G-quadruplex, a structurally unique structure in nucleic acids present all throughout the human genome, has sparked great attention in therapeutic investigations. Targeting G-quadruplex structure is a new strategy for the drug development. Flavonoids are found in almost all dietary plant-based beverages and food products; therefore, they are ingested in significant proportions through the human diet. Although synthetically developed drug molecules are used vigorously but they have various adverse effects. While on the other hand, nature supplies chemically unique scaffolds in the form of distinct dietary flavonoids that are easily accessible, less poisonous, and have higher bioavailability. Because of their great pharmacological effectiveness and minimal cytotoxicity, such low molecular weight compounds are feasible alternatives to synthetic therapeutic medicines. Therefore, from a drug-development point of view, investigation on screening the binding capabilities of quadruplex-interactive small natural compounds like dietary flavonoids are expected to be highly effective, with a particular emphasis on the selectivity towards polymorphic G-quadruplex structures. In this respect, quadruplexes have scintillated research into their potential interaction with these dietary flavonoids. The purpose of this review is to offer an up-to-date close-up look at the research on their interaction with structurally varied dietary flavonoids with the goal of providing newer perspectives to construct novel therapeutic agents for next-generation disease managements.  •Humans consume considerable amounts of dietary flavonoids through their diet, for making them viable substitutes for conventional medicinal drugs.•Functional characterization and novel interactions between different types of dietary flavonoids with G-quadruplexes has been explored.•Interactions between G-quadruplex DNA structures and dietary flavonoids studied as potential molecular targets for prospective disease control.•Enlighten the structural variation between dietary flavonoids with respect to their binding to G-quadruplex DNA structures for providing knowledge that can be used to develop novel drugs for next-generation disease managements. A G. A G7]. Generally, cellular DNA exists in the B-form, with the regular H-bonding as seen in the Watson-Crick DNA model. Alternative nucleic acid structure that differs from double stranded structure have essential biological functions. G-quadruplexes are common in the human genome, and also in viruses, bacteria and plants. The distribution of G-quadruplexes shows regulatory functions in a variety of cellular activities such as recombination, replication, transcription, translation, and telomere preservation. Furthermore, the polymorphism of G quadruplexes makes it difficult to interpret and design particular recognition of appropriate structure by ligands. The polymorphism results from alterations in strand stoichiometry, location and orientation of the loops linking guanines, and reciprocal orientations of G-tracts in the structure's interior . In humaGenerally, naturally available plant chemicals have been shown to be extremely effective anti-cancer drugs that are far less toxic than synthesized derivatives, and they are commonly utilized in a variety of pharmacological functions with greater biological benefits. Flavonoids are polyphenolic substances that naturally occur in medicinal plants, vegetables, fruits, flowers, and a range of beverages  ,12. FlavBased on the existing research, in this review, we highlighted and explored the interactions between several types of dietary flavonoids with G-quadruplexes. This systematic review demonstrates that the structural variations between dietary flavonoids cause these chemicals to interact differently with distinct DNA structures. Furthermore, the goal of this review is to provide a structural underpinning for the association and maintenance of the G-quadruplexes by the most prevalent naturally occurring dietary flavonoids. This study will serve as a solid foundation for future research and the production of safer, more effective medications for a wide range of diseases.2According to a bioinformatics study, the human genome contains 700,000 potential G-quadruplex producing sequences . The fasA resulted in much reduced replication origination performance as compared to wild type cells [G-quadruplex forming sequences were discovered in telomeric DNA of humans for the first time. The stability as well as structure of telomeres are linked to aging, cancer, and genetic stability. G-quadruplex works as a tumour suppressor in telomeres by suppressing telomerase and so preserving the telomere . Telomerpe cells .Fig. 2ScIn-silico investigations revealed that G-quadruplex motifs are typically found in the promoter sequences of over 20,000 genes [00 genes . A poten00 genes . In most00 genes . Likewis00 genes ,26.Stylonychia lemnae provided direct indication for the presence of G-quadruplexes at telomere regions. The prescriptive and quantifiable visualisation of DNA G-quadruplex structure in mammalian cells has been recently discovered. The G-quadruplex was detected in telomere regions and outside of the telomeres using the structure-specific antibody BG4, and it generated in a replication-dependent way during the cell cycle [There are numerous more instances that support the notion that G-quadruplexes are important regulatory gatekeepers inside the cell. A genome-wide assessment of G-quadruplex motifs in numerous species found that they are evolutionarily conserved, confirming their crucial functional significance in biology. Targeted antibodies towards G-quadruplexes in the telomeric DNA of the ciliate ll cycle . More crll cycle . All thi33 to completely alter its fold from a hybrid-1 to an antiparallel chair-type configuration, in which the ligand intercalates between a 2-tetrad unit and a pseudo-tetrad unit, expelling one potassium ion. This exceptional high-resolution NMR (Nuclear magnetic resonance) structure reveals for the very first time a genuine ligand complexation into an intramolecular G-quadruplex [The prevalence of G-quadruplex within and around important oncogenes, as seen above, gives it a promising location for therapeutic targeting. Numerous ligands attach to G-quadruplex, either stabilising or destabilising it . Terminaadruplex .499 firefly luciferase mRNA in vitro after exposure with the TMPyP4 ligand [Ligand-mediated structural regulation of G-quadruplex-DNA in promoter sequences of genes might be a strategy for controlling detrimental gene expression. Investigations to increase G-quadruplex-DNA production and stability have revealed the possibility for transcriptional regulation, perhaps downregulation or upregulation of expression of genes, based on G-quadruplex-DNA associations with transcription-regulating proteins. There are several reviews accessible on ligand targets of promoter-specific G-quadruplex-DNA production. G-quadruplex-DNA structural destabilisation, on the other side, is a largely unexplored method of controlling gene expression. This was demonstrated by the enhancement in translation effectiveness of (CGG)4 ligand .in vivo because of non-specific adherence to other proteins [Through chemical fluorophores to metallic complexes, the number of described G-quadruplex-DNA-stabilising chemicals offers ligands that can be employed as possible probes of G-quadruplex-DNA conformation . In paraproteins ,41. Furtin vivo have encountered similar difficulties. There are many G-quadruplex-selective fluorescent probes that depend on an end-stacking detection process. These probes' recurring motifs comprise heterocyclic compounds like benzothiazole, the retention of charged centres, and prolonged conjugation. Given the significantly restricted collection of G-quadruplex-selective fluorescent agonists, it is possible that such drugs might have a dual theranostic function, allowing for more confirmation of G-quadruplex-targets in clinical situations.Throughout the last decade, significant progress has been achieved in the discovery of G-quadruplex-specific compounds effective for secondary structure stabilisation ,43. The 5Human health and physiology have been positively impacted by flavonoids. Research concentrating on the pharmacodynamic, and physiological actions of flavonoids have increased because of the recent discovery of several flavonoids with a variety of activities. Plant vacuoles contain secondary compounds called flavonoids. Flavonoids are naturally existing polyphenolic substances present in many vegetables, citrus fruits, seeds, and so on, and are thus absorbed in significant quantities into the human diet . The numThe worldwide variation in the number and varieties of flavonoids ingested Is determined by differences in lifestyle, gender, dietary preferences, and socioeconomic position, all of which led to disparities in disease prevalence when combined with environmental variables . Estimat6Flavonoids have several positive benefits, ranging from control of different enzymatic activities to prevention of cellular proliferation, although the underlying processes are not entirely known. Increasing data suggests that flavonoids may target G-quadruplex structures ,52. LuteIn vitro experiments on HeLa cells have shown that quercetin causes apoptosis-mediated cellular death and decreases gene expression of c-myc. This investigation highlights flavonoids' potential as a prospective option for hitting the c-myc promoter area and therefore serving as a possible anti-cancer drug [Styphnolobium japonicum (L.), with G-quadruplex DNA was studied using molecular docking and ESI-MS. While rutin and quercetin had comparable G-quadruplex binding propensity values, rutin was distinguished by its improved preference for G-quadruplex over ds DNA. Furthermore, CID (Collision-Induced Dissociation) experiments revealed that rutin more effectively stabilises the G-quadruplex structure, and molecular docking suggested stacking as the preferable interaction type [The most prevalent dietary plant flavonoid, quercetin, has been discovered to have various advantageous benefits on human health . Furthercer drug . Researccer drug . In a reion type . Rutin wNaringenin and fisetin are dietary flavonoids found in citrus fruits . Naringenin could execute several biological processes in order to cure a wide variety of inflammatory disorders and pathological ailments caused by oxidative stress. Naringenin's immunomodulatory properties have recently been studied as a viable alternative therapy for autoimmune illnesses ,61. FiseResearchers used spectroscopic as well as docking experiments to investigate the affinities of the structurally similar plant flavonols morin and kaempferol with distinct duplex DNA and G-quadruplex-DNA sequences . The findings suggest that kaempferol interacts preferentially with VEGF G-quadruplex-DNA over duplex DNA and other G-quadruplex sequences. Furthermore, kaempferol improves the thermal durability of VEGF G-quadruplex-DNA. Morin, on the other hand, has a significantly lesser amount of affinity with both different G-quadruplex-DNAs and duplex DNA, with no notable structural specialization. The differing DNA binding behaviours point to a critical role for the 2′-OH functional group in the flavonol moiety's B-ring. While kaempferol has a comparatively planar shape, morin has a considerably non-planar configuration due to steric hindrance from the extra 2′-OH group. This little structural change appears to be critical for kaempferol and morin's capacity to engage with VEGF G-quadruplex-DNA. Therefore, kaempferol (but not morin) appears to be an efficient ligand for VEGF G-quadruplex-DNA, suggesting that it might be used to regulate gene transcription in cancer cells .Quercetin, a dietary flavonoid, known to interact with the Tel7 G-quadruplex sequences d- (TTAGGGT). A study has been performed to focus on the interaction of 4 distinct flavonoids, quercetin, genistein, rutin, and, luteolin, with the Tel7 G-quadruplex sequences. To explore the binding mechanism of these dietary flavonoids to Tel7 G-quadruplex DNA, NMR experiments with additional biophysical methods including as uv–visible absorption, steady-state & time-resolved fluorescence spectroscopy, circular dichroism were studied. According to the findings, all of the flavonoids interact to Tel7 G-quadruplex DNA. Quercetin also binds to Tel7 G-quadruplex DNA by intercalation between both the G6pT7 and T1pT2 stages, according to comprehensive structural investigations . In an eCurcuma longa which may function as a G-quadruplex binding agent. Curcumin's G-quadruplex binding affinity was explored using structural characteristics shared through other effective G-quadruplex binder