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BackgroundVitamin B12 is essential for neurological function, red blood cell formation, and DNA synthesis. Deficiency can lead to diverse health conditions, including megaloblastic anemia and neurological issues. Oral supplementation is a standard treatment for B12 deficiency. The Sucrosomial® carrier system offers an innovative approach that enhances supplemental nutrient absorption and bioavailability.ObjectivesThis study aimed to compare the effectiveness of oral Sucrosomial® vitamin B12 formulation vs various conventional B12 supplements, randomly selected from local pharmacies, in increasing and maintaining circulatory B12 levels in healthy deficient adults (200–300 pg/mL).MethodsA randomized, double-blind clinical trial was conducted across three centers in Pakistan from April to July 2024. At KEMU, participants received either Sucrosomial® vitamin B12 or Mecogen SL B12; at LRH, Sucrosomial® B12 or B-SUB B12; and at LUMHS, Sucrosomial® B12, Evermin B12, or Neuromax B12. Participants took a daily single dose of 1,000 μg of the assigned B12 formulation for 7 days. Serum B12 levels were measured at baseline (day 0) and on days 1, 3, 5, and 7.ResultsSucrosomial® B12 was significantly more effective than conventional B12 formulations in increasing and maintaining higher serum B12 levels across all time points. At KEMU, it reached a peak concentration of 454 ± 3.9 pg/mL by day 5, compared to 274 ± 11.1 pg/mL with Mecogen SL B12. At LRH, it peaked at 496 ± 34.4 pg/mL by day 5 versus 304 ± 49.4 pg/mL for B-SUB B12. At LUMHS, it reached 592.7 ± 74.3 pg/mL by day 7, compared to 407.24 ± 41.6 pg/mL for Evermin B12 and 263.82 ± 23.8 pg/mL for Neuromax B12. Sucrosomial® B12 was the only formulation to surpass the deficiency-borderline threshold (200–300 pg/mL) within 24 h of the first dose and was well tolerated with no reported side effects.ConclusionSucrosomial® vitamin B12 demonstrated superior efficacy in rapidly and consistently elevating and maintaining higher circulatory B12 levels compared to conventional supplements. Its characteristic absorption mode and proven efficacy suggest it could effectively address B12 deficiency in a broad range of populations, including those with gastrointestinal conditions and pernicious anemia, thereby supporting overall health.Clinical trial registrationclinicaltrials.gov, NCT06376591.

BackgroundVitamin D (Vit D) deficiency (VDD), associated with diverse health conditions, is commonly treated with Vit D3 supplements. However, the gastrointestinal (GI) absorption of Vit D3 in different formulations has not been well studied.ObjectiveWe aimed to compare the absorption of an innovative phospholipids-sucrester matrix biodelivery vehicle-based (sucrosomial®) orodispersible Vit D3 preparation against a reference chewable tablet and soft gel capsule (SGC) Vit D3 formulations in Vit D-deficient healthy adults.MethodsIn study 1, 25 subjects were randomized to receive a weekly single dose of 200,000 IU of sucrosomial® Vit D3 (n = 12) or chewable tablet Vit D3 (n = 13) for 3 weeks. In study 2, 20 subjects were randomized to receive a single dose of 200,000 IU every other week of sucrosomial® Vit D3 (n = 10) or SGC Vit D3 (n = 10) for 6 weeks. Circulatory 25-hydroxyvitamin D3 [25(OH)D] levels were reassessed after 2, 3, and 6 weeks in study 1 and after 4 and 6 weeks in study 2.ResultsIn study 1, after 2 weeks, circulatory 25(OH)D levels increased significantly in both Vit D3 treatment groups (p < 0.0001) but improved markedly in the sucrosomial® Vit D3 group, with no further considerable change after 3 and 6 weeks in both groups. Overall, at all three follow-ups, sucrosomial® Vit D3 treatment achieved significantly higher and sustained 25(OH)D levels (p < 0.001). In study 2, after 4 weeks, both Vit D3 treatment groups showed significant improvement in circulatory 25(OH)D levels (p < 0.0001) but substantially higher in the sucrosomial® group with statistically significant differences between the two treatment groups (p = 0.02). At the 6-week follow-up, only subjects in the sucrosomial® Vit D3 group showed a further increase in circulatory 25(OH)D levels (p = 0.049), but no further significant changes in the levels of the SGC Vit D3 group (p = 0.062), showing a statistically significant difference between the two treatment groups (p = 0.002). The Vit D3 treatment was well tolerated by all participants, and no treatment-emergent effects or serious adverse events were reported.ConclusionOur results suggest that the sucrosomial® Vit D3 preparation absorbs efficiently in the GI system, achieving adequately higher and sustained circulatory Vit D levels in VDD, and thus can effectively contribute to the body protection against VDD-associated health conditions.Clinical trial registrationclinicaltrials.gov, identifier: NCT05706259.

