Diaphorina citri Kuwayama, is one of the most important citrus pests because it transmits the bacterium that causes citrus Huanglongbing during feeding. The saliva of herbivorous insects can modulate plant defenses and, in turn, impact insect fitness, which is mostly studied in insects feeding on herbaceous plants. The role of saliva in the relationship between the Asian citrus psyllid and its woody host plant citrus, on the other hand, is unknown. Because two citrus aphid species, Aphis spiraecola Patch and Aphis (Toxoptera) citricidus , have a contrasting impact on the performance of subsequently infested D. citri, we explored the role of their saliva on D. citri feeding behavior and host plant defenses. We found that the infiltrating saliva of A. spiraecola into the host citrus leaves of the psyllid disrupted the subsequent feeding behavior of D. citri and also activated the expression of genes involved in plant salicylic acid (SA) and jasmonic acid (JA) defense pathways. By contrast, saliva infiltrations of A. citricidus  promoted D. citri feeding, activated the expression of one gene involved in the SA pathway, and repressed several genes involved in the JA pathway. We demonstrate that the saliva of aphids can affect D. citri performance, possibly by modulating plant defenses. This is the first study to show that insect saliva can influence D. citri feeding behavior by changing plant defenses.The Asian citrus psyllid, Aphis spiraecola Patch and Aphis (Toxoptera) citricidus  exhibited positive and negative effects on the performance (feeding and reproduction) of Diaphorina citri Kuwayama. We explored the role of saliva in this plant-mediated interaction by infiltrating fresh and boiled aphid saliva into plants and detecting D. citri feeding behavior and citrus plant defense response. Leaf infiltration of A. spiraecola saliva disrupted the subsequent feeding of D. citri, indicated by prolonged extracellular stylet pathway duration and decreased phloem sap ingestion duration. By contrast, infiltration of A. citricidus saliva decreased the duration of the extracellular stylet pathway and phloem sap ingestion of D. citri. Furthermore, gene expression analysis showed that several salicylic acid (SA)- and jasmonic acid (JA)-pathway-related genes were activated by A. spiraecola saliva infiltration. However, two SA-pathway-related genes were activated and three JA-pathway-related genes were suppressed following A. citricidus saliva infiltration. Treatment with boiled saliva did not similarly impact D. citri feeding behavior or plant defense response. This study suggests that salivary components (those that can be inactivated by heating) from two citrus aphid species differently affect plant defenses and that they were responsible for the contrasting plant-mediated effects of two citrus aphids on the feeding behavior of D. citri. This study indicates a novel three-way citrus aphid–plant–citrus psyllid interaction.While herbivorous insect saliva plays a crucial role in the interaction between plants and insects, its role in the inter-specific interactions between herbivorous insects has received little attention. Pre-infestation of citrus plants with  Plant-initiated defenses against herbivores may reflect cues from herbivore-associated molecular patterns (HAMPs) present in oral secretions , oviposition secretions, cuticle, pheromones, or feces . These dMyzus persicae (Sulzer) secrete cysteine protease cathepsin B3 (CathB3) into tobacco plants, which activates a reactive oxygen species defense response and inhibits the feeding of M. persicae [M. persicae saliva or salivary components (in the range of 3–10 kDa) into Arabidopsis plants activates defense signaling and decreases M. persicae offspring number [Acyrthosiphon pisum (Harris) secretes a macrophage migration inhibitory factor (MIF) in its saliva, which inhibits the expression of defense-related genes, callose deposition, and hypersensitive cell death, and promotes aphid survival and feeding [Bemisia tabaci (Gennadius) salivary protein ferritin BtFer1, which inhibited JA-dependent signaling, and facilitated whitefly host exploitation [M. persicae [A. pisum [B. tabaci [Nilaparvata lugens (Stål) [Molecular studies of genes and proteins involved in plant–herbivore interactions suggest that insect saliva are primary HAMPs that trigger plant defenses . Piercinpersicae . The infg number . In addi feeding . Similaroitation . Despitepersicae , A. pisuA. pisum , B. taba. tabaci , and Nils (Stål) , which aDiaphorina citri Kuwayama is a primary pest of citrus due to its transmission of the pathogen causing Huanglongbing, a devastating and uncurable citrus disease [D. citri salivary gland gene expression and salivary secretions [D. citri and host plants has not been investigated. In citrus groves, D. citri are sympatric with other herbivorous insects, including citrus aphids [D. citri, many citrus aphids preferentially feed and develop on young shoots and leaves. We previously reported that the citrus aphids Aphis spiraecola Patch and Aphis (Toxoptera) citricidus  manipulated citrus phytohormone-dependent defenses in different ways [A. spiraecola and A. citricidus decrease and increase D. citri reproduction and feeding, respectively [The Asian citrus psyllid  disease . Althougcretions ,22,23, ts aphids . Similarent ways  and alsoectively . D. citri performance and that saliva from two citrus aphid species may differently affect plant defense responses. In this study, we collected saliva of citrus aphids using an artificial diet; then, saliva was infiltrated into citrus leaves to investigate subsequent effects on D. citri feeding and plant defense. The electrical penetration graph (EPG) technique was used to detect D. citri feeding behavior, and reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expression of genes involved in SA- and JA-dependent pathways. We hypothesized that the saliva of citrus aphids might play a role in the plant-mediated effect of citrus aphids on Citrus sinensis (L.) Osbeck) grafted on Citrus tangerine Tanaka rootstock, obtained from a nursery at Guangxi province, China, was used for experiments. Plants  were planted in 8 L plastic pots and maintained at 25 °C, 16 h light: 8 h dark, and 60–70% humidity. Plants were watered twice a week, fertilized weekly with 1 g/L NPK 20-10-20 , and pruned three weeks before experiments to promote flushing.Sweet orange  to prevent dispersion. D. citri were collected from Murraya paniculata (L.) Jack from Sun Yat-sen university at Guangzhou, China in 2019, and maintained on M. paniculata (in 8 L plastic pots) in a separate climate-controlled room. Plants and insects were maintained under the same conditions described above.A laboratory colony of eristics ,28. PartA. spiraecola or A. citricidus were infested on young citrus shoots (4 cm in length) containing flushing (not expanded leaves). Infested and uninfested control shoots were covered with an 80-mesh nylon net (one shoot per plant). Aphids were allowed to feed for 24 h, after which they were removed, and the prior infested and uninfested shoots used to quantify D. citri feeding behavior. Ten fourth instar A. spiraecola and A. citricidus), mixed life stages (starved for 12 h) were allowed to feed from 1 mL artificial diet (15% w/v sucrose) between two layers of Parafilm. The diet was sterilized with a millipore membrane filter (0.22 μm). Parafilm was stretched across a PVC tube (3 cm diameter and 4 cm height), and aphids were transferred into the tubes (150 aphids per tube). Equivalent tubes without aphids served as controls. The tubes were placed in a climate chamber , and the sucrose diet collected 48 h later. Some saliva was boiled at 95 °C for 30 min to inactivate proteins [C. sinensis leaves using a 1 mL syringe without the needle. For each leaf (2.5 cm in width and 5.5 cm in length), 200 µL diet was infiltrated at the abaxial side [D. citri feeding behavior analysis after 24 h. Aphid saliva was collected using the double layer membrane method . For eacD. citri were investigated using an electrical penetration graph (EPG) technique [D. citri at the pronotum with conductive silver glue, with the other side of the wire connected to the EPG amplifier. Another electrode