compounds, such as the electron charged aromatic surface offered by curcumin's alkoxy-side extremities, and the flat aromatic rings that are stuck by an intrinsic hydrogen bonding crosslink. Curcumin comprises 2 aromatic ring configurations with o-methoxy phenolic groups which are linked by a 7-carbon coupler made up of an alpha, beta-unsaturated beta-diketone molecule. Curcumin has an effective electron transfer capacity owing to its unusual structure and many functional units, which include a beta-diketone and multiple π-electrons capable of conjugating between 2 phenyl rings [Beside flavonoids, there are also other dietary compounds that are found to interact with G-quadruplex DNA structures. A biological active substance Curcumin (turmeric) is a hydrophobic phenolic compound derived from the rhizome of yl rings ,68. Curcyl rings . Curcumiin vitro as well as in vivo, paving the way for G-quadruplex structure targeting phenolics (72). Nutrigenomics is concerned with the current molecular connections between genes and nutrition. Researchers discovered how a natural biologically active molecule found in our food, gallic acid, might influence gene expression via interactions with G-quadruplex structures. Investigations with cells in vitro and, more crucially, animals have deduced the stabilising qualities of gallic acid, which might be generalised to humans [Gallic acid (GA) is a phenolic compound found in a variety of dietary sources, including many vegetables and fruits. GA is more important than other phenols because of its powerful and specific antitumoral activity in colorectal cancer ,71. The henolics . Nutrigeo humans . Gallic The spectroscopic investigations on dietary flavonoid-G-quadruplex DNA interactions presented here provide a potent strategy for investigating their DNA binding by using the flavonoids' very reactive intrinsic fluorescence capabilities as their individual “reporter” for their associations with macromolecular targets. Therefore, a potential strategy for controlling the expression of different genes may be illustrated by the unique binding of certain dietary flavonoids to the G-quadruplex DNA structures.7(I)It must be able to tell the difference between quadruplex and duplex and adhere to it.(II)It must be able to distinguish between distinct G-Quadruplex structures.(III)It ought to have a minimal off-target impact.(IV)It must be cell permeable.(V)In the cell, it must not break down into a harmful metabolite.G-quadruplex, which regulates gene expression, has emerged as an attractive anti-cancer target. Because quadruplexes serve a variety of biological functions, targeting them has emerged as a popular field of drug discovery study. A new viewpoint on these motifs has resulted in the discovery of ligand compounds that engage with them. We've seen in this study that various dietary flavonoids have been designed to target G-quadruplex structures. In reality, the majority of these dietary flavonoids are unable to differentiate between distinct quadruplexes and exhibit preference to more than one G-quadruplex. Because G-quadruplex structures have a high polymorphism and dynamic shape, designing ligands that can recognise and react with these entities is tough. The main disadvantage of using such compounds as quadruplex targeting drugs is their off-target action and toxicity. These compounds can break down and create hazardous metabolites, resulting in poor body clearance. All these disadvantages must be resolved to produce a molecule as a medication. Any compound that wants to be a G-quadruplex targeted drug must meet the following criteria:A molecule that meets all of these qualities has yet to be identified. Despite the availability of various anti-cancer drugs, the multi-drug resistant characteristic of cancer posed a challenge to traditional treatment. As a result, drug research necessitates the development of an alternate therapy, which is why recombinant compounds and peptides as drugs are thriving. Investigators are gradually concentrating more on developing G-quadruplex stabilising peptides. The current multimodal chemistry and phage display libraries approach enables one to analyse several peptides against a target and choose the best from lots all at once. Because peptides can fold back and forth on their own, they can adapt to changing G-quadruplex landscapes while attaching to them, enabling increased specificity and tighter adherence.The unfortunate disadvantages that make G-quadruplex a difficult target can be addressed by merging computational and bioinformatics approaches, which can lead to the identification and creation of more new ligands capable of distinguishing various topologies of G-quadruplex with much greater accuracy. Using structure-based drug discovery and virtual database-based ligand screenings is the next stage in obtaining ligands that are specific to a quadruplex . The X-r8Quadruplexes are involved in a variety of biological activities, including the translation and transcription of many oncogenes and tumour suppressors, as well as telomere stability and genomic instabilities. In this respect, quadruplexes have sparked research into their potential significance in cancer biology as well as the assessment of small-molecule ligands as prospective therapeutic drugs ,76. ExpeDietary flavonoid's ability to act as anti-proliferative and anti-tumorigenic agents has earlier been documented but is not completely comprehended. One of the methods through which these flavonoids exhibit anticancer activities might be by targeting human telomeric G-quadruplex DNA. According to scientific investigations, a flavonoid-rich diet is associated with a lower risk of breast cancer. Although the potential relevance of nutritional and dietary aspects in cancer treatment, and diagnosis has been demonstrated ,78, the Despite several investigations on these flavonoids’ anti-proliferative, anti-tumour and anti-apoptotic effects, the primary cellular mechanism of their activity remains unknown. Furthermore, it has been demonstrated that ligands containing planar aromatic domains intercalate and stabilise the G-quadruplex structure. The flavonoid skeleton has a planar chromophore with an extra carboxyl group for charge transfer and may successfully intercalate into the G-tetrad planar scaffold. Previous study focuses on the structural characteristics of flavonoid attachment to G-quadruplex structures generated by the telomeric DNA sequence of human, as well as the possibilities of flavonoids as anti-cancer treatments by modulating the telomeric G-quadruplex organization . Dietaryin vitro stability and cell activities is not apparent. A G-quadruplex target identified in vitro, for example, might not be the only G-quadruplex directed in cells. In addition, there is intrinsic cell variability that influences the correlation between in vivo and in vitro observations. Another issue for the bulk of the ligands identified so far is that they have higher molecular mass and protonated side groups that might impact their cell absorption. Furthermore, the main barrier to practical use of quadruplex ligands appears to be connected to selectivity. The selectivity patterns of some quadruplex ligands are dose dependent. While universal or numerous G-quadruplex targeting strategies may be useful, targets must be explicitly identified ahead of time. Other possible impediments include the ligands' potential adverse effects on normal tissues. Furthermore, predicted response indicators must be established in order to provide customised anticancer treatment. Nonetheless, given the enormous collection of data on quadruplex architectures and the biological roles associated with them, as well as the fast evolution of ligands, we are hopeful that these restrictions may be addressed. A plethora of novel compounds with lesser cytotoxicity and higher selectivity will evolve in the near future in this approach.Despite the fact that there is still a long way to go before the production of powerful drugs, some possible lead substances have been developed; nonetheless, the outcomes have been restricted thus far. To begin, the diversity of binding locations for these ligands, as well as the variation in their impacts on quadruplex structures, start making it difficult to determine how quadruplexes affect biological functionality, i.e., whether quadruplex destabilisation or stabilisation helps promote or hinders gene expression. Second, the relationship between The molecular influence of G-quadruplex structures-flavonoids interaction on essential cancer processes is anticipated to offer up new pathways in cancer treatment and detection. In this respect, the interaction of two specific regions is critical: (A) G-quadruplex structures generated in cancerous cells that can serve as possible molecular targets; and (B) small compounds capable of binding, stabilising, and perhaps visualising G-quadruplex complexes. Investigating the interactions and mechanisms of flavonoids with G-quadruplex DNA would aid in the development of appropriate dietary treatments and adjuvant medications for cancer therapy, which is critical to medical and people's health. To describe the mechanism of action by which dietary flavonoids are interacted with G-quadruplexes, more research at the proteome and genome levels is required. Insights into the processes by which dietary flavonoids affect cellular activities may be gained via mapping the direct interactions of flavonoids with G-quadruplex DNA. This work may also open the door for understanding, forecasting, and managing flavonoid responses in human.9ex vivo, in vivo and in vitro models is recommended. The application of this integrative research methodology provides a prospective avenue for the development of novel flavonoid-based phytomedicines. Several research suggests that there is no universal rule that describes the interactions between ligands and G-quadruplex-DNA, and that a sequence-dependent interaction may be essential as well. The role of the flavonoids' structure and substitution patterns on their affinities and binding modes to their target DNAs can be precisely assessed through the complementary application of additional experimental biophysical strategies and theoretical (molecular modelling) investigations. Further research into the mechanics of interactions between dietary flavonoids with G-quadruplex structures will be necessary in the future. Finally, G quadruplex-dietary flavonoid interactions will pave the way for the next generation of biology and revolutionary molecular medicine.The present review compiles information from the literatures and from the most recent studies and then compares the interactions of various flavonoids with their targets and concentrating on the links between dietary flavonoid structure and activity with G quadruplexes. Additionally, the various approaches for assessing interactions between dietary flavonoids and G-quadruplex DNA are highlighted. Natural substances have traditionally been useful companions in the fight against many diseases. The search for quadruplex-specific ligands has progressed from the early problem of identifying quadruplexes from canonical DNA to the current challenge of differentiating quadruplex structures from each other. Flavonoids are reported to influence a diverse panel of carcinogenesis processes, making them promising candidates for both tumor prevention and therapy. Flavonoids, in fact, were shown to control apoptosis as well as inhibit proliferation and migration, both of which are important processes in the development of cancer. Since G-quadruplex-DNA structures have evolved as a unique class of molecular target for anticancer medications, it is critical to examine correlations between dietary flavonoids and G-quadruplex-DNA in the search for G-quadruplex-DNA targeting therapies. Overall, these flavonoids have promising potential, and further research is needed to properly define its pharmaco-toxicological characteristics and analyse their prospective utility in chemoprevention and complementary therapy regimens. This was concluded that G-quadruplex-DNA-flavonoids interactions are a viable target for therapeutic development and that additional pharmacology research using All authors listed have significantly contributed to the development and the writing of this article.University Grants Commission, India,{201610001623}, Mangal Deep Burman was supported by 10.13039/501100001412CSIR, India, {09/028(1154)/2020-EMR-I}, Dr. Sudipta Bhowmik was supported by SBV Intramural Seed Money Research Committee, {SBV/IRC/10.13039/501100001845SEED MONEY/134/2022}Sagar Bag was supported by Data included in article/supp. Material/referenced in article.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.