Iron deficiency (ID) and iron deficiency anemia (IDA) are highly prevalent worldwide. Oral iron salts, especially ferrous sulfate, are commonly used for the treatment of iron deficiency (ID). However, its use is associated with gastrointestinal side effects, thus compromising treatment compliance. Intravenous iron administration is a more costly and logistically complex alternative and is not risk-free, as infusion and hypersensitivity reactions may occur. Sucrosomial® iron is an oral formulation consisting of ferric pyrophosphate conveyed by a phospholipid and sucrester matrix (sucrosome®). Intestinal Sucrosomial® iron absorption is mediated by enterocytes and M cells, through the paracellular and transcellular routes, and occurs mostly as intact particles. These pharmacokinetic properties of Sucrosomial® iron result in higher iron intestinal absorption and excellent gastrointestinal tolerance compared to oral iron salts. The evidence derived from clinical studies supports the use of Sucrosomial® iron as a valid first option for the treatment of ID and IDA, especially for subjects who are intolerant or refractory to conventional iron salts. Newer evidence also demonstrates the effectiveness of Sucrosomial® iron, with a lower cost and fewer side effects, in certain conditions usually treated with IV iron in current clinical practice.

Background: Athletic pubalgia (AP), a frequent problem among professional roller hockey players (PRHPs), consists of lower abdominal and groin pain, without the presence of true hernia. Aims: We assessed cetylated fatty acids (CFAs) in association with conservative therapy for treatment of AP in PRHPs. Methods: Ultrasound examination was performed before and after treatment. Strength tests were performed and AP-related pain was measured during the treatment period. Findings: Nine of 10 enrolled PRHPs completed a 12-week treatment with CFAs in association with conservative therapy, consisting of manual therapy, diathermy or ultrasonography. Initial ultrasound examination showed AP signs in 7 (70%) of 10 PRHPs. After 12 weeks of therapy, these signs could only be detected by ultrasound in 2 (22.2%) of 9 PRHPs. An increase in muscle strength (already after first week of treatment) and a reduction of AP-related pain were also observed during the treatment. Conclusion: The association of CFA treatment with a conservative rehabilitation therapy improves muscle strength and pain and may accelerate recovery from AP.

Iron deficiency (ID) is usually treated with oral iron salts, but up to 50% of patients complain of gastrointestinal side effects, leading to reduced compliance with treatment. Intravenous (IV) iron formulations are increasingly safe, but there is still a risk of infusion, hypersensitivity reactions and the need for venous access and infusion monitoring. Sucrosomial® Iron (SI) is an innovative oral iron formulation in which ferric pyrophosphate is protected by a phospholipid bilayer plus a sucrester matrix (sucrosome), which is absorbed through para-cellular and trans-cellular routes (M cells). This confers SI’s unique structural, physicochemical and pharmacokinetic characteristics, together with its high iron bioavailability and excellent gastrointestinal tolerance. The analysis of the available evidence supports oral SI iron as a valid option for ID treatment, which is more efficacious and tolerable than oral iron salts. SI has also demonstrated a similar effectiveness, with lower risks, in patients usually receiving IV iron (e.g., chronic kidney disease, cancer, bariatric surgery). Thus, oral SI emerges as a valuable first option for treating ID, especially for subjects with intolerance to iron salts or those for whom iron salts are inefficacious. Moreover, SI should also be considered as an alternative to IV iron for initial and/or maintenance treatment in different patient populations.