was inserted into the soil at the base of the tested plant. The D. citri was placed on the abaxial side of the leaf, and tested plants were placed in a Faraday cage to prevent external electrical disruption. Only one leaf was used per plant. The stylet activity was recorded continuously for 8 h, with 13 replicates performed on aphid pre-infestation treatments, and 11 replicates performed for each saliva treatment.The effects of both direct aphid infestation and the aphid saliva infiltration on the subsequent feeding behavior of echnique . A gold D. citri [Psyllid feeding behaviors were quantified using a 4-channel direct-current EPG system  and the signals analyzed by Stylet+ software . Six waveforms are reported for D. citri : non-proD. citri ,33. TM Real-Time PCR System . The temperature protocol was: 95 °C for 30 s, followed by 40 cycles at 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 30 s. The expression of five genes involved in the SA pathway and five genes involved in the JA pathway were detected. CitGAPC1 was used as an internal control. Sequences and description of primers are listed in The saliva collection and infiltration treatments were as described in p < 0.05.One-way analysis of variance (ANOVA) and Dunnett’s test was used to compare different EPG variable values, as well as phytohormone-dependent defense-associated gene expression . Where necessary to meet assumption of normality and homogeneity of variances, data were log transformed. Results were considered significantly different at D. citri feeding on C. sinensis shoots varied, based on prior aphid infestation. For the number of waveform events per insect (NWEI), none of the six waveforms were significantly affected by A. spiraecola or A. citricidus pre-infestation when compared with controls (D. citri spent a significantly longer time on pathway phase (C), and less time on phloem ingestion (E2) on A. spiraecola pre-infested plants. However, pre-infestation by A. citricidus decreased the duration of C, and increased the duration of E2 and xylem ingestion (G), when compared with controls. The non-probing (Np), first phloem contact (D), and phloem salivation (E1) were not affected by any aphid pre-infestation when compared with controls  and controls were observed for C and E2 duration. The duration per insect of waveform Np, D, and E1 did not show significant differences between saliva treatments and controls, while G could not be statistically analyzed due to the low sample number  showed significantly increased expression after 6 h  were upregulated in C. sinensis leaves 24 h after infiltration with fresh A. spiraecola saliva when compared with control  at 24 h when compared with controls. However, A. citricidus fresh saliva did not affect JA pathway gene expression at 6 h after infiltration. In addition, exposure of C. sinensis shoots to boiled saliva of either aphid species did not result in significant changes in JA pathway gene expression relative to controls  saliva into wheat disrupted the feeding of subsequent infested S. avenae [A. spiraecola, different results were observed following A. citricidus feeding or saliva infiltration. In this case, the extracellular stylet pathway phase duration was decreased and the phloem ingestion duration was increased, suggesting better feeding efficiency. Although aphid saliva infiltration and aphid feeding caused a similar subsequent effect on D. citri feeding, there were some differences. For example, A. citricidus feeding increased G of WDI, and E2 and G of WDE, but A. citricidus saliva did not. Similarly, while A. citricidus saliva decreased C of WDE, the aphid feeding did not. This suggests that the plant–insect interaction is more complex than the saliva–plant interaction, and insect cues other than saliva may also modulate plant defense responses [A. spiraecola and A. citricidus fresh saliva on D. citri feeding were not observed when boiled saliva was used. This finding indicates that aphid salivary proteins may be responsible for the plant-mediated effects on D. citri. However, saliva also contains non-protein compounds, such as benzoic acid, phytohormones, and disulfooxy fatty acids [M. persicae to host plants, which promoted aphid fecundity [The EPG technique used in this study records the stylet activity and location of piercing–sucking insects in different host plant tissues, with such feeding behavior variables known as indicators of host plant suitability ,40. For sistance . Such di. avenae . By contesponses . Proteinesponses ,42. In tty acids ,44. Receecundity . TreatinA. spiraecola saliva activated the expression of two genes at 6 h and five genes at 24 h, while A. citricidus saliva only activated the expression of two genes at 24 h. For the genes involved in JA-dependent defense, A. spiraecola saliva activated the expression of one gene at 6 h and four genes at 24 h, while A. citricidus saliva inhibited three genes’ expression at 24 h. This indicates that A. spiraecola saliva-induced phytohormone-dependent defense is faster and stronger than that induced by A. citricidus saliva. This modulation of plant defense responses by saliva has also been reported in other piercing–sucking insects, including B. tabaci [S. avenae [M. persicae [A. spiraecola activated and A. citricidus repressed genes involved in JA-dependent defenses [A. spiraecola is polyphagous  [A. citricidus is oligophagous (feeding on members of Rutaceae) [The EPG data suggest that saliva from two citrus aphid species differently affected plant defense response. To verify this further, we examined saliva infiltration events on phytohormone-dependent defenses. For the genes involved in SA-dependent defense, . tabaci , S. aven. avenae , and M. persicae . Moreovedefenses . It was defenses . In thisus spp.) , while Autaceae) . This deS. avenae [D. citri feeding behavior and reduced its number of offspring [coi1 mutant, which has impaired JA defenses, accelerated B. tabaci nymph development [A. spiraecola saliva-infiltrated plants with activated JA-dependent defenses disrupted the feeding behavior of D. citri, while A. citricidus saliva-infiltrated plants with suppressed JA-dependent defense enhanced the feeding efficiency of D. citri. This indicates that citrus aphids may differently affect subsequently infested D. citri by inducing and repressing JA signaling through injecting saliva during feeding. Determining the JA phytohormone content may help elucidate the role of JA in this interaction. On the other hand, SA is less effective in conferring resistance against piercing–sucking insects, including D. citri [D. citri feeding behavior were different, suggesting that the SA pathways are less important when compared with the JA pathways.The SA- and JA-dependent defense influence plant resistance against herbivores, with JA signaling playing a central role . For exa. avenae . Foliar ffspring . By contelopment . ConsistD. citri . Some heD. citri . In thisD. citri in C. sinensis. Moreover, two citrus aphids’ saliva differently modulated the expression of genes involved in plant SA and JA defense pathways, and this difference may explain this contrasting effect. This study provides new information regarding the function of saliva in plant-mediated inter-specific herbivore interactions. Further study will focus on identifying the salivary components, as well as elicitor screening. This work may help develop plant defense inducers and develop D. citri-resistant cultivars.In summary, we showed that saliva was involved in the contrasting plant-mediated effects of two citrus aphids on the feeding behavior of