Gene-gene co-expression correlations measured by mRNA-sequencing (RNA-seq) can be used to predict gene annotations based on the co-variance structure within these data. In our prior work, we showed that uniformly aligned RNA-seq co-expression data from thousands of diverse studies is highly predictive of both gene annotations and protein-protein interactions. However, the performance of the predictions varies depending on whether the gene annotations and interactions are cell type and tissue specific or agnostic. Tissue and cell type-specific gene-gene co-expression data can be useful for making more accurate predictions because many genes perform their functions in unique ways in different cellular contexts. However, identifying the optimal tissues and cell types to partition the global gene-gene co-expression matrix is challenging.Here we introduce and validate an approach called PRediction of gene Insights from Stratified Mammalian gene co-EXPression (PrismEXP) for improved gene annotation predictions based on RNA-seq gene-gene co-expression data. Using uniformly aligned data from ARCHS4, we apply PrismEXP to predict a wide variety of gene annotations including pathway membership, Gene Ontology terms, as well as human and mouse phenotypes. Predictions made with PrismEXP outperform predictions made with the global cross-tissue co-expression correlation matrix approach on all tested domains, and training using one annotation domain can be used to predict annotations in other domains.via a user-friendly web interface, a Python package, and an Appyter. AVAILABILITY. The PrismEXP web-based application, with pre-computed PrismEXP predictions, is available from: https://maayanlab.cloud/prismexp; PrismEXP is also available as an Appyter: https://appyters.maayanlab.cloud/PrismEXP/; and as Python package: https://github.com/maayanlab/prismexp.By demonstrating the utility of PrismEXP predictions in multiple use cases we show how PrismEXP can be used to enhance unsupervised machine learning methods to better understand the roles of understudied genes and proteins. To make PrismEXP accessible, it is provided  Gene annotations can be automatically inferred by combining established annotations with patterns of mRNA co-expression based on the concept of guilt-by-association. It is generally accepted that genes and proteins that co-express may be functionally related . ProteinOver decades of biochemistry, pharmacology, molecular and cell biology research, many mammalian genes have been thoroughly characterized. However, there are still many genes that remain obscured due to a lack of research interest and as a result of focus research biases. Extensive known gene annotations can be accessed from resources such as the Gene Ontology , the HumI. When making predictions about a specific annotation, the correct cellular context of the particular annotation has to be considered. With potentially hundreds of relevant tissues and cell types to choose from, tissue and cell type-specific predictions require significant domain knowledge.II. While some gene expression resources are well organized into manually annotated tissues and cell types, for example, GTEx  or TCGA III. Gene annotations prediction algorithms that utilize gene-gene co-expression data are applied to predict thousands of annotations. Thus, manual input for each case is not feasible.Computational methods that use known gene annotations to impute annotations for understudied genes are a useful way for generating hypotheses that can lead to new discoveries. High-content genome-wide data, such as RNA-seq, includes information about under-studied genes and is not biased towards the well-studied genes. Significant efforts are made to produce data, and develop tools, to shed light on understudied genes, particularly those genes that can serve as drug targets . One sucThe Simpson’s paradox  is a staTo apply an unsupervised machine learning approach called PRediction of gene Insights from Stratified Mammalian gene co-EXPression (PrismEXP) we generated a high dimensional feature space from large unlabeled RNA-seq mRNA gene expression data. The generated feature space automatically encodes tissue and cell type-specific information and produces tissue and cell type-specific correlation patterns, thus facilitating improved gene annotation predictions . As demoTo compute pairwise gene-gene mRNA co-expression correlations, we used the mouse ARCHS4 RNA-seq gene expression dataset . We chose mouse over human because benchmarking  demonstrG. The threshold to include genes in G requires that a gene has at least 20 reads in 1% of all samples. This filtering step is intended to remove pseudo-genes and genes with low level of transcript abundance. PrismEXP automatically identifies a set of N clusters defined as C using a k-means clustering algorithm, where N is specified by the user. For each cluster, we apply quantile normalization on the log2 transformed gene counts to correct for library size variability. For each resulting normalized gene expression matrix, we calculate the pairwise gene-gene correlations for genes in G using the Pearson correlation coefficient method. Additionally, we calculate the Pearson correlation coefficients between genes across a random selection of 10,000 samples from all aligned samples in ARCHS4, Genes with low gene expression are first filtered out, resulting in a set of genes, denoted as Gene × gene-set features are scores for the strength of association between a gene and a gene-set. For a gene N gene-gene co-expression matrices S, the corresponding gene-annotation association features Given N gene-gene co-expression matrices, each pair of gene and gene-set-annotation-term has the features,Alternatively, a gene-set library can also be defined as a non-binary adjacency matrix. Given PrismEXP takes the features on model  to accomon model . All theC to partition the global expression matrix. For the web-based interface, we precomputed PrismEXP predictions with 100 gene expression clusters for 38,721 genes and eight gene-set libraries from Enrichr. These libraries are: ChEA 2022, GO Biological Process 2021, GWAS Catalog 2019, Human Phenotype Ontology (HPO), KEA 2015, KEGG 2021 Human, MGI Mammalian Phenotype Level 4 2021, and huMAP. The results from such predictions can be accessed through the PrismEXP web server available at: https://maayanlab.cloud/prismexp. In addition, PrismEXP is also available as an Appyter (https://appyters.maayanlab.cloud/PrismEXP/). Appyters convert Jupyter Notebooks to web-based bioinformatics applications  curve for each gene. These AUROCs are used to compare the quality of the predictions by different methods, model parameters, and underlying datasets.Alternatively, we can rank the genes for each annotation by the most likely to the least likely. Similarly, we can calculate the AUROCs by labeling the known associations as true positives. Both sets of AUROC scores, ranking gene for likely annotations, and ranking annotations for likely genes, are critical to consider when evaluating and validating various sources of data and model parameter settings.PrismEXP is optimized to run on standard desktop computers utilizing multiple cores for the clustering and correlation calculations. Since PrismEXP is using large matrices, it has significant memory requirements. For a realistic use-case, using the ARCHS4 human gene-gene co-expression dataset, the memory requirement is 22 GB. PrismEXP was benchmarked using an Intel i7-8750H with six cores and 64 GB of RAM using Python 3.8.5. PrismEXP was also tested on an AWS r5ad.2xlarge instance with eight cores and 64 GB of RAM running Ubuntu. Both tests produce comparable performance . The memThe number of gene-gene co-expression matrices impacts the final prediction quality. We compared the prediction quality for partitioning the global matrix into 5, 10, 25, 50, 100, and 300 clusters . These rg2(bx)+c . This fiBy projecting the high dimensional feature-space of PrismEXP using t-SNE  we obserIt is expected that a model trained with a specific gene-set library will produce high prediction performance when predicting gene annotations from that same library. However, we next asked how well a model trained with one gene library can predict terms from another library . A heatmThe MGI Phenotype Database is a comprehensive resource for characterizing single gene knockout phenotypes in mice . PrismEXThe Illuminating the Druggable Genome (IDG) project is focused on furthering our knowledge about understudied potential protein targets from the ion channel, kinase, and GPCR gene families . Here wend CCL19 , and the}p<10−5) . Loss of}p<10−5) . Hence, The predictions produced by PrismEXP can be used to stimulate hypothesis generation and guide the design of functional wet-bench experiments. Specifically, PrismEXP generates ranks for all human genes based on annotated gene sets, including over 400,000 annotated gene sets from Enrichr , or thosAs with all machine learning applications in this domain, there is a risk of over-fitting the PrismEXP model to patterns embedded within one specific gene-set library used for training. Hence, we evaluated the performance of models trained to predict annotations for one library, to make annotation predictions for another unrelated library. We confirmed that the models are, in general, generalizable. In addition, we observed a significant improvement in the predictions for hu.MAP and KEA due to the partitioning of the samples for implementing the PrismEXP approach. One possibility to explain this observation is that protein-protein interactions and kinase-substrate phosphorylation reactions are highly context-specific.It should be highlighted that all the predictions made by PrismEXP were made with features derived from RNA-seq mRNA gene-gene co-expression data. However, other omics data such as those from proteomics and epigenomics could be incorporated into the same framework. By adding other sources of gene-gene similarity, additional features can be added to the model without much modification. We have explored the use of other gene-gene similarity matrices for annotation predictions for other applications. For example, Geneshot  containsvia a dedicated website and an Appyter. The Appyter and webserver application provide access to gene annotation predictions for users without programming skills. In the future, we plan to add the PrismEXP predictions to the ARCHS4 (Finally, to make the PrismEXP algorithm accessible to the community, we developed a Python package that enables computational biologists to access the PrismEXP method programmatically. In addition, we also provide access to the predictions that are made by PrismEXP e ARCHS4  gene pagHere we show that by automatically partitioning the ARCHS4 gene-gene co-expression correlation matrix we can significantly improve gene annotation predictions from co-expression data. Such automated partitioning is likely positively correlated with tissue and cell type-specific partitioning, but without the need for manual metadata labeling. The method treats each gene expression cluster as a source for features to construct unsupervised machine learning models for gene annotation prediction. This approach is different from prior work that requires correct labeling of the gene expression profiles, for example, by text mining the metadata . Another10.7717/peerj.14927/supp-1Supplemental Information 1Click here for additional data file.