n-Propyl gallate (PG) is a phenolic compound that influences enzymatic processes, mostly involving AOX, PTOX, LOX, POD, and PPO. Here, analyses of different PG concentrations (1, 2.5, and 5 mM) during cowpea seed germination at 16, 32, and 48h showed that 2.5 mM PG partially inhibited seed germination at 16 and/or 32h, but by 48h the germination re-established. Thus, this PG concentration was chosen to study the molecular and biochemical mechanisms linked to the PG inhibitory effects and germination recovery. PG inhibition was related to lower H 2 O 2 , higher antioxidant activity, and downregulation of genes linked to cell cycle progression, energy status, and the Krebs cycle at 16 and/or 32h, but these changes were reversed at 48h. In general, genes associated with detoxification, germination-related phytohormones, and NAD(P)H metabolism were highly up-regulated across the time points. AOX1 and Pgb1 were continuously up-regulated along the time points, and linked to NR transcript level increase only at 48h. These findings indicated that AOX and the phytoglobin cycle, both systems involved in NO levels regulation, worked efficiently in germination re-establishment. However, genes other than AOX associated with potential target enzymes of PG, such as LOX, POD, PTOX and PPO (except at 48h), were mostly unchanged or down-regulated. Genes linked to glycolysis (PFK and PK) and acetate synthesis (PDC and ALDH) connected with AOX via NAD(P)+ were up-regulated under PG mainly at 48h. The data are discussed in light of AOX's role in cell reprogramming to reverse PG-induced inhibition of germination in cowpea seeds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier GmbH. All rights reserved.)

Key Message: AOX gene family in motion marks in-born efficiency of respiration adjustment; can serve for primer screening, genotype ranking, in vitro-plant discrimination and a SMART perspective for multiple-resilient plant holobiont selection. The bacteria Xylella fastidiosa (Xf) is a climate-dependent, global threat to many crops of high socio-economic value, including grapevine. Currently designed breeding strategies for Xf-tolerant or -resistant genotypes insufficiently address the danger of biodiversity loss by focusing on selected threats, neglecting future environmental conditions. Thus, breeding strategies should be validated across diverse populations and acknowledge temperature changes and drought by minimizing the metabolic-physiologic effects of multiple stress-induced oxygen shortages. This research hypothesizes that multiple-resilient plant holobionts achieve lifelong adaptive robustness through early molecular and metabolic responses in primary stress target cells, which facilitate efficient respiration adjustment and cell cycle down-regulation. To validate this concept open-access transcriptome data were analyzed of xylem tissues of Xf-tolerant and -resistant Vitis holobionts from diverse trials and genetic origins from early hours to longer periods after Xf-inoculation. The results indicated repetitive involvement of alternative oxidase (AOX) transcription in episodes of down-regulated transcripts of cytochrome c oxidase (COX) at various critical time points before disease symptoms emerged. The relation between transcript levels of COX and AOX ('relCOX/AOX') was found promising for plant discrimination and primer screening. Furthermore, transcript levels of xylem-harbored bacterial consortia indicated common regulation with Xf and revealed stress-induced early down-regulation and later enhancement. LPS priming promoted the earlier increase in bacterial transcripts after Xf-inoculation. This proof-of-principle study highlights a SMART perspective for AOX-assisted plant selection towards multiple-resilience that includes Xf-tolerance. It aims to support timely future plant diagnostics and in-field substitution, sustainable agro-management, which protects population diversity and strengthens both conventional breeding and high-tech, molecular breeding research. Furthermore, the results suggested early up-regulation of bacterial microbiota consortia in vascular-enriched tissues as a novel additional trait for future studies on Xf-tolerance., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Peroxidases (EC 1.11.1.7) are involved in a wide range of physiological processes, hence their broad distribution across biological systems. These proteins can be classified as haem or non-haem enzymes. According to the RedOxiBase database, haem peroxidases are approximately 84 % of all known peroxidase enzymes. Class III plant peroxidases are haem-enzymes that share similar three-dimensional structures and a common catalytic mechanism for hydrogen peroxide degradation. They exist as large multigene families and are involved in metabolizing Reactive Oxygen Species (ROS), hormone synthesis and decomposition, fruit growth, defense, and cell wall synthesis and maintenance. As a result, plant peroxidases gained attention in research and became one of the most extensively studied groups of enzymes. This review provides an update on the database, classification, phylogeny, mechanism of action, structure, and physiological functions of class III plant peroxidases., Competing Interests: Declaration of competing interest The authors confirm that the contents of this article pose no conflicts of interest., (Copyright © 2024. Published by Elsevier B.V.)