Mitochondria are critical organelles that form networks within our cells, generate energy dynamically, contribute to diverse cell and organ function, and produce a variety of critical signaling molecules, such as cortisol. This intracellular microbiome can differ between cells, tissues, and organs. Mitochondria can change with disease, age, and in response to the environment. Single nucleotide variants in the circular genomes of human mitochondrial DNA are associated with many different life-threatening diseases. Mitochondrial DNA base editing tools have established novel disease models and represent a new possibility toward personalized gene therapies for the treatment of mtDNA-based disorders. Mitochondria are semi-autonomous organelles essential for the correct functioning of eukaryotic cells . They arNuclear-mitochondrial genome interaction plays a key role in cellular metabolism ,5, as ovHeteroplasmic conditions linked to point mutations can cause various clinical manifestations, including mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), Leigh syndrome, and myoclonic epilepsy with ragged red fibers (MERRF), among others  Figure . MDs canPrimary mtDNA-based disorders are currently incurable . These dGene editing technologies can introduce targeted DNA modifications in cells or tissues to correct a genetic defect, and have already been successfully used to correct pathogenic mutations in the nuclear genome ,18. MitoBroadly, two distinct modalities are currently used for mtDNA manipulation: (i) nuclease-based and (ii) base editing approaches. Conceptually, nuclease-based methods can be utilized to decrease the amount of variant mtDNA in mitochondria by specifically targeting and cleaving the mutant mtDNA molecules ,23. ThisDespite their potential usefulness for shifting heteroplasmy, mitochondrially targeted nucleases are unable to correct variant genomes ,22. TherIn contrast to notable developments in nDNA manipulation since the repurposing of CRISPR as a gene editing platform ,42, precBurkholderia cenocepacia, revolutionized the field of mtDNA editing . F. Ftox  derived from COX8A, a FLAG tag, a TALE domain, a 2-amino-acid linker, a DddA and UGI A 39]. T. Ttox haCs positioned 4–7 nucleotides upstream of the 3′ end of the spacer . F. Ftox had T1413I ,73. Additox orientations [ZF-DdCBE design guidelines for mtDNA editing recommend using the v8 architecture and ZF scaffold X1 . For ZF ntations . Once anntations . Furtherntations .MT-TK in HEK293T cells with an efficiency of 31% [Nd1 in mouse C2C12 cells, with an efficiency of 39% [HS1 ZF-DdCBE pairs were packaged into single AAV2/9 vectors in order to facilitate in vivo mtDNA base editing in newborn P1 mice [Interestingly, an optimized 3ZF + 3ZF ZF-DdCBE pair with the AGKS scaffold was able to install the m.8340G>A pathogenic variant within y of 31% . Similary of 39% . Moreove P1 mice . Each ZF P1 mice . This st P1 mice . These r P1 mice . However P1 mice .In conclusion, comprehensively optimized ZF-DdCBEs were developed . These cTo better understand the genome-wide specificity of DdCBEs, Lei et al.  conductetox, or UGI to reduce the undesired nuclear localization of DdCBE pairs [A with canonical DdCBEs to inhibit their unwanted activity on nDNA [A is a naturally occurring protein that occludes the active site of DddAtox [tox to decrease its DNA binding affinity [A co-expression were the most effective strategies in preventing nDNA off-target mutagenesis [Lei et al.  tested tBE pairs . These mBE pairs . The sec on nDNA . DddIA i DddAtox . This st DddAtox . The thiaffinity . A globaagenesis .Nd5, was microinjected as mRNA into mouse zygotes, which were then cultured to the blastocyst stage [Lee et al.  tested tst stage . The spest stage . Additiost stage . Furtherst stage .Nd5 via DdCBE-NES, Lee et al. [TrnA and Rnr2 sites in mouse embryos. On average, DdCBE-NES resulted in higher on-target editing efficiencies than canonical DdCBEs at both target regions [In addition to installing the m.12918G>A mutation within e et al.  compared regions . Interes regions . No addi regions . Notably regions . However regions . It is a regions .In their report of a DdCBE library for the ablation of all protein-coding genes in mouse mtDNA, Minczuk lab  tested tAtp6-specific DdCBE pair or a corresponding T2A-linked construct [As hypothesized, the modified DdCBEs led to significant reductions in mtDNA off-target mutagenesis, as compared to canonical DdCBEs in vitro . Additioonstruct . Whole-gonstruct . Notablyonstruct .A co-expression [tox variants with reduced binding affinity [tox halves [tox halves with weakened associations [In summary, several strategies have been reported for limiting DdCBE-induced nuclear and mitochondrial off-target mutagenesis. These include NES addition ,74, DddIpression , incorpoaffinity , and HHRaffinity . Other saffinity , split-daffinity , truncatx halves , and incciations . Thus fatox protein. This unique nucleic acid deaminase proved to be capable of accepting dsDNA as a substrate, thus catalyzing cytidine deamination as an intrinsic activity—a combination of biochemical properties that had not been previously observed [tox. First, the intrinsic deaminase activity of DddAtox could be significantly impaired by mutating the active site (E1347A). Second, the fusion of a UGI molecule downstream of DddAtox led to a marked increase in net C-to-T editing efficiencies from this intrinsic cytidine deaminase, and a reduction in indel byproducts [Mougous lab  made a gobserved . Mok et observed  reportedproducts .tox or full-length DddAtox E1347A as potential dsDNA unwinders, and (4) TadA8e, an ssDNA-specific adenine deaminase variant enzyme derived from E. Coli  [C motif was completely flipped out of the double helix, potentially allowing DddAtox to locate it and deaminate it [These various TALED architectures demonstrated a dual functionality for the DddA protein . In partng dsDNA . Thus, tactivity ,40. In areported . The strThe single nucleotide sequence requirement inherent in the use of TadA8e means the potential off-target effects of TALED constructs will need to be critically evaluated before they can be utilized for human gene therapy. Whole mitochondrial genome sequencing detected notably low levels of off-target editing, at frequencies of 0.019% ± 0.002% for sTALEDs, 0.009% ± 0.001% for mTALEDs, and 0.008% ± 0.001% for dTALEDs . With reBase editing techniques for correcting point mutations in nuclear DNA have been extensively used in animal models ,81,82. TAnimal models are valuable tools for understanding the underlying molecular mechanisms of disease progression and developing new treatments. Multiple research groups have created various gene edited animal models of mitochondrial diseases ,87,88. MNd5 gene was associated with multiple MDs, such as MELAS, LHON, and Leigh syndrome [Nd5 gene [Lee et al.  microinjsyndrome ,95. AddiNd5 gene . The injNd5 gene . A totalNd5 gene . The autNd5 gene . HoweverNd5 gene . SimilarNd5 gene . They geNd5 gene . HoweverNd5 gene .A similar approach was used to create rat models of mitochondrial dysfunction . Qi et aMicroinjection of DdCBE-encoding mRNA into zebrafish embryos was reported by two groups, including ours, to create various disease models, such as MELAS and LHON ,49. ShenAtp6 gene, followed by selective breeding for several generations [MT-ATP6 mutations had heterogeneous clinical symptoms, as the ATP6 protein is a subunit of Complex V, and suboptimal expression resulted in impaired mitochondrial function [MT-ATP6 mutations, demonstrating the clinical relevance of in vivo models of mtDNA dysfunction [Animal models with knockouts of protein-coding genes in mtDNA are needed for the systematic and comprehensive investigation of mtDNA-related pathways. To this end, we previously developed TALE Writer, a computational tool for the efficient design of mitochondrial base editors, aimed at introducing premature termination codons in mitochondrial protein coding genes. This tool was tested in human and zebrafish mtDNA . Recentlerations . Patientfunction . The molfunction . It is wfunction . In the Despite recent progress, options for generating animal models of mitochondrial diseases remain limited. Notably, the TALED system has not yet been used to create such models. One potential avenue for future research is the creation of conditional models with inducible, organ-specific mtDNA editing systems to better understand the etiology of mitochondrial diseases. Where rodent models do not accurately model human mitochondrial diseases, it may soon be possible to generate nonhuman primate models with mtDNA mutations that more closely resemble human conditions. This possibility is supported by recent reports of DdCBE-mediated base editing in 3PN human embryos by two different groups ,52.In particular, Chen et al.  used DdCThe groundbreaking development of