With the global rollout of mother-to-child prevention programs for women living with HIV, vertical transmission has been all but eliminated in many countries. However, the number of children who are exposed in utero to HIV and antiretroviral therapy (ART) is ever-increasing. These children who are HIV-exposed-but-uninfected (CHEU) are now well recognized as having persistent health disparities compared to children who are HIV-unexposed–and-uninfected (CHUU). Differences reported between these two groups include immune dysfunction and higher levels of inflammation, cognitive and metabolic abnormalities, as well as increased morbidity and mortality in CHEU. The reasons for these disparities remain largely unknown. The present review focuses on a proposed link between immunometabolic aberrations and clinical pathologies observed in the rapidly expanding CHEU population. By drawing attention, firstly, to the significance of the immune and metabolic alterations observed in these children, and secondly, the impact of their healthcare requirements, particularly in low- and middle-income countries, this review aims to sensitize healthcare workers and policymakers about the long-term risks of in utero exposure to HIV and ART. The widespread use of antiretroviral therapy (ART) and the ongoing commitment to end the human immunodeficiency virus (HIV) pandemic have seen a 31% decrease in new HIV infections and a 47% reduction in HIV-associated mortality across the globe in the past decade 2010–2020) ) on CD8+ T-cells from birth up to 12 months and increased numbers of CD38+ HLA-DR+ CD4+ and CD8+ T-cells at 12 months . IncreasBased on the hypothesis that in utero exposure to HIV can shape the adaptive immune response of an unborn child, a study assessed the TCR repertoire in cord blood samples of CHEU from the pre-ART era, using next-generation sequencing. The authors demonstrated less clonal diversity of the TCR beta chain of CHEU compared to those of CHUU from the same community. Reduced diversity is associated with a greater number of high abundance clonotypes and is indicative of clonal expansions. In this study, the expanded clonotypes were identified as HIV-1-specific in 6 out of 7 CHEU cord blood samples, suggesting that the clonal expansion was due to exposure to HIV antigen in utero .1 response rather than the preferred immunoregulatory Th2 and T regulatory (Treg) responses usually observed in new-borns [+ T cells to acquire a Th1 phenotype in vitro, 50 HEU new-borns were enrolled in a study in New Mexico. The CHEU group had smaller subsets of Th1, Th2, Th17, and CD4+ CD25 + + (presumably Treg) T-cells compared to their unexposed counterparts. The reduced proportions of these Th cells found in CHEU resulted in impaired effector cytokine production [Some chronic maternal viral infections, such as hepatitis B virus, can predispose naïve T-cells to be driven towards a pro-inflammatory T-helper (Th)ew-borns . To inveoduction .0 cells, defined as antigen-primed T-cells that do not gain an effector function despite stimulation from the TCR or locally associated cytokines, were elevated in CHEU. This would imply that the stimulation received by the T-cells is insufficient for them to differentiate into Th effector cells [Brito-Pérez et al. observed that the number of Thor cells . They poor cells . In addior cells . It is tThe effect of CHEUs’ reduced ability to produce effector Th cytokines is also evident in their deficient cell-mediated immune response towards vaccines ,69. For Recently, the differences in maternal microchimerism between CHEU and CHUU were investigated as a potential mediator of altered T-cell responses . MicrochDysfunctional B-cells could also contribute to suboptimal responses to vaccines. While similar B-cell subsets have been found in CHEU and CHUU during the first 18 months of life , CHEU haMycobacterium tuberculosis infection [A dysregulated immune environment, mostly characterized by a pro-inflammatory milieu, has also been described in CHEU. For instance, Kashara et al. reported increased plasma levels of the pro-inflammatory cytokines, IL-1β and TNF-α, in CHEU . Furthernfection .As shown by Kashara et al., in addition to the consequences of in utero exposure to HIV, the effect of ART exposure cannot be discounted. Perinatal exposure to ART has been shown to impair adaptive immune cells and responses. A South African study evaluated the effect of ART exposure on Th cell responses in CHEU. Both mothers and neonates received a short course of zidovudine-lamivudine. Weaker HIV-stimulated T-helper cell reactivity was observed in CHEU exposed to ART, suggesting that ART exposure might negatively influence HIV-stimulated T-cell responses . AnotherThe underlying mechanisms causing adverse events in CHEU are complex and multifactorial requiring more harmonized collaborative clinical cohorts of adequate size to identify and link the mechanisms involved as well as the clinical impact thereof . One areThe association between immune cell function and cellular metabolism has been well established ,82. In t2+ signaling, iron metabolism, innate immunity, and apoptotic cell death [Mitochondrial dysfunction, or mitochondrial toxicity, including mutations in mitochondrial DNA (mtDNA), alterations in mtDNA levels, and aberrant mitochondrial histological morphology, has been reported in children perinatally exposed to HIV and ART ,84,85. tll death . Mitocholl death . mtDNA ill death . Not onlll death .Mitochondrial dysfunction further affects immune function, where fatty acid oxidation both modulates macrophage inflammatory function and regulates T-cell responses while the metabolism and availability of many amino- and organic acids play a vital role in the effective functioning of the immune system . In CHEUJao et al. evaluated 37 acylcarnitines and three branched-chain amino acids in 6-week-old CHEU exposed in utero to ART (zidovudine/lamivudine/nevirapine) and postnatal zidovudine or nevirapine, as indicators of changes in biochemical fuel metabolism and insulin sensitivity. They found a strong correlation between long-chain acylcarnitines and lower levels of mtDNA content as well as between short-chain and branched-chain amino acid-related acylcarnitines and higher levels of mtDNA content in CHEU exposed specifically to AZT . AdditioIn a Canadian cohort of CHEU exposed to cART (a combination of two NRTIs and either a NNRTI or a PI) in utero and to AZT at delivery or for 6 weeks postnatally, plasma lactate levels were found to be increased above the normal limit at least once during the first 6 months of life as compared to CHUU. The conversion of pyruvate to lactate is the most well-known metabolic perturbation in mitochondrial dysfunction , therefoAbnormal acylcarnitine profiles in newborn CHEU exposed to AZT were also demonstrated in a study by Kirmse et al. . This fiTwo specific metabolites, hypoxanthine and 3-hydroxybutyric acid, have also been shown to differ between CHEU and CHUU at birth, with hypoxanthine levels increased and 3-hydroxybutyric acid levels decreased in CHEU vs. CHUU . Both hyThe investigation of CHEU outcomes has not only been challenging due to the various contributing factors, but also because of repeatedly changing interventions and ART guidelines. Also, the medium-to long-term health impacts can best be studied through longitudinal cohort studies, which are labor intensive, time-consuming, and limited by participants becoming lost to follow-up. In addition, growth, neurodevelopmental, and immunological outcomes are intricately linked, adding further complexity.Multiple studies have shown that growth deficits in CHEU are already appreciable at birth . SuboptiUnfortunately, our understanding of the clinical impact of peripartum HIV and ART exposure remains incomplete. Several studies have reported on clinical outcomes, such as increased susceptibility to infection in CHEU, however, these studies did not perform immunological analysis to explore the possible causes ,109,110.While the reasons for worse clinical outcomes amongst CHEU remain unclear, this review aimed to shed light on the interconnectedness of the immunological and metabolomic aberrations in CHEU that culminate in altered innate and adaptive immune responses, causing immune activation, on the one hand, and increased susceptibility to infection, on the other. Concurrently, metabolomic aberrations manifest as mitochondrial toxicity, altered insulin metabolism, hyperlactatemia, and abnormal amino acid profiles that lead to growth delays. It is essential that the long-term risks of in utero exposure to HIV and ART in CHEU be fully appreciated so that suitable mitigation strategies can be employed on an individual as well as public health basis.More studies are required to clarify the link between immunometabolic alterations and clinical outcomes in CHEU. In addition, the growth and development of CHEU need to be followed-up for extended periods of time in sufficiently large cohorts to build on the current knowledge and assist in developing intervention strategies to improve the morbidity and mortality of this vulnerable population. Research in diverse contexts and populations will be needed to create a comprehensive and representative picture of the long-term health prospects of CHEU.


