margarine. The increased SDS and RS contents might mainly be due to the hydrophobic nature of OSA-wheat flour, which could promote the formation of lipid-amylose complexes with GMS and peanut diacylglycerol oil. XRD pattern suggested that the presence of GMS in DEC-based oleogels facilitated the formation of lipid-amylose complexes. The DSC analysis revealed that the addition of GMS resulted in a significant increase in gelatinization enthalpy, rising from 249.7 to 551.9 J/g, which indicates an improved resistance to gelatinization. The FTIR spectra indicated that the combination of GMS could enhance the hydrogen bonding forces and short-range ordered structure in DEC-based cakes. The rheological analysis revealed that an increase in GMS concentration resulted in enhanced viscoelasticity of DEC-based cake compared to TEC-based cakes. The DEC-based cakes exhibited a more satisfactory texture profile and higher overall acceptability than those of TEC-based cakes. Overall, these findings demonstrated that the utilization of DEC-based oleogel presented a viable alternative to commercial margarine in the development of cakes with reduced starch digestibility.
PubMed: 38860263
DOI: 10.1016/j.crfs.2024.100770 -
Frontiers in Nutrition 2024Research on the physicochemical properties of rice-derived endo-sperm high resistant starch (RS) with low amylose content (AC) is limited. In this study, we evaluated...
Research on the physicochemical properties of rice-derived endo-sperm high resistant starch (RS) with low amylose content (AC) is limited. In this study, we evaluated the physicochemical characteristics of such a starch variety and revealed that the starch granules exhibit a smoother, more refined surface with distinct edges, increased compactness, higher order of surface, and fewer cavities compared to those of a low RS rice variety. The starch crystal was classified as an A-type, which may be connected to the high amylose-lipid complex content. The branched internal long chains (B2 + B3) were abundant, allowing for easy entanglement with other molecular chains and a compact structure. Differential scanning calorimetry revealed the need for high temperature and energy to disrupt the double helix structure within the crystallization region of starch. Furthermore, starch viscosity analysis revealed a high cold paste viscosity, consistency, and setback value, with recrystallization yielding a stable structure, increased viscosity, and enhanced hydrolysis resistance to enzymes.
PubMed: 38860156
DOI: 10.3389/fnut.2024.1413923 -
BMC Plant Biology Jun 2024Amylose, a prebiotic found in yams is known to be beneficial for the gut microflora and is particularly advantageous for diabetic patients' diet. However, the genetic...
BACKGROUND
Amylose, a prebiotic found in yams is known to be beneficial for the gut microflora and is particularly advantageous for diabetic patients' diet. However, the genetic machinery underlying amylose production remains elusive. A comprehensive characterization of the genetic basis of amylose content in yam tubers is a prerequisite for accelerating the genetic engineering of yams with respect to amylose content variation.
RESULTS
To uncover the genetic variants underlying variation in amylose content, we evaluated amylose content in freshly harvested tubers from 150 accessions of Dioscorea zingibensis. With 30,000 high-quality single nucleotide polymorphisms (SNP), we performed a genome-wide association analysis (GWAS). The population structure analysis classified the D. zingiberensis accessions into three groups. A total of 115 significant loci were detected on four chromosomes. Of these, 112 significant SNPs (log10(p) = 5, q-value < 0.004) were clustered in a narrow window on the chromosome 6 (chr6). The peak SNP at the position 75,609,202 on chr6 could explain 63.15% of amylose variation in the population and fell into the first exon of the ADP-glucose pyrophosphorylase (AGPase) small subunit gene, causing a non-synonymous modification of the resulting protein sequence. Allele segregation analysis showed that accessions with the rare G allele had a higher amylose content than those harboring the common A allele. However, AGPase, a key enzyme precursor of amylose biosynthesis, was not expressed differentially between accessions with A and G alleles. Overexpression of the two variants of AGPase in Arabidopsis thaliana resulted in a significantly higher amylose content in lines transformed with the AGPase-G allele.
CONCLUSIONS
Overall, this study showed that a major genetic variant in AGPase probably enhances the enzyme activity leading to high amylose content in D. zingiberensis tuber. The results provide valuable insights for the development of amylose-enriched genotypes.
Topics: Amylose; Dioscorea; Genome-Wide Association Study; Plant Tubers; Polymorphism, Single Nucleotide; Plants, Genetically Modified; Genes, Plant
PubMed: 38853253
DOI: 10.1186/s12870-024-05122-4 -
Rice (New York, N.Y.) Jun 2024Cooking quality is the main factor determining the market value of rice. Although several major genes and a certain number of QTLs controlling cooking quality have been...