DdCBEs , the firPrecise editing of the human mitochondrial genome opens up the possibility of modeling mtDNA-based disorders, as well as developing gene therapies for a currently incurable set of diseases. Furthermore, the ability to make single edits in mtDNA could help improve our understanding of mitochondrial biology. For instance, introducing a premature stop codon to knock out a protein-coding gene could be of aid in elucidating the role of a specific mtDNA-related pathway on organismal homeostasis. To date, mtDNA base editing has been used to introduce a number of disease-associated variants in cultured human cell lines and animal models ,49,63,75tox E1347A and other accessory effector domains could broaden the scope of possible mtDNA modifications. Metagenomic approaches could aid in discovering additional dsDNA-specific deaminases with unique properties. It is worth noting that current mtDNA editing technologies are limited by bystander editing. For example, targeting the most common human mtDNA pathogenic variant, m.3243A>G in the MT-TL1 locus, remains challenging, given its sequence context, in which multiple bystander editing events are likely to occur. Thus, deaminase domains with unique sequence context preferences and more general strategies for the mitigation of bystander editing would significantly improve the applicability of mitochondrial base editors.In addition to advances on targeting scope, activity, specificity, and applications of existing mitochondrial base editors, developing new tools would be beneficial. Similar to TALEDs, novel technologies that rely on the combined activity between full-length DddAtox or developing tandem deaminase domains. Similar to the ZF scaffold engineering efforts reported for ZF-DdCBEs, it would be beneficial to further characterize or advance TALEs in the context of mtDNA binding. For example, it remains to be determined whether mtDNA base editors containing TALEs that can recognize any base at the 5′ terminus of a binding site are as efficient and specific as their canonical counterparts. Furthermore, the effects of TALE length on the activity and specificity of TALE-based mtDNA base editors have yet to be thoroughly examined. In addition to ZF and TALE engineering, alternative all-protein DNA-binding moieties could be fused to dsDNA-specific effector domains, such as DddAtox or other related molecules, to create alternative mtDNA base editors. These efforts would further expand the mitochondrial genome engineering toolbox.Thus far, advances in mtDNA base editing have remained focused on effector domain engineering, e.g., mutating DddAFinally, the elucidation of a reproducible pathway for the import of nucleic acids into the mitochondrial matrix would enable further mtDNA manipulation, including CRISPR-based editing and other approaches. Such an advancement would significantly democratize the field of mitochondrial genome engineering, catalyzing the development of technologies for basic research, disease modeling, and gene therapy. In the meantime, all-protein technologies will remain at the forefront of the mtDNA editing revolution.


H. pylori infection positivity rates, this review aims to assess the influence of vitamin D deficiency and/or insufficiency upon the prevalence of H. pylori infection and its eradication success. Three major databases were searched for articles that analyzed a relationship between vitamin D status and H. pylori infection. The literature search retrieved a total of 37 reports, after the article selection process. Hypovitaminosis D emerged as a potential risk factor for H. pylori infection, given the higher prevalence of vitamin D deficiency and/or insufficiency among H. pylori-positive subjects. Furthermore, the same type of micronutrient deficiency has been directly linked to H. pylori eradication failure. An inverse linear relationship between vitamin D status and gastric cancer risk exists, but the additional involvement of H. pylori in this correlation is still in question. The potential benefit of oral supplements in enhancing the success of classical therapeutic regimens of H. pylori still requires future research. Future population-based studies from larger geographical areas are warranted to address this subject in more depth.Taking into account previous data that sustain a relationship between vitamin D deficiency and higher  Vitamin D represents one of the essential micronutrients in the human body, the main function of which is to ensure calcium hemostasis and bone mineralization . AvailabHelicobacter pylori (H. pylori), a spiral-shaped Gram negative bacterium, is ubiquitously spread, infecting more than half of the world’s population [H. pylori represents a major causative factor of peptic ulcer disease, an important precursor lesion of gastric cancer, and possesses virulence factors such as cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A, as well as various outer membrane proteins which have been directly linked to the activation of pathways involved in aberrant cell proliferation and gastric carcinogenesis [H. pylori infection, but not without treatment-associated side effects [H. pylori eradication failure represents a threat to human health, which can lead to a higher incidence of gastric cancer [H. pylori therapeutic failure, with studies proposing its supplementation as an adjuvant to standard medication [H. pylori positivity rates seem to be higher among populations with low serum vitamin D levels [H. pylori infection has been found that constitutes grounds for further research.pulation . H. pyloogenesis ,17. Henc effects . The incc cancer . Vitamindication ,21. MoreD levels ,23. ThusH. pylori infection, its complications, and its eradication success.This review aims to highlight the association between vitamin D deficiency and/or insufficiency and the prevalence of H. pylori infection. Search terms included “Helicobacter pylori” OR “Helicobacter” AND “vitamin D” OR “cholecalciferol” OR “calcitriol”. The inclusion criteria were human-based population studies, literature data published in the English language, and research articles that analyzed a correlation between serum vitamin D levels and H. pylori incidence and therapeutic management. Papers in-extenso were included, consisting of randomized controlled trials (RCTs), prospective cohort studies, retrospective cross-sectional studies, and longitudinal studies. We excluded studies that did not meet our article objective, case reports, editorials, review articles, and meta-analyses, as well as non-English records, articles without available abstracts, and duplicates/triplicates. Furthermore, abstracts and proceeding papers were excluded from our search results due to the insufficiently detailed information regarding the characteristics of the study population included, diagnostic methods applied for H. pylori infection, and the eradication regimen used.Web of Science, Scopus, and PubMed databases were searched for articles published until 15 April 2023 that analyzed a relationship between vitamin D status and The Rayyan web app aided in the article selection process. Initially, duplicates and triplicates were removed, and afterwards each of the authors examined the records’ titles and their abstracts in order to exclude irrelevant articles. Authors SM and KAM assessed the full-length text of each manuscript to ensure its compliance with inclusion criteria. Potential disagreements between authors were debated and discussed by all authors, who afterwards mutually agreed with the inclusion of each individual record within this review.H. pylori infection.The search retrieved a total of 465 records, which further translated into 319 results after the exclusion of duplicates. The article selection process has been represented in more detail in H. pylori infection [H. pylori infection according to two studies conducted on Turkish and Lebanese adult populations [H. pylori infection prevalence, which suggested that adequate vitamin D levels might offer protection against H. pylori [H. pylori infection increases by 1.1 [H. pylori was proposed, suggesting that H. pylori might actually be the risk factor for vitamin D level decrease [H. pylori colonization of the gastric