Radiotherapy (RT) is a traditional therapeutic regime that focuses on ionizing radiation, however, RT maintains largely palliative due to radioresistance. Factors such as hypoxia, the radiosensitivity of immune cells, and cancer stem cells (CSCs) all come into play in influencing the significant impact of radioresistance in the irradiated tumor microenvironment (TME). Due to the substantial advances in the treatment of malignant tumors, a promising approach is the genetically modified T cells with chimeric antigen receptors (CARs) to eliminate solid tumors. Moreover, CAR-T cells targeting CSC-related markers would eliminate radioresistant solid tumors. But solid tumors that support an immune deserted TME, are described as immunosuppressive and typically fail to respond to CAR-T cell therapy. And RT could overcome these immunosuppressive features; thus, growing evidence supports the combination of RT with CAR-T cell therapy. In this review, we provide a deep insight into the radioresistance mechanisms, advances, and barriers of CAR-T cells in response to solid tumors within TME. Therefore, we focus on how the combination strategy can be used to eliminate these barriers. Finally, we show the challenges of this therapeutic partnership. Radiotherapy is the localized cancer treatment of cancer patients through high-energy radiation. The principle is to induce double-stranded DNA damage, single-strand breaks, incomplete repairs, and chromosomal aberrations in cancer cells to accomplish local tumor control and reduce the outcome of disease transmission . In addi+ T cell toxicity, promoting Treg transformation, and inhibiting NK cell proliferation. Concurrent radiation damage can enhance pro-inflammatory responses after irradiation and recruit CAFs. Activated CAFs may secrete TGF-β and matrix metalloproteinases (MMPs), extracellular matrix modulators, to promote conversion to radioresistant cancer stem cells (CSCs)  . TME hass (CSCs) , 9. Radis (CSCs) Figure 1in vivo -T cell therapy has been considered one of the most successful approaches in tumor immunotherapy, particularly in treating hematological tumors. The principle is to engineer synthetic receptor CARs to redirect T cells to recognize and eradicate tumor cells expressing specific target antigens independent of the MHC receptor, which causes the activation of powerful T cells and a more robust anti-tumor response in vivo . For cerin vivo . Howeverin vivo . CombiniThis paper reviews the mechanisms of tumor radioresistance in irradiated TME. Here, we also summarize the role of CAR-T cells in the solid tumor microenvironment and experimental data on combination therapy, which provide a basis for further exploration of combination therapies to improve the outcomes of tumor radioresistance.In irradiated TME, there are dynamic interactions and crosstalk between these components within TME. Changes in these TME mediate various immunosuppressive mechanisms or the development of immune suppressor cells to promote tumor radioresistance.The majority of solid tumors exhibit hyperoxygenation, which results in areas of permanent or transient hypoxia being developed. Cellular adaptation to these hypoxic conditions is mediated through a family of hypoxia-inducible transcription factors (HIFs) , 14, whiHeat shock protein (HSP) serves as a molecular chaperone rapidly upregulated when exposed to harmful stimuli under oxidative stress, contributing to the correct folding of proteins, degradation, and removal of denatured proteins. It is well known that the heat shock transcription factor 1 (HSF1) can confer tumor cell radioresistance by upregulating HSP27, HSP70and HSP90 protein levels, suppressing post-radiation cell apoptosis, and correlates with poor prognosis in patients . HSP27 cKip1 and p21waf1, and dysregulation of CDK activity is a common characteristic of numerous cancers , vascular cell adhesion molecule 1 (VCAM1), and E-selectin  and immunosuppressive MDSCs are more radioresistant. Shi et al. evaluated the impact of 10, 20, or 30 Gy local irradiation in cervical cancer patients. They found that the number of CD8+ T cells has dramatically reduced, whereas not affecting Tregs. The accumulation of T cells after ablative radiotherapy and exhaustion of CD8+ T cell infiltration is an essential mechanism of radioresistance (+ and CD8+ T cell activation. Studies have shown that MDSC and TAM can express high levels of arginase-1 (Arg-1), which reduces the pool of arginine for T-cell activation, and they also sequester a cysteine that is essential for T-cell proliferation, thereby restricting cysteine availability and consequently disrupting the T-cell receptors (TCRs) by generating ROS  have achieved promising outcomes in patients with hematologic malignancies However, it still has some challenges that need to be resolved for solid tumors. We detail a series of considerations for the improvement of the CAR-T cell approach in order to make CAR-T cell therapy more widely available Table 1.CARs are modular synthetic receptors that can confer target antigen specificity. For structure, it consists of four main components: (i) the antigen-binding domains are derived from a single-chain variable fragment (scFv), (ii) the hinge or spacer region, (iii) the CD3ζ, CD8α, or CD28 transmembrane domain, and (iv) one or more intracellular signaling domains . First-gAlthough the principle of CAR-T cells is to achieve tumor lysis through direct interaction between T cells and tumor cell surface-specific antigens, tumor heterogeneity is challenging for CAR-T cells . In receThe effectiveness of CAR-T cell therapy in solid tumors is significantly hampered by poor immune cell infiltration. The vascular endothelium can be considered a dynamic cellular organ and a barrier to CAR T-cell entry, which controls the passage of nutrients, maintains blood flow, and regulates leukocyte trafficking by controlling the chemokine and cytokine composition of the TME . Thus, cin vivo to their higher tumor tissue homing activity  showed that T cells homed to CCL2-expressing neuroblastoma and malignant pleural mesothelioma xenografts, respectively . Similaractivity . Indeed,activity , 63.+/CD4+ T cells in the TME , inhibits the cytotoxicity of CAR-T cells and induces non-responsiveness. Thus, CAR-T cells must inhibit the PD-1/PD-L1 axis . Constru the TME , 67. In sistence . Therefo+ T cells proliferation and anti-tumor function, preventing the development of anti-tumor immune responses. Recent studies have shown that a novel strategy of integrating the HIF structural domain to the intracellular domain of the CAR or introducing the HRE region on the promoter of the construct contributes to CAR hydroxylation and degradation in the presence of oxygen  system has been reported to mediate the secretion of relative proinflammatory cytokines and enhance T cell proliferation when targeting high-level radioresistant head and neck squamous cell carcinoma (HNSCC) in vitro (in vivo. In GBM or other treatment-resistant primary cancers, CAR-T cells targeting CD133-positive tumor-initiating cells, a marker of radioresistance in multiple aggressive cancers, demonstrated superior efficacy (Developed as a promising strategy for overcoming the TME barriers, CAR-T cell therapy regimens likely hold applied value in irradiated TME. many clinical trials are being carried out Table 2.in vitro , 87. Alsefficacy . It is nefficacy .in vitro, DeSelm et al. illustrated that TRAIL could be produced by CAR-T cells after binding to antigen-positive tumor cells, and the clearance of antigen-negative tumor cells has been previously exposed to a low dose of local or systemic radiation. Whole-body irradiation of complete lymphatic depletion is suggested to boost survival and proliferation via upregulation of IL-7 and IL-15 of CAR-T cells without competing with endogenous lymphocytes in the mouse model (Additionally, low-dose radiation-exposed tumors present cells susceptible to TRAIL, a death-inducing ligand-mediated by RNA sequencing analysis . Throughse model . In addise model .Overall, these studies successfully demonstrated that radioresistant cancer cells could be eradicated by combining CAR-T cells in a highly efficient and antigen-specific manner.+CD38- (The focus of the successful implementation of radiotherapy combined with the CAR-T cell therapeutic approach is to analyze the effect of CAR-T cells on radioresistant tumor cells. On the one hand, radioresistance and tumor recurrence are mainly associated with CSCs and are considered targets for novel anticancer therapeutic agents . Identif+CD38- .On the other hand, RT may cause an increase in Treg infiltration while increasing the enrichment of T cells into the tumor, and Treg infiltration may grow dose-dependent. Treg cells are more resistant to radiation than other T cell subsets, which may also be a potential obstacle to a strategy of combining RT with CAR-T cells . Further+ T cells and NK cells with antitumor effects were eliminated, whereas Tregs and MDSCs were left. A low dose of 2 Gy of radiation was reported to stimulate nitric oxide synthase through tumor-associated macrophages and generate an immunogenic environment. By contrast, high doses of radiation more significant than 5 to 10 Gy have been shown to promote severe vascular damage, limit the infiltration of CD8+ T cells into the tumor, and increase the area of hypoxia, which results in radioresistance (The average tissue volume in routine RT is usually considerably more significant than the tumor volume itself due to the absence of dose delivery techniques that restrict regular tissue exposure in clinical practice. Therefore, applying fractionated irradiation regimens is the only alternative for providing high tumor doses . After ssistance . A 6 Gy sistance . In addisistance . Howeversistance . Indeed,The successful CAR-T cell therapy for hematological malignancies signals a new era of immunotherapy in treating malignant diseases. Still, this review has pointed out emphatically that attaining effective clinical outcomes for radioresistant patients will require detailed consideration of this combination therapy. For example, according to the description of Sim et al., in the case of patients receiving CAR-T cell therapy, adequate local disease control before CAR-T cell infusion could cause the lowest acute toxicity . In addiAdditionally, CAR-T cells are subject to a complex immunosuppressive microenvironment in radioresistant patients, which has hampered progress. Most tumor treatments failed and relapsed due to individual immunity and comorbidity conditions. Therefore, an integration of next-generation sequencing technologies provides newer opportunities to understand the dynamic antigen landscape and other immune-related factors of tumor cells in the TME. In terms of RT combination with CAR-T cell therapy, at least for now, those are lofty goals that are far, far away Figure 4TH researched data and wrote the article. HL and BT contributed to scientific discussion and critical review of the manuscript. Contributions to the article were made by all three authors, and the submitted version was approved.Health Research Project of Huai’an City in 2020, General Program, Jiangsu, China. Grant number 33.We would like to thank Jinhu County People’s Hospital and Jiangsu University for their support.