Xylella fastidiosa ( Xf ) is a global bacterial threat for a diversity of plants, including olive trees. However, current understanding of host responses upon Xf -infection is limited to allow early disease prediction, diagnosis, and sustainable strategies for breeding on plant tolerance. Recently, we identified a ma jor c omplex t rait for e arly d e novo programming, named CoV-MAC-TED , by comparing early transcriptome data during plant cell survival with SARS-CoV-2-infected human cells. This trait linked ROS/RNS balancing during first hours of stress perception with increased aerobic fermentation connected to alpha-tubulin-based cell restructuration and control of cell cycle progression. Furthermore, our group had advanced concepts and strategies for breeding on plant holobionts. Here, we studied tolerance against Xf -infection by applying a CoV-MAC-TED -related gene set to (1) progress proof-of-principles, (2) highlight the importance of individual host responses for knowledge gain, (3) benefit sustainable production of Xf -threatened olive, (4) stimulate new thinking on principle roles of secondary metabolite synthesis and microbiota for system equilibration and, (5) advance functional marker development for resilience prediction including tolerance to Xf -infections. We performed hypothesis-driven complex analyses in an open access transcriptome of primary target xylem tissues of naturally Xf -infected olive trees of the Xf -tolerant cv. Leccino and the Xf -susceptible cv. Ogliarola. The results indicated that cyanide-mediated equilibration of oxygen-dependent respiration and carbon-stress alleviation by the help of increased glycolysis-driven aerobic fermentation paths and phenolic metabolism associate to tolerance against Xf . Furthermore, enhanced alternative oxidase (AOX) transcript levels through transcription Gleichschaltung linked to quinic acid synthesis appeared as promising trait for functional marker development. Moreover, the results support the idea that fungal endophytes strengthen Xf -susceptible genotypes, which lack efficient AOX functionality. Overall, this proof-of-principles approach supports the idea that efficient regulation of the multi-functional AOX gene family can assist selection on multiple-resilience, which integrates Xf -tolerance, and stimulates future validation across diverse systems.

Pepper ( Capsicum annuum L.) is a vegetable consumed worldwide, primarily used for vitamin C uptake and condiment purposes. Ascorbate (Asc) is a multifunctional metabolite, acting as an antioxidant and enzymatic cofactor involved in multiple cellular processes. Nevertheless, there is no evidence about the contribution of biosynthesis pathways and regulatory mechanisms responsible for Asc reserves in pepper plants. Here, we present a genome- and transcriptome-wide investigation of genes responsible for Asc biosynthesis in pepper during fruit development, stresses, and phytohormone exposures. A total of 21 genes, scattered in ten of twelve pepper chromosomes were annotated. Gene expression analyses of nine transcriptomic experiments supported the primary role of the L-galactose pathway in the Asc-biosynthesizing process, given its constitutive, ubiquitous, and high expression profile observed in all studied conditions. However, genes from alternative pathways generally exhibited low expression or were unexpressed and appeared to play some secondary role under specific stress conditions and phytohormone treatments. Taken together, our findings provide a deeper spatio-temporal understanding of expression levels of genes involved in Asc biosynthesis, and they highlight GGP2 , GME1 and 2 , and GalLDH members from L-galactose pathway as promising candidates for future wet experimentation, addressing the attainment of increase in ascorbate content of peppers and other crops.

Plants subjected to stress need to respond rapidly and efficiently to acclimatize and survive. In this paper, we investigated a selected gene set potentially involved in early cell reprogramming in two rice genotypes with contrasting salinity tolerance (Pokkali tolerant and IR29 susceptible) in order to advance knowledge of early molecular mechanisms of rice in dealing with salt stress. Selected genes were evaluated in available transcriptomic data over a short period of 24 h and involved enzymes that avoid ROS formation (AOX, UCP and PTOX), impact ATP production (PFK, ADH and COX) or relate to the antioxidant system. Higher transcript accumulation of AOX (ROS balancing), PFK and ADH (alcohol fermentation) was detected in the tolerant genotype, while the sensitive genotype revealed higher UCP and PTOX transcript levels, indicating a predominant role for early transcription of AOX and fermentation in conferring salt stress tolerance to rice. Antioxidant gene analyses supported higher oxidative stress in IR29, with transcript increases of cytosolic CAT and SOD from all cell compartments (cytoplasm, peroxisome, chloroplast and mitochondria). In contrast, Pokkali increased mRNA levels from the AsA-GSH cycle as cytosolic/mitochondrial DHAR was involved in ascorbate recovery. In addition, these responses occurred from 2 h in IR29 and 10 h in Pokkali, indicating early but ineffective antioxidant activity in the susceptible genotype. Overall, our data suggest that AOX and ADH can play a critical role during early cell reprogramming for improving salt stress tolerance by efficiently controlling ROS formation in mitochondria. We discuss our results in relation to gene engineering and editing approaches to develop salinity-tolerant crops.