mucosa. Therefore, vitamin D intake is probably influencing the prevalence of this particular infection [Among various dietary habits, decreased vitamin D intake has emerged as a possible concern related to nfection . Vitaminulations ,27. More. pylori . Furthers by 1.1 . In a stdecrease . Howevernfection .H. pylori infection and concluded that those individuals with vitamin D values below 20 ng/mL stand the best chances of acquiring this particular bacterial infection [H. pylori, despite including a significant number of patients with levels under 20 ng/mL among the control group as well [H. pylori infection was put into question. Other authors proposed that vitamin D insufficiency, rather than deficiency, is the major risk factor for H. pylori infection [H. pylori serum antibody positivity only in patients with vitamin D deficiency, while the presence/absence rate of antibodies directed against the same bacterial infection was not influenced by vitamin D insufficiency [Shafrir et al. found an inverse association between vitamin D serum levels and nfection . However as well . Hence, nfection . In contficiency .H. pylori infection. One study that analyzed the impact of concomitant vitamin D deficiency and H. pylori infection upon the prevalence of metabolic syndrome concluded that vitamin D levels were similar among the enrolled individuals, independent of H. pylori infectious status. The authors reported that those subjects with both H. pylori infection and vitamin D deficiency were the most susceptible to developing metabolic syndrome [H. pylori infectious status, confirmed through analysis of gastric mucosa specimens [H. pylori. However, these differences did not reach statistical significance [Several studies failed to identify an association between serum vitamin D levels and syndrome . Furtherpecimens . Contradificance .H. pylori co-infection in the setting of other infectious diseases or chronic conditions seems to be additionally associated with vitamin D deficiency. One study conducted on 800 dengue fever patients, divided into two equal groups based on H. pylori co-infection status, revealed that those individuals who were positive for the infection presented significantly lower vitamin D serum levels than dengue fever controls [H. pylori immunoglobulin G (IgG) serum antibodies was discovered [H. pylori specific antibodies, vitamin D levels were significantly lower than in healthy controls [H. pylori stool antigen test, significantly lower values of vitamin D levels were reported [controls . In patiscovered . Moreovecontrols . Similarreported .H. pylori infection rates within their pediatric study group [H. pylori colonization [H. pylori infection in infants and toddlers. This study assessed H. pylori infection based on serum antibody positivity, which cannot distinguish present from past H. pylori infection [H. pylori was performed, revealed that vitamin D levels are significantly higher in children with with non-H. pylori gastro-duodenal conditions, as opposed to infected individuals. Moreover, the same study identified an inverse association between vitamin D levels and CagA positive H. pylori strains, which are known to increase gastric cancer risk even further [H. pylori, have been depicted in In children, results have been scarce, contradictory, and did not match those obtained in adult populations. Agin et al. found an association between lower vitamin D levels and peptic ulcer in children, but concluded that vitamin D levels did not significantly impact dy group . Moreovenization . One stunfection . Another further . This fi further . The stuH. pylori eradication failure [H. pylori positive patients have been linked to vitamin D sufficiency, which translated into serum levels exceeding at least 20 ng/mL. Significantly lower mean serum vitamin D levels have been reported among patients with therapeutic failure [H. pylori eradication success [H. pylori infected patients that responded to the same therapeutic regimen and those who did not [H. pylori infection in more than 80% of patients with vitamin D levels below 30 ng/mL [H. pylori eradication failure [3 supplements to a classical regimen consisting of PPPIs, amoxicillin, and clarithromycin, and to report superior eradication rates of H. pylori [Vitamin D deficiency has been regarded as a risk factor for  failure ,45. The  did not . Similar30 ng/mL . However failure . Therefo. pylori .H. pylori eradication. In the study of Yildirim et al., over 80% of the patients in whom H. pylori eradication attempt was unsuccessful had serum vitamin D levels below 10 ng/mL [H. pylori eradication rates in patients with vitamin D levels under 10 ng/mL and the best therapeutic success rates for populations with vitamin D serum values ranging between 30 and 34.9 ng/mL [H. pylori eradication success [A lower vitamin D serum level of 10 ng/mL was proposed as referential for its impact on 10 ng/mL . Another.9 ng/mL . This 10H. pylori infection/positive serum antibodies [H. pylori therapeutic success in patients with type 2 diabetes mellitus. Vitamin D levels under 20 ng/mL significantly negatively impacted H. pylori eradication, whereas levels ranging between 20 and 30 ng/mL did not influence therapeutic success. Furthermore, the authors acknowledged that vitamin D deficit might also have enhanced dyslipidemia in their study cohort, which, together with prolonged evolution of the glycemic imbalance, could have also contributed to eradication failure [As previously described, lower vitamin D levels have been found in patients with diabetes and tibodies ,39. Howe failure .H. pylori eradication rates are currently limited to a few studies. Zhang et al. found the lowest vitamin D serum values among a pediatric population with H. pylori recurrence, despite adequate treatment. Within their study, significantly higher values of the same parameter were found in those patients in whom bacterial eradication was successful and in healthy controls, but each of the analyzed study groups had a low percentage of vitamin D-deficient and -insufficient individuals [H. pylori eradication success are still very limited. Only one population-based study has managed so far to identify a correlation between a higher vitamin D intake, positively correlated with a higher dietary fish intake, and efficacy of H. pylori eradication [In children, data on the role of vitamin D in ividuals . Sorokmaividuals . In factdication .H. pylori infection, recent research tried to evaluate the impact of VDR gene’s polymorphisms as well. Martins et al. found a differential distribution of BsmI genotypes of the VDR gene within a study population, divided based on H. pylori positivity within gastric mucosa biopsy specimens. The other three polymorphisms studied—namely FokI, ApaI, and TaqI—presented no significant genotype distribution variation among the two study groups [H. pylori infection was found [H. pylori induces increased expression of VDRs, which have been shown to play an important role in innate immunity and to render antimicrobial activity. One study conducted on adult patients showed that cells of gastric mucosa specimens harvested from H. pylori-infected patients presented a significant elevation in VDR expression, as opposed to healthy controls . Startiny groups . Howeveras found .H. pylori [H. pylori infection [H. pylori gastritis, a group of patients diagnosed with non-specific lymphocytic gastritis, and a control group of healthy subjects. The study group of patients with atrophic, autoimmune-induced gastritis presented the lowest vitamin D levels, followed by the H. pylori gastritis group, which also exhibited significantly lower values of serum vitamin D than the other two study groups [H. pylori infection [Vitamin D intake seems to decrease gastric cancer risk, as reflected through a study conducted on a Vietnamese population. The same study also revealed that vitamin D intake is inversely associated with both positive and negative IgG serum antibody titers directed against . pylori . Thus, tnfection . The posy groups . Contrarnfection .H. pylori infection has been associated with a decrease in serum levels of various vitamins, including vitamin B12, vitamin C, and vitamin D. Higher serum vitamin D