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Helicobacter pylori infection according to antibiotic resistance has been frequently recommended. However, information on its real effectiveness is scarce.Treating H. pylori.The aim of this study is to perform a meta-analysis comparing empirical vs. susceptibility-guided treatment of Selection of studies: Studies comparing empirical versus susceptibility-guided treatment were selected. Search strategy: electronic and manual up to August 2021. Data synthesis: by intention-to-treat (random-effects model).H. pylori eradication rate was 86 vs. 76%, respectively . Similar results were found when only RCTs were evaluated  and when susceptibility testing was assessed by culture  or PCR . For first-line treatments , better efficacy results were obtained with the susceptibility-guided strategy . However, for empirical first-line quadruple regimens, in particular , not based on CYP2C19 gene polymorphism, no differences in efficacy were found compared with the susceptibility-guided group ; this lack of difference was confirmed in RCTs . For rescue therapies , similar results were demonstrated for both strategies, including all studies  and when only RCTs were considered .Overall, 54 studies were included . H. pylori infection could not be demonstrated, either in first-line (if the most updated quadruple regimens are prescribed) or in rescue therapies.The benefit of susceptibility-guided treatment over empirical treatment of  Helicobacter pylori (H. pylori) infection affects billions of people worldwide, which is the main cause of gastritis, peptic ulcer disease, and gastric cancer  were reviewed, and those dealing with the susceptibility-guided treatment of H. pylori. Patients could be treated with any of the available eradication treatments for H. pylori infection in any line of treatment. Trials had to compare the efficacy of an H. pylori eradication treatment based on a previous susceptibility-guided diagnostic test with that of empirical treatment. Pre-treatment diagnostic methods for H. pylori detection should comprise one or more of the most commonly validated tests: urea breath test, histology, rapid urease test, and stool antigen test; for susceptibility-guided treatments, studies should include methods to test antimicrobial susceptibility on gastric biopsies such as PCR or culture.The studied population included adults or children diagnosed as positive for Eligible studies should include accessible data on successful eradication rates in both tailored and empirical groups.H. pylori eradication rate). Reported efficacy was considered as the rate (proportion) of patients cured among the total of treated patients. Trials were included if they reported the number of patients with H. pylori eradication in each treatment arm; otherwise, the numerator was calculated from the percentage of eradication reported and the intention-to-treat (ITT) sample size.The primary endpoint was intention-to-treat efficacy : pylori AND [ OR ].Reference lists of the articles selected by electronic searching were examined in detail to further identify relevant studies. In addition, references of articles retrieved, significant reviews, and the personal databases of the authors were also checked for eligible publications.www.prismastatement.org) was used to develop a diagram to schematize the different steps of study selection  approach  performed the screenings independently; disagreements were resolved by consensus with a third reviewer (JPG). The reason for the exclusion of a given study was reported in the second phase only as appropriate.A pre-tested data extraction form was used in a pilot test before the final collection of data to test its reliability. The following information was extracted from each study: first author; year of publication of the study; country; population (adult or children); study design (RCT or non-RCT); treatment line; susceptibility test; clarithromycin resistance rate (%); metronidazole resistance rate (%); levofloxacin resistance rate (%); type of empirical regimen; eradication rate with the empirical regimen; and eradication rate with the susceptibility-guided regimen. Two reviewers (OPN and ME) performed the data extraction independently; disagreements were resolved by consensus with a third reviewer (JPG).The risk of bias was assessed independently by two reviewers (OPN and ME); disagreements were resolved by consensus with a third reviewer (JPG) in accordance with the Cochrane Collaboration's current recommendations  tool was used and the six quality items were evaluated: random sequence generation (selection bias), allocation concealment (selection bias), blinding of participants and personnel (performance bias), blinding of outcome assessment (detection bias), incomplete outcome data (attrition bias), and selective reporting (reporting bias). A study was considered to be an RCT if it was explicitly described as “randomized.” This should include the use of words such as “random,” “randomly,” or “randomization.” We then rated the potential randomized trial as truly random, pseudo-random (randomization was mentioned but the method used was not reported), or non-random, based on the definitions by the Cochrane Handbook  and non-RCTs, the RoB criteria for EPOC Reviews  advocated by the Cochrane was used. The same quality domains (as for RCTs) were assessed, but that related to the evaluation of randomization was reported as “high risk of bias” as no allocation of the sequence was generated as per the study design.2 test for heterogeneity for each combined analysis, where P < 0.10 indicated significant heterogeneity between studies  of the individual studies with their corresponding 95% confidence intervals (CIs), using a random effects model (Mantel–Haenszel). Additional sensitivity analyses were performed to check the robustness of the results , treatment line (naïve vs. rescue), susceptibility testing (culture vs. PCR), RCT by treatment line, and RCT by susceptibility test. The last group evaluating the empirical first-line quadruple treatments only was also included, to perform the most equitable comparison according to the most updated recommended first empirical quadruple treatments   version 5.4.1 (2020).In total, 17,383 citations were retrieved from the following electronic databases: PubMed and EMBASE, up to August 2021. After removing duplicates, a total of 13,941 citations were screened. After reviewing the abstracts and full texts, 47 studies . Overall, Results of meta-analyses comparing the effectiveness of the empirical and the susceptibility-guided treatments between different groups  are detailed below.I2: 83%; A total of 35 studies were included in this analysis, with 10,894 patients treated with first-line treatment. Statistically significant differences were found in cure rates in favor of susceptibility-guided therapy (87%) vs. empirical treatment (78%); however, results were highly heterogeneous  and also in those with low clarithromycin resistance .Sensitivity analyses confirmed that susceptibility-guided therapy was also superior to first-line clarithromycin-based triple therapy, in areas with high  clarithromycin resistance  and analyzed together , no differences were reported  therapy. When patients receiving a second-  or third-line (152) treatment were evaluated separately, no differences were found between groups. Likewise, when all rescue lines were grouped (from 2I2: 83%) or PCR ; however, no significant variation in the mean effects was found between the different subgroups (P = 0.64).Similar results were reported when culture  were included in the meta-analysis; that is, 7,325 patients  were evaluated. I2: 86%). Likewise, the exclusion of one study .In non-RCTs, 8,000 patients  were analyzed. In this sub-group, eradication was also higher in the susceptibility-guided group than in the empirical group , with moderate heterogeneity between arms  or subsequent rescue treatment lines; that is, when participants received more than one eradication treatment . Two RCTs  any bacterial antibiotic resistance . OverallI2: 65%; I2: 81%). However, when rescue treatments (more than one treatment failure) were considered, no statistically significant differences were found between treatment arms .Among studies with culture testing, results were moderately heterogeneous . In this subgroup, sensitivity analyses performed among naïve patients showed similar results . One study .Among studies using PCR, no statistically significant differences were found between treatment groups  in this same subgroup meta-analysis also confirmed the lack of difference in effectiveness between both groups . Moreover, excluding the RCT by Zhou et al. (I2: 75%).In total, 12 studies (RCTs and non-RCTs) including 2,762 naïve patients  were evaluated. No statistically significant differences were found in cure rates between the guided therapy (87%) and the empirical treatment (78%), with low to moderate heterogeneity between treatment arms  with the RoB tool.H. pylori is an objective measurable endpoint. Therefore, these items were considered as introducing a low risk of bias, even for open-labeled studies.The RoB for both the randomization and allocation items was either unclear or high-risk in 50 to 75% of the studies . The quaAll studies reported complete outcome data with no imbalance between arms in the patient's participation flow; therefore, no attrition bias was identified. Likewise, no selective reporting bias was detected [except for one study . Therefore, this study presents the highest number of studies in each subgroup published so far. Furthermore, the subgroup analyses performed in the present meta-analysis were more comprehensive than those of previous systematic reviews, and our protocol established no language restrictions.H. pylori infection is not better than empirical treatment in first-line if the most updated bismuth or non-bismuth quadruple regimens are empirically prescribed, in agreement with a previous study , although the results were borderline statistically significant . However, when prescribing only empirical quadruple regimens—that is, excluding the suboptimal triple therapies—no differences in efficacy were found vs. the susceptibility-guided group; this lack of difference was confirmed when only RCTs were considered. Therefore, we may conclude that susceptibility-guided treatment of The results of our meta-analysis are in agreement with the well-known high effectiveness of bismuth quadruple therapy, even in patients with clarithromycin or metronidazole resistance. In particular, when a bismuth quadruple therapy [either with tetracycline  and the agar dilution method   culture implies the performance of endoscopic exploration, which is uncomfortable, expensive, and not free of risk. In addition, as endoscopy centers have been facing increasing demands, the technique frequently involves prolonged waiting times. As a consequence of the aforementioned problems, several diagnostic policies have been proposed for selecting patients with symptoms of dyspepsia, the most outstanding being the so-called “test-and-treat” strategy. Several prospective studies and decision analyses support the use of the test-and-treat strategy for dyspeptic patients  as well as attrition or reporting biases (as per the robustness of the outcome).Some relevant limitations affect studies comparing empirical vs. susceptibility-guided strategies, and consequently also the reliability of our meta-analysis. A major limitation of the current evidence regarding susceptibility-guided therapy is that comparative studies of susceptibility-guided therapy randomized patients after diagnostic endoscopy or even after successful culture  of our clinical practice should be continuously audited to confirm that we always maintain a high success rate.In summary, we think that susceptibility tests (culture or PCR) should be routinely performed, even before prescribing first-line treatment, in specialized centers with an interest in The original contributions presented in the study are included in the article/Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.OPN and JPG interpreted the data. OPN performed the analysis and prepared the first draft of the manuscript. ME, JPG, and OPN contributed to the literature search and data extraction. ME reviewed and approved the final draft of the manuscript. JPG analyzed and reviewed the draft manuscript. All authors contributed to the article and approved the submitted version.JPG has served as a speaker, consultant, and advisory member for or has received research funding from Mayoly, Allergan, Diasorin, Gebro Pharma, and Richen. OPN has received research funding from Mayoly and Allergan. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Metasurfaces, the ultra-thin media with extraordinary wavefront modulation ability, have shown great promise for many potential applications. However, most of the existing metasurfaces are limited by narrow-band and strong dispersive modulation, which complicates their real-world applications and, therefore require strict customized dispersion. To address this issue, we report a general methodology for generating ultra-broadband achromatic metasurfaces with prescribed ultra-broadband achromatic properties in a bottom-up inverse-design paradigm. We demonstrate three ultra-broadband functionalities, including acoustic beam deflection, focusing and levitation, with relative bandwidths of 93.3%, 120% and 118.9%, respectively. In addition, we reveal a relationship between broadband achromatic functionality and element dispersion. All metasurface elements have anisotropic and asymmetric geometries with multiple scatterers and local cavities that synthetically support internal resonances, bi-anisotropy and multiple scattering for ultra-broadband customized dispersion. Our study opens new horizons for ultra-broadband highly efficient achromatic functional devices, with promising extension to optical and elastic metamaterials. This paper reports inversely designed metasurfaces with customized dispersion through bottom-up topology optimization to realize frequency-independent and ultra-broadband acoustic functionalities including directional energy transmission and convergence and ultrasound particle levitation. Metasurfaces have offered tremendous possibility in the fields of optical devices , carpet To achieve an arbitrarily defined broadband functionality, each metasurface element must provide the required local amplitude and/or phase modulation, which is controlled over the entire operating bandwidth. This modulation depends on the specific functionality and leads to the customization of the dispersion of each element. Recent studies have shown that broadband  optical To address the aforementioned challenges, we propose a systematic bottom-up inverse-design methodology for implementing ultra-broadband and customized metasurfaces, and explore the physics behind different functionalities. Our study demonstrates the feasibility of developing a systematic inverse-design model for constructing ultra-broadband achromatic metasurfaces, the dispersions of which can be tactically tailored. Optimized elements can be either non-dispersive or dispersive through customization, exhibiting many asymmetric scatterers and cavities within the elements. By combining these elements into specific patterns, we consistently achieve a class of metasurfaces that entail ultra-broadband, achromatic beam deflection, focusing and levitation, beyond the relative bandwidth of existing achromatic metasurfaces. Finally, we also reveal the ultra-broadband mechanism that results from a combination of integrated internal resonances, bi-anisotropy and multiple-scattering effects. We design three types of achromatic metasurfaces with customized microscopic and macroscopic dispersions, which entail ultra-broadband frequency-independent acoustic beam functionalities that have not been achieved before. The discovered asymmetric and bi-anisotropic microstructural topological characteristic and synthetical ultra-broadband achromatic mechanism offer a new basic platform for designing broadband frequency-independent metasurfaces. Meanwhile, we demonstrate a metasurface that supports ultra-broadband, stable and single-sided ultrasound levitation that can hardly be achieved with traditional ultrasound levitation technologies.The presented metasurfaces can simultaneously provide the customized specific dispersion of elements and non-dispersive nature of functionality within the ultra-broadband range, which leads to ultra-broadband achromatic metasurfaces, which, as expected, will allow them to be used in noise-control systems, medical ultrasonics and contactless particle control assembly. Meanwhile, the proposed ultra-broadband achromatic strategy can be extended to optical and elastic metamaterials for highly efficient frequency-independent functional devices.Figure Unlike traditional top-down topology-optimization approaches, the proposed bottom-up inverse-design paradigm is based on achromatic dispersion tailoring and offers the following salient advantages: (i) it is based on the responses of metasurface elements rather than the entire physical field, thus simultaneously reducing the search space, optimization complexity and computational cost; (ii) it avoids the blindness of the empirical design; (iii) its element-based design philosophy embraces and combines multiple physical mechanisms, thus maximizing the ultimate performance such as broadband, high efficiency and achromatism, etc.; and (iv) it reveals novel topological configurations of the elements with broadband features.We start with anomalous refraction to validate the proposed inverse-design strategy. To achieve broadband negative refraction with a fixed angle of 20.2°, we perform topology optimization of 2D metasurface elements  based onHere we use the aforementioned inverse-design strategy to systematically construct a metasurface for ultra-broadband focusing at a constant depth of focus Fig. . The optAs shown in Fig. To understand the physics behind the achieved ultra-broadband operations, we closely examine phase, effective impedance matrices and multiple-scattering effects for representative metasurface elements Fig. . RegardlThe space-coiling and Helmholtz-resonator structures do not naturally accommodate bi-anisotropy and multiple scattering. However, all three mechanisms are important in our analyses , which further leads to achromatic functionality. On the one hand, most Helmholtz-resonator and space-coiling structures can only produce high transmissions near resonant frequencies. But our elements provide relatively high transmission over a broadband frequency range. This is the essential difference between our elements and traditional resonant ones. On the other hand, most of the existing Helmholtz-resonator and space-coiling metasurfaces are limited by a certain dispersion property—that is, the characteristic of the wave functionality  changes markedly with frequency. But our metasurfaces can support the non-dispersive nature of the wave functionality, which is another distinctive feature. Third, the effective metamaterial index of the space-coiling structure can be numerically characterized by the length of the propagation path. However, this approach fails to characterize the effective movements of our elements. In other words, the effective index is not directly related to the length of the propagation path. Therefore, we believe that the proposed elements differ from the previous ones using Helmholtz-resonator (HR) or coiling structures.In short, we believe that the physical mechanisms of our inverse-designed elements are different from previous Helmholtz-resonator and space-coiling structures. Only the synergistic effect of internal resonances, bi-anisotropy and multiple scattering can simultaneously contribute to the ultra-broadband achromatic wave functionalities with a high transmission that has never been realized before. This discovery would provide a way to achieve ultra-broadband functionalities of metamaterials (though not yet attempted) through the combined effects of multiple mechanisms described above.According to the acoustic intensity distributions , it is tBy integrating phase modulation, transmission, geometric features and wave functionalities into the design, we have methodically demonstrated the deflection of an ultra-broadband acoustic beam and the focusing and levitation of achromatic metasurfaces following a bottom-up topology optimization. All optimized structures can represent the new category of AMs, which can support asymmetric multiple scatterers, curve air channels and local cavities simultaneously. We have found that the optimized asymmetric metasurfaces can appropriately combine integrated internal resonances, bi-anisotropy and multi-scattering effects simultaneously, resulting in ultra-broadband customized dispersions. This highly efficient ultra-broadband mechanism is expected to become a new and versatile benchmark for broadband AMs. In view of the robustness and versatility of the proposed metasurfaces, the reported ultra-broadband topological features and tunable dispersion mechanisms can provide a new impetus to the entire field of optical  Supplem, acoustiBased on the intrinsic physical parameters, our constructed topology-optimization model enhances the existing design capability and the applicability of the AMs. The proposed design paradigm can be extended to other waves  for their manipulation as well as the realization of more diversified functionalities such as absorption, acoustic insulation, vibration control and underwater acoustic manipulation, etc. Whenever a target functionality requires the tuning of specific dispersion, the proposed approach should be applicable to generate various broadband achromatic meta-devices. Meanwhile, the principle of stable single-sided ultrasound levitation can be extended to the area of acoustic tweezers. The dispersion-based wave manipulation method can also be generalized to vortex acoustics, holographic, 3D acoustic field control, etc.A limitation of the proposed approach may be the complex geometry of inverse-designed structures. However, with the advent of advanced additive manufacturing capability, this could be mitigated in the foreseeable future. Limited by computational capacity, the current optimization process is applied to a 2D system. In terms of 3D design, dramatically increased design freedom will allow more complex wave functionalities with better performance and even wider bandwidth. Finally, combing our designs with existing active control and deep learning techniques will make large-scale, digital, reconfigurable and integrated achromatic metasurface devices available.3 and Young's modulus of 2370–2650 MPa. The metasurface for levitation was manufactured by a Micro Scale 3D Printing System nanoArch® S140  using a low-viscosity high-temperature resistant resin with a mass density of 1150 kg/m3 and Young's modulus of 4.2 GPa.Metasurfaces for deflection and focusing were manufactured using a UnionTech Lite800HD 3D-printing machine  using a low-viscosity photosensitive resin with a mass density of 1250 kg/mnwac030_Supplemental_FilesClick here for additional data file.