Cooking quality is the main factor determining the market value of rice. Although several major genes and a certain number of QTLs controlling cooking quality have been identified, the genetic complexity and environmental susceptibility limit the further improvement for cooking quality by molecular breeding. This research conducted a genome-wide association study to elucidate the QTLs related to cooking quality including amylose content (AC), gel consistency (GC) and alkali spreading value (ASV) by using 450 rice accessions consisting of 300 indica and 150 japonica accessions in two distinct environments. A total of 54 QTLs were identified, including 25 QTLs for AC, 12 QTLs for GC and 17 QTLs for ASV. Among them, 10 QTLs were consistently observed by the same population in both environments. Six QTLs were co-localized with the reported QTLs or cloned genes. The Wx gene for AC and GC, and the ALK gene for ASV were identified in every population across the two environments. The qAC9-2 for AC and the qGC9-2 for GC were defined to the same interval. The OsRING315 gene, encoding an E3 ubiquitin ligase, was considered as the candidate gene for both qAC9-2 and qGC9-2. The higher expression of OsRING315 corresponded to the lower AC and higher GC. Three haplotypes of OsRING315 were identified. The Hap 1 mainly existed in the japonica accessions and had lower AC. The Hap 2 and Hap 3 were predominantly present in the indica accessions, associated with higher AC. Meanwhile, the GC of accessions harboring Hap 1 was higher than that of accessions harboring Hap 3. In addition, the distribution of the three haplotypes in several rice-growing regions was unbalanced. The three traits of cooking quality are controlled by both major and minor genes and susceptible to environmental factors. The expression level of OsRING315 is related to both AC and GC, and this gene can be a promising target in quality improvement by using the gene editing method. Moreover, the haplotypes of OsRING315 differentiate between indica and japonica, and reveal the differences in GC and AC between indica and japonica rice.
PubMed: 38849622
DOI: 10.1186/s12284-024-00718-8 -
Physicochemical characterization of a composite flour: Blending purple sweet potato and rice flours.Food Chemistry: X Jun 2024In this study, the physicochemical characterization of different ratios of purple sweet potato flour (PSPF) and rice flour was investigated to improve the nutritional...
In this study, the physicochemical characterization of different ratios of purple sweet potato flour (PSPF) and rice flour was investigated to improve the nutritional value and enrich the variety of rice-based staple food. The results showed that adding PSPF increased total dietary fiber and anthocyanin content whereas decreased amylose content of the composite flours. Additionally, the composite flours exhibited lower thermodynamic parameters and displayed darker, redder, and bluer colors. There were no noticeable changes in the functional group structure of the composite flours. The addition of PSPF decreased the crystallinity and water-holding capacity of the composite flours, whereas increased the average particle size and iodine blue value. PSPF increased the pasting temperature of the flours whereas decreased the breakdown and setback values. Overall, the addition of PSPF significantly affects the nutrition, color and physicochemical properties of the composite flours.
PubMed: 38832302
DOI: 10.1016/j.fochx.2024.101493 -
Scientific Reports Jun 2024In the field of biotechnology, the utilization of agro-industrial waste for generating high-value products, such as microbial biomass and enzymes, holds significant...
In the field of biotechnology, the utilization of agro-industrial waste for generating high-value products, such as microbial biomass and enzymes, holds significant importance. This study aimed to produce recombinant α-amylase from Anoxybacillus karvacharensis strain K1, utilizing whey as an useful growth medium. The purified hexahistidine-tagged α-amylase exhibited remarkable homogeneity, boasting a specific activity of 1069.2 U mg. The enzyme displayed its peak activity at 55 °C and pH 6.5, retaining approximately 70% of its activity even after 3 h of incubation at 55 °C. Its molecular weight, as determined via SDS-PAGE, was approximately 69 kDa. The α-amylase demonstrated high activity against wheat starch (1648.8 ± 16.8 U mg) while exhibiting comparatively lower activity towards cyclodextrins and amylose (≤ 200.2 ± 16.2 U mg). It exhibited exceptional tolerance to salt, withstanding concentrations of up to 2.5 M. Interestingly, metal ions and detergents such as sodium dodecyl sulfate (SDS), Triton 100, Triton 40, and Tween 80, 5,5'-dithio-bis-[2-nitrobenzoic acid (DNTB), β-mercaptoethanol (ME), and dithiothreitol (DTT) had no significant inhibitory effect on the enzyme's activity, and the presence of CaCl (2 mM) even led to a slight activation of the recombinant enzyme (1.4 times). The Michaelis constant (K) and maximum reaction rate (V), were determined using soluble starch as a substrate, yielding values of 1.2 ± 0.19 mg mL and 1580.3 ± 183.7 μmol mg protein min, respectively. Notably, the most favorable conditions for biomass and recombinant α-amylase production were achieved through the treatment of acid whey with β-glucosidase for 24 h.
Topics: alpha-Amylases; Whey; Anoxybacillus; Detergents; Hydrogen-Ion Concentration; Enzyme Stability; Recombinant Proteins; Starch; Temperature
PubMed: 38830978
DOI: 10.1038/s41598-024-63606-7 -
Journal of Oleo Science 2024Glucosylceramide (GlcCer), a major sphingolipid in plants, possesses various food functions, including improvement of intestinal impairments. This study evaluated rice...
Glucosylceramide (GlcCer), a major sphingolipid in plants, possesses various food functions, including improvement of intestinal impairments. This study evaluated rice cooking conditions and cultivars based on GlcCer levels transferred into the digestive juice using an in vitro digestion model to investigate the factors related to GlcCer availability. GlcCer levels transferred into the digestive juice were higher in rice gruel than in boiled rice. The GlcCer levels in the digestive juice of boiled rice varied based on the rice cultivar, whereas those in rice gruel had no difference. Thus, GlcCer in rice was not fully utilized via digestion. Further, bioaccessibility was related to the amylose ratio and added water content.
Topics: Oryza; Glucosylceramides; Cooking; Digestion; Biological Availability; Water; Amylose
PubMed: 38825542
DOI: 10.5650/jos.ess24057