and vitamin B12 levels were warranted to improve H. pylori eradication rates, whereas vitamin C supplementation helped achieve the same result [H. pylori infection. Despite the heterogenicity of the included studies, the authors concluded that lower vitamin D levels might account for a higher prevalence of H. pylori infection and might negatively influence bacterial eradication [H. pylori infection occurrence and its subsequent eradication. As seen in H. pylori infection and eradication rates. However, despite the important number of relevant studies on this topic, their heterogeneity cannot be neglected and hinders the performance of a meta-analysis. Thus, H. pylori detection methods vary greatly, whereas the data were mostly limited to Asian studies, which might significantly impact the interpretation of results due to the miscellaneous dietary and nutrition patterns across the globe. Moreover, a handful of studies found no significant relationship between vitamin D and H. pylori, and very few of the studies included in this review assessed the importance of vitamin D supplementation [H. pylori, and to evaluate whether the addition of vitamin D3 to therapeutic regimens influences bacterial eradication. Cofounding factors such as age, interfering in the relationship between vitamin D supplementation and H. pylori prevalence and eradication, are also subject to future debate, given the lack of regular vitamin D intake among adult populations and the possible inverse relationship between H. pylori and vitamin D deficiency, as proposed by Bahși et al. [One meta-analysis of 48 studies pointed out that e result . This mee result . One othdication . Similarentation ,35,49,52entation ,42,43,51i et al. .H. pylori in mice, the administration of oral vitamin D3 produced a significant decrease in colonization rates, as well as an upregulation of VDRs [H. pylori activity of vitamin D3 is further enhanced by the activation of the VDR-Cathelicidin antimicrobial peptide (CAMP) pathway [H. pylori through the autolysosomal pathway [H. pylori [H. pylori action through induction of the bacteria’s cell membrane structure collapse [H. pylori has been proposed, after the compound was proven to protect against H.pylori-induced apoptosis of gastric epithelial cells [H. pylori infection and in facilitating its eradication. Pediatric studies could provide more insight into this matter, given that vitamin D supplements are routinely administered in children [Our review excluded experimental data, due to the significant abundance of clinical data and the limited number of studies that included animal populations. However, some important aspects can be retained from experimental studies as well, which might aid future clinical research. After artificial infection of the stomach with  of VDRs ,61. The  pathway ,61. Anot pathway . Further. pylori ,64. Morecollapse . A syneral cells . This thal cells . Thus, ichildren . The datchildren ,42,43,51H. pylori infection, still requires more research. Two review articles clearly underlined an inverse correlation between vitamin D serum levels and gastric cancer risk, morbidity, and mortality, but most of the studies included in these articles did not perform a separate analysis on H. pylori infection status [H. pylori, might present lower vitamin D serum levels, but research on this matter needs to be expanded [The link between vitamin D intake and gastric cancer risk, while taking into consideration the presence/absence of n status ,70. As pexpanded ,57. Furtexpanded ,70.H. pylori infection. The bacteriolytic action of hydrophobic lipid structures, such as indene compounds, vitamin D, and 3-carbonyl steroids, has been recently highlighted [H. pylori’s survival, pathogenicity, and eradication, vitamin D distinguished itself as an important regulator of host immune response [H. pylori treatment based on vitamin D supplements [A few previous reviews also provided some important take-home messages on the role of vitamin D in hlighted . Among mresponse . Hence, plements . HoweverH. pylori infection and its eradication failure. Vitamin D-deficient subjects might be more prone to developing H. pylori infection, but the influence of H. pylori infection upon vitamin D status is still puzzling. The role of vitamin D intake is still in question, as most of the reviewed studies failed to analyze whether the addition of oral supplements influences the performance of current therapeutic regimens. Thus, the screening of vitamin D levels in subjects with H. pylori infection might establish the need for its supplementation in ensuring therapeutic success. Vitamin D deficit might be connected to an increase in gastric cancer risk, but the additional influence of H. pylori in this linear relationship is still unclear. Furthermore, the currently available data are hindered by the enrollment of populations from restricted geographical regions, as well as by the heterogenicity of H. pylori infection detection methods and the reduced number of pediatric populations included so far. Future population-based studies and randomized clinical trials from larger geographical areas are warranted to address this subject in more depth and to analyze the synergistic effect of vitamin D supplementation and classical therapeutic schemes.This comprehensive review highlights the impact of vitamin D deficiency and/or insufficiency upon an ascending prevalence of


Salvia miltiorrhiza (S. miltiorrhiza) have not been thoroughly investigated. In this study, we employed bioinformatics methods to identify 23 Aux/IAA genes within the genome of S. miltiorrhiza. These genes were classified into typical IAA and atypical IAA based on their domain structure. Our analysis of the promoter regions revealed that the expression of these genes is regulated not only by auxins, but also by other hormones and environmental factors. Furthermore, we found that the expression patterns of these genes varied across various tissues of S. miltiorrhiza. While our initial hypothesis suggested that the primary function of these genes was the interaction between SmIAA and ARF, gene co-expression network analysis revealed that they are also influenced by various other transcription factors, such as WRKY and ERF. The findings establish a sturdy basis for future investigations into the function of the Aux/IAA gene family and exhibit promising prospects for enhancing the genetics of this medicinal flora and its associated species.The auxin/indole-3-acetic acid (Aux/IAA) gene family serves as a principal group of genes responsible for modulating plant growth and development through the auxin signaling pathway. Despite the significance of this gene family, the identification and characterization of members within the well-known Chinese medicinal herb  Auxin, the first identified plant hormone, is present ubiquitously in all plants and participates in every stage of plant growth and development . Indole-TIR1 complex breaks down the dimer formed by Aux/IAA protein and ARF protein through ubiquitin  contains 29 IAA proteins (Oryza sativa (O. sativa) contains 31 members (Solanum tuberosum (S. tuberosum) and Vitis vinifera (V. vinifera) all contain 26 members , a member of the genus Salvia within the Labiatae family, is well known for its dried roots and rhizomes, which serve as a traditional Chinese medicine . Subsequently, we performed bioinformatic analyses to evaluate the physicochemical properties and sequence features of these genes. In addition, we utilized RNA-seq data to investigate the tissue-specific expression profiles of the SmIAAs, and further established a co-expression network to uncover the co-expression relationships between the SmIAAs and transcription factors.In the present investigation, we executed a comprehensive analysis of the Aux/IAA gene family in A. thaliana and O. sativa were downloaded from The Arabidopsis Information Resources (TAIR) database and Rice Genome Annotation Project (RGAP) database respectively. The genomic data of S. miltiorrhiza was downloaded from National Genomics Data Center (NGDC) database (project number GWHAOSJ00000000).The Aux/IAA protein sequences of SmIAAs. First, we utilized the A. thaliana Aux/IAA protein sequence as a seed sequence for