Key Message: We found 34 and 71 key genes potentially involved in flavonoid biosynthesis and cell wall disassembly, respectively, which could be associated with specific peel coloration and softening of each genotype. Cashew apple (Anacardium occidentale) has a great economic importance worldwide due to its high nutritional value, peculiar flavor and aroma. During ripening, the peduncle develops different peel color and becomes quickly fragile due to its oversoftening, impacting its consumers' acceptance. In view of this, the understanding about its transcriptional dynamics throughout ripening is imperative. In this study, we performed a transcriptome sequencing of two cashew apple genotypes (CCP 76 and BRS 265), presenting different firmness and color peel, in the immature and ripe stages. Comparative transcriptome analysis between immature and ripe cashew apple revealed 4374 and 3266 differentially expressed genes (DEGs) to CCP 76 and BRS 265 genotypes, respectively. These genes included 71 and 34 GDEs involved in the cell wall disassembly and flavonoid biosynthesis, respectively, which could be associated with firmness loss and anthocyanin accumulation during cashew apple development. Then, softer peduncle of CCP 76 could be justified by down-regulated EXP and up-regulation of genes involved in pectin degradation (PG, PL and PAE) and in cell wall biosynthesis. Moreover, genes related to flavonoid biosynthesis (PAL, C4H and CHS) could be associated with early high accumulation of anthocyanin in red-peel peduncle of BRS 265. Finally, expression patterns of the selected genes were tested by real-time quantitative PCR (qRT-PCR), and the qRT-PCR results were consistent with transcriptome data. The information generated in this work will provide insights into transcriptome responses to cashew apple ripening and hence, it will be helpful for cashew breeding programs aimed at developing genotypes with improved quality traits., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Ascorbate-glutathione (AsA-GSH) cycle plays an important role in tuning beneficial ROS accumulation for intracellular signals and imparts plant tolerance to oxidative stress by detoxifying excess of ROS. Here, we present genome-wide identification of AsA-GSH cycle genes (APX, MDHAR, DHAR, and GR) in several leguminous species and expression analyses in G. max during stress, germination and tissue development. Our data revealed 24 genes in Glycine genus against the maximum of 15 in other leguminous species, which was due to 9 pars of duplicated genes mostly originated from sub/neofunctionalization. Cytosolic APX and MDHAR genes were highly expressed in different tissues and physiological conditions. Germination induced genes encoding AsA-GSH proteins from different cell compartments, whereas vegetative phase (leaves) stimulated predominantly genes related to chloroplast/mitochondria proteins. Moreover, cytosolic APX-1, 2, MDHAR-1a, 1b and GR genes were the primary genes linked to senescence and biotic stresses, while stAPX-a, b and GR (from organelles) were the most abiotic stress related genes. Biotic and abiotic stress tolerant genotypes generally showed increased MDHAR, DHAR and/or GR mRNA levels compared to susceptible genotypes. Overall, these data clarified evolutionary events in leguminous plants and point to the functional specificity of duplicated genes of the AsA-GSH cycle in G. max., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Understanding into acerola (Malpighia emarginata) molecular and biochemical bases is still obscure, despite it is one of the most important natural source of vitamin C for humans. Recently, our research group published the first data on acerola transcriptome generating valuable information to identify reference genes for RT-qPCR in this species. Hence, this study aimed to identify the most stably expressed genes based on acerola transcriptome data, and further to evaluate the suitability of F-box, U3, Merad50-ATPase, TGD4, NOB1, PA-RNA, RCC1, RBL and PGAL candidates for accurate gene expression normalization in leaf, flower and fruit at 12, 16 and 20 days after anthesis using RT-qPCR analysis. Three algorithms, geNorm, NormFinder, and BestKeeper confirmed the expression stability of all nine candidate reference genes, whereas RefFinder consensually summarized a comprehensive gene ranking. Based on geNorm, the combination of the most stable reference genes RBL and U3 for leaf/flower group, TGD4, F-box and PGAL (fruit developmental stages or fruit/leaf), RCC1, PGAL and RBL (fruit/flower) and RCC1, RBL, TGD4 and PGAL (total samples) were required for accurate normalization. Moreover, the use of these reference genes to assess the expression profile of GMP1 and NAT3 genes confirmed the reliability of ranking and defined the best combination of genes recommended by geNorm and RefFinder. This work will benefit further RT-qPCR studies in these acerola organs by offering a foundation for accurate normalization of gene expression profiling.