BLASTP homologous alignment in the S. miltiorrhiza local database. We then validated the domain structure of the resulting sequences using the Pfam database  approach implemented in MEGA X software (http://web.expasy.org/protparam/) and predicted their subcellular localization using the WoLF PSORT software (https://wolfpsort.hgc.jp/). To further investigate the chromosomal distribution of SmIAAs, we obtained relevant information from the S. miltiorrhiza genome annotation file and visualized it using the MG2C software (http://mg2c.iask.in/mg2c_v2.0/).To explore the evolutionary relationships among software , employiSmIAA gene sequence was analyzed by TBtools software according to the GFF3 file and CDS file of S. miltiorrhiza (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi). Finally, Tbtools software was used to visually analyze the results , with the results of this analysis being visualized through TBtools software, as detailed in The structure of iorrhiza . The pro results . Furtherhttps://www.evolgenius.info/evolview/) (https://meme-suite.org/meme/), was used to assess conserved motifs.In order to explore the genetic and evolutionary relationships among members of the Aux/IAA gene family in various species, a multiple sequence alignment of 23 SmIAA proteins, 29 AtIAA proteins, and 31 OsIAA proteins was performed using the online program Clustal Omega, followed by MEGAX to create the phylogenetic tree , with tholview/) . MEME, aSmIAAs. The MCScanX software, developed by SmIAAs. For each identified homologous gene pair by MCScanX, the synonymous substitution rate (Ks) and non-synonymous substitution rate (Ka) were computed using KaKs_Calculator software . The received sequence read archive (SRA) files were first converted to the fastq format using the fastq-dump program. The downloaded raw data was then trimmed and preprocessed using TrimGalore , and the resulting clean data was utilized for downstream analysis. To construct a genome index with default settings, the Hisat2-build command in Hisat2 (version 2.3.0) (SmIAAs was extracted and a corresponding heat map was generated using the TBtools software (The RNA-seq data of n 2.3.0)  was usedn 2.3.0) . Finallysoftware .Salvia miltiorrhiza using the iTAK online software developed by Transcription factors were identified in the entire protein sequence of S. miltiorrhiza seedlings were collected and frozen in a −80 °C refrigerator. The roots, stems, leaves, and flowers were used for specific expression detection. Fluorescent quantitative specific primers  and reverse transcribed using the PrimeScriptTM reverse transcriptase kit . Real-time PCR reactions were performed using the PerfectStart Green qPCR SuperMix Kit , and fluorescence signals were collected using a qTOWER 3G PCR instrument. Each sample was analyzed in triplicate, and relative expression was calculated using the 2−ΔΔCT method (http://sangerbox.com/) (Two-year-old  primers  were desT method . The corox.com/) .S. miltiorrhiza. The initial approach involved using Arabidopsis Aux/IAA protein sequences to conduct a BLASTp search on the proteomic data of S. miltiorrhiza. Subsequently, the results were subjected to domain detection via submission to the Pfam database , while the lowest was on chromosomes 6 and 8 (one gene). Moreover, the genes on chromosomes 2 and 5 were more densely distributed compared to those on other chromosomes, which were more sparsely and randomly distributed .The GO categorization system is a widely used method for characterizing genes and proteins in various organisms. The system is organized into three distinct categories, including molecular function, biological process, and cellular component, with each category corresponding to specific functions or properties. To investigate the potential functions of the SmIAAs are associated with 10, five, and three GO terms related to biological process, cellular component, and molecular function, respectively. The majority of SmIAAs are believed to play a role in various biological activities. Their molecular functions include transcription regulator activity, DNA-binding transcription factor activity, and identical protein binding. The cell composition mainly includes the nucleus, nuclear, intracellular membrane-bounded organelle, membrane-bounded organelle, and other cellular components. Furthermore, SmIAAs are primarily involved in biological processes related to the response to auxin, organic substance, endogenous stimulus, and hormone, among others.SmIAAs in the growth and development of S. miltiorrhiza, we analyzed the cis-acting elements in the promoter region of the related genes, which is 2,000 bp upstream of the initiation codon  (SmIAA4 and SmIAA6) that were primarily expressed in the leaves of S. miltiorrhiza. Group B included four genes  that showed similar expression levels in the leaves, stems, and flowers of S. miltiorrhiza, and relatively low expression levels in the roots. Group C comprised five genes  that were predominantly expressed in the flowers and roots of S. miltiorrhiza. Group D encompassed six genes  that showed the highest expression levels in the stems, roots, and flowers of S. miltiorrhiza, but minimal expression in the leaves. Group E consisted of three genes  that were primarily expressed in the stems and roots of S. miltiorrhiza. However, the three genes in group E  were significantly expressed only in the stem of S. miltiorrhiza. In summary, SmIAAs were expressed in clusters in various tissues of S. miltiorrhiza, with the majority of SmIAAs exhibiting high expression levels in roots, stems, and particularly flowers.The temporal and spatial expression of a gene may provide insights into its functional features . In thisps (A~F) . Group AARFs  to examine additional transcriptome data from S. miltiorrhiza flower tissue at different stages . Our analysis identified 68 TFs, including ARF, ERF, MYB, and others , suggesting that SmIAAs may have other important functions beyond their role in the auxin pathway. However, more research is needed to confirm this hypothesis.Although the Aux/IAA gene family is crucial for auxin signal transduction, prior studies have suggested that its members may interact with other transcription factors (TFs) besides ARFs . To expld others  and S6. d others . The outd others . Furtherub genes . We seledatabase . SurprisSmIAAs, four hub genes selected by co-expression modules, and four transcription factors chosen randomly  technique to evaluate the expression levels of 12 genes, comprising of four randomly . The finS. miltiorrhiza. On the whole, the number of SmIAAs is similar to that of Aux/IAA gene family members in A. thaliana , so it is inferred that the transcriptional inhibitory function of these six members of S. miltiorrhiza on downstream ARF may be missing  to regulate stem growth, while SmIAA22 and the DBB (EVM0017797) were observed to antagonize each other and regulate root development. These results suggest that SmIAAs perform various unknown functions in different tissues and during different growth and development processes of S. miltiorrhiza. The outcomes of this research provide a foundation for further elucidation of the function of the SmIAAs and offer valuable resources for improving the characteristics of S. miltiorrhiza and breeding new S. miltiorrhiza varieties through genetic engineering.In this study, a comprehensive analysis was conducted on the Aux/IAA gene family in 10.7717/peerj.15212/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.15212/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.15212/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.15212/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.15212/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.15212/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.15212/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.15212/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.15212/supp-9Supplemental Information 9Click here for additional data file.10.7717/peerj.15212/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.15212/supp-11Supplemental Information 11Click here for additional data file.10.7717/peerj.15212/supp-12Supplemental Information 12Click here for additional data file.


Lipoprotein(a) (Lp[a]) is a risk factor for cardiovascular disease. The burden of thrombus in ST-segment elevation myocardial infarction (STEMI) has implications on treatment and outcomes. However, the association between Lp(a) and atherothrombosis in STEMI remains unclear.The aim of the study was to determine the association between Lp(a) and culprit artery thrombus burden in younger patients with STEMI.This was a single-center study of 83 patients aged <65 years with STEMI between 2016–2018 who underwent percutaneous coronary intervention and measurement of Lp(a); those receiving thrombolytic therapy were excluded. Thrombus burden in the culprit artery was determined angiographically using the Thrombolysis In Myocardial Infarction score and classified as absent-to-small, moderate, or large. Elevated Lp(a) was defined as plasma mass concentration >30 mg/dL. Multivariate analysis was performed adjusting for cardiovascular risk factors.p = 0.024). Elevated Lp(a) was associated with moderate or large thrombus in univariate  and multivariate analysis . Lp(a) was not associated with culprit artery or stenosis location according to culprit artery.The mean age was 48.0 ± 8.4 years, and 78.3% were male. Thirteen (16%), 9 (11%), and 61 (73%) patients had small, moderate, or large thrombus burden, respectively, and 34 (41%) had elevated Lp(a). Elevated Lp(a) was associated with greater thrombus burden compared to normal Lp(a)  is associated with greater thrombus burden in younger patients with STEMI. The finding of this observational study accords with the thrombotic and anti-fibrinolytic properties of Lp(a). A causal relationship requires verification. Lipoprotein(a) (Lp[a]) is a genetically determined low-density lipoprotein (LDL)-like particle bound covalently to apolipoprotein(a) (apo[a]) . MendeliAt present, there is limited literature evaluating Lp(a) levels and its association with coronary artery thrombosis in vivo . In the Patients who presented with STEMI between January 2016 and December 2018, who underwent primary PCI, were aged <65 years, and who had a Lp(a) measurement performed during the index admission were retrospectively identified by case record review at Royal Perth Hospital, Australia. Approval was granted by the Local Institutional Review Board. This age group was chosen to study the effects of Lp(a) in patients with premature coronary artery disease. In the year 2016, Lp(a) was measured in consecutive patients over a 6-month period for a separate study. Lp(a) measurement was performed using an automated latex-enhanced immunoassay (Quantia Lp[a]) on an Abbott Architect c16000 Analyzer . This assay employs a rabbit polyclonal antibody and is sensitive to variation in apo(a) isoform size.PCI for STEMI is performed by certified cardiologists with training in intervention, and coronary angiography is performed using standard techniques and angulated views. Images were reviewed retrospectively by a cardiologist blinded to Lp(a) results. The burden of thrombus in the culprit coronary artery was determined on angiographic images performed prior to PCI, using the validated Thrombolysis In Myocardial Infarction (TIMI) thrombus grade, and classified as absent to small, moderate, or large depending on the angiographically apparent luminal filling defect . A priort test, Pearson's χ2 test, or Fisher's exact test. An elevated Lp(a) was prospectively defined as a plasma mass concentration >30 mg/dL, as this level has been shown to be associated with increased risk of myocardial infarction [p value <0.05.Statistical analyses were performed using SPSS . Descriptive data are presented as number (%) or mean ± standard deviation, as the Kolmogorov-Smirnov test was not significant for the continuous variables. Differences between groups were analyzed using Student's farction . As Lp(an = 65 (78%) male were included. Elevated Lp(a) was detected in 34 (41%). Baseline characteristics stratified according to Lp(a) status are presented in Table Eighty-three patients with mean age 48.0 ± 8.4 years and d in 34 4%. Baselip = 0.024), where large thrombus burden was seen in 85% with versus 65% of those without elevated Lp(a). Log-transformed Lp(a) was associated with an increased risk of moderate or large thrombus burden in univariate  and multivariate analysis (Table p = 0.037). Elevated Lp(a) was associated with increased risk of moderate or large thrombus burden in univariate analysis  and multivariate analysis (Table p = 0.034). There were no significant associations between elevated Lp(a) and left ventricular ejection fraction, culprit artery, or location of stenosis according to coronary artery segment.Overall, 13 (16%), 9 (11%), and 61 (73%) patients had small, moderate, and large burden of thrombus in the culprit artery, respectively. Patients with elevated Lp(a) were more likely to have greater severity of thrombus burden p = 0.04, where This study is the first to report that elevated levels of Lp(a) are associated with a greater thrombus burden in the culprit coronary artery in patients presenting with STEMI. This supports the hypothesis that arterial thrombus formation following plaque rupture in STEMI may be influenced by Lp(a).A previous study reported that higher Lp(a) levels were associated with lower coronary artery patency rates in patients with STEMI not treated with reperfusion therapy, while another study found an association with lower spontaneous recanalization of the infarct artery on diagnostic angiography prior to primary PCI in patients with STEMI , 8. InteThe current findings may have potential clinical implications with respect to the intensity and duration of anti-platelet or anti-thrombotic therapies for patients with STEMI and elevated Lp(a). A pro-thrombotic state contributes to residual cardiovascular risk. In a large retrospective registry, patients with stable coronary artery disease and Lp(a) >30 mg/dL were found to have reduced cardiovascular events with prolonged duration (>1 year) of dual anti-platelet therapy after PCI . ClinicaThe results of the current study provide further support for the routine measurement of Lp(a) in younger patients with STEMI. An Lp(a) level of 30 mg/dL is approximately the 75th percentile in white populations, but elevated Lp(a) (defined as >30 mg/dL) was detected in 41% of patients in this Australian study [Limitations of this study include its small sample size and retrospective analysis at a single center. In addition, Lp(a) was not measured routinely in all patients with STEMI during the study period; thus, this cohort may be affected by selection bias. Lp(a) was measured at a single time point, as Lp(a) concentration is considered relatively stable throughout one's lifetime . HoweverIn conclusion, an elevated Lp(a) level is associated with greater thrombus burden in culprit arteries in younger patients presenting with STEMI. The finding of this observational study concurs with the pro-thrombotic and anti-fibrinolytic properties of Lp(a) and may have important clinical implications if a causal relationship is verified.The study complies with the Declaration of Helsinki and has been approved by the Royal Perth Hospital Institutional Review Board (GEKO 40393). The need for informed consent was waived due to retrospective analysis of de-identified data.NSRL has received research funding from Sanofi as part of a Clinical Fellowship in Endocrinology and Diabetes, education support from Amgen, Bayer, Boehringer Ingelheim, and Eli Lilly, and speaker honoraria from Sanofi, Boehringer Ingelheim, and Eli Lilly. GFW has received honoraria for advisory boards and research grants from Amgen, Arrowhead, Esperion, Novartis, Pfizer, Sanofi, and Regeneron.This study was not funded.Conceptualization and design, and methodology and project administration: D.M.S. and C.J.S. Data curation: D.M.S., P.G., E.Z., and S.R. Formal analysis: N.S.R.L. and D.C.C. resources: G.S.H., D.C.C., J.P., G.F.W., and C.J.S. Interpretation of data: D.M.S., N.S.R.L., D.C.C., J.P., G.S.H., G.F.W., and C.J.S. Writing − original draft: D.M.S. and N.S.R.L. Writing − review and editing: All authors.Data are not publicly available due to ethical reasons. Further inquiries can be directed to the corresponding author.