Ductal adenocarcinoma of the pancreas is the most common pancreatic cancer, but undifferentiated carcinoma of the pancreas with osteoclast-like giant cells (UC-OGCs) is an exceedingly rare tumor. Microscopically, this tumor is characterized by the presence of two different cellular elements, namely, spindle or ovoid mononuclear cells and osteoclast-like giant cells (OGCs). Here, we report a rare case of UC-OGCs in a 79-year-old male with a one-month history of epigastric abdominal pain and unintentional weight loss. A blood workup revealed new-onset type 2 diabetes mellitus, and a computed tomography scan of the abdomen showed acute pancreatitis with a hypodense lesion in the head of the pancreas concerning for malignancy. He underwent an endoscopic ultrasound that also revealed a mass in the head of the pancreas, but no lymphadenopathy was observed. Biopsy was obtained and histopathology revealed UC-OGCs. We present this case to increase awareness of this rare clinical entity in patients presenting with acute-onset pancreatitis. Pancreatic cancer has emerged as the seventh most common cause of cancer-related death worldwide. Even though pancreatic ductal adenocarcinoma is the most common pancreatic cancer, undifferentiated carcinoma of the pancreas with osteoclast-like giant cells (UC-OGCs) is an exceedingly rare exocrine tumor, accounting for less than 1% of all pancreatic malignancies .A 79-year-old male initially presented to his primary care provider for evaluation of month-long epigastric pain and unintentional weight loss of 10 lbs. His medical history was significant for coronary artery disease, hypertension, and atrial fibrillation. His vital signs were not significant except for an irregularly irregular pulse. Physical examination was normal except for epigastric tenderness. His blood work showed glycated hemoglobin (HbA1C) of 8.3% , blood glucose of 383 mg/dL , bilirubin of 1.2 mg/dL , aspartate aminotransferase (AST) of 25 U/L , alanine aminotransferase (ALT) of 19 U/L , lipase of 271 U/L , amylase of 100 U/L (range: 0-130 U/L), blood urea nitrogen (BUN) of 34 mg/dL , creatinine of 1.6 mg/dL , white blood cell count (WBC) of 7.9 k/µL , hemoglobin of 10.5 g/dL , and platelet count of 169,000 k/µL . Cancer antigen 19-9 was 534 U/mL . The patient underwent computed tomography (CT) scan of the abdomen and pelvis with intravenous and oral contrast which was suggestive of a 2 cm hypodense lesion in the head of the pancreas  Figure , and othPancreatic cancer represents the second most common gastrointestinal malignancy, with adenocarcinoma being the most common subtype . UndiffeUDC is a highly malignant variant consisting of high mitotic activity with early local and systemic invasion. Histologically, it consists of large eosinophilic pleomorphic cells and ovoid-to-spindle-shaped cells that grow in cohesive formation. Compared to UDC, UC-OGC is composed of both pleomorphic-to-spindle-shaped cells and carries a better prognosis. A mixed subtype has been proposed by Loya et al. and Ezenekwe et al. with features resembling both UDC and UC-OGC ,8.From previous reports, UC-OGC tends to occur more commonly in elderly women, with a mean age of presentation of approximately 63 years. The most common clinical presentations of UC-OGC are similar to any other types of pancreatic tumors including upper abdominal pain and weight loss . Our patUC-OGC can be either pure or associated with other more common pancreatic tumors such as pancreatic ductal adenocarcinoma and mucinous cystic neoplasm. The origin of OGCs in UC-OGC is not well understood. It was thought to originate from mononuclear histiocytes/macrophages due to their nuclear features along with expression of CD68, vimentin, and lack of reactivity to cytokeratin. Their migration was thought to be due to chemotactic factors produced by the cancerous cells. Areas of necrosis, calcifications, and osteoid bone formation can be observed as well . On immuTreatment guidelines are limited and most of the information is obtained from isolated case reports/series. Surgery is usually the first line with unpleasant outcomes in most cases due to early recurrence and mortality. The role of radiation and neoadjuvant chemotherapy is extremely limited due to the rarity of the tumor. Limited evidence exists on the use of cisplatin and gemcitabine due to the epithelial origin of the tumor and favorable response. Our patient was referred to higher centers for surgical resection and was subsequently lost to follow-up -14.The prognosis of UC-OGC was found to vary widely with time from diagnosis to death varying from four months to ten years in a study by Togawa et al. . They obPancreatic osteoclast-like giant cell tumor is an extremely uncommon and complex type of pancreatic cancer with unique characteristics and histopathology. Currently, surgery is the first-line treatment, but the role of radiotherapy and adjuvant/neoadjuvant chemotherapy is not well elucidated. Performing randomized trials is not feasible due to the rarity of the tumor type; hence, maintaining an international registry might help to provide more information to devise potential treatment strategies for this tumor type.


Pulled-in-two syndrome is one of the significant complications of strabismus surgery. This study aimed to report a case of pulled-in-two syndrome of the contractured medial rectus muscle (MR) that occurred during strabismus surgery for strabismus fixus due to high myopia, and to describe a rescue of this complication.A woman in her 60s presented to our Ophthalmology Department with the main complaint of unilateral high myopia and severe myopic strabismus fixus. Esotropia exceeded 45° and hypotropia exceeded 15° in her right eye in the Hirschberg test. Right eye duction was markedly limited in every gaze direction. Orbital magnetic resonance images showed rupture of the superior rectus to lateral rectus band ligament and lengthening of the distance between the SR and LR muscles in the right eye.Due to the patient's ophthalmic examination and imaging results, she was diagnosed with high myopic strabismus fixus.We performed MR recession and Yokoyama surgery to correct right eye hypoesotropia. In the MR recession procedure, pulled-in-two syndrome (MR muscle tear) occurred. Thus, no additional procedure was performed on the MR. After the surgery, she presented 45 prism diopter exotropia and 18 prism diopter residual right hypotropia in a Krimsky test. We performed a second surgery, combining MR muscle advancement and inferior rectus (IR) muscle recession, 3 months after the first surgery.One and a half years after the second surgery, she presented exotropia of 14 prism diopters without hypotropia in the Krimsky test and was satisfied with her ocular position and improved motility.We experienced pulled-in-two syndrome in a case with severe myopic strabismus fixus and achieved a good outcome by performing additional surgery 3 months later, in which the lost MR muscle was advanced. This case underscores that, if the lost muscle cannot be found during surgery, one should maintain composure and perform a reoperation a few months after the initial surgery, if necessary. This case report can aid in making rescue treatment decisions when pulled-in-two syndrome occurs. The clinical manifestations of pulled-in-two syndrome are related to limited duction in the field of action of the lost muscle. Major risk factors for pulled-in-two syndrome are cranial nerve palsy, prior surgery, and advanced age.Pulled-in-two syndrome is characterized by extraocular muscle rupture under strong tension during strabismus surgery. This is extremely rarely encountered in clinical practice, but is one of the significant complications of strabismus surgery.We experienced a case of pulled-in-two syndrome of the contractured medial rectus (MR) muscle that occurred during strabismus surgery for treatment of severe myopic strabismus fixus. Furthermore, we rescued the complication 3 months after the surgery.A 60s woman presented with unilateral high myopia and myopic strabismus fixus. She showed esotropia of more than 45° and hypotropia of more than 15° in the right eye in the Hirschberg test  in the right eye. We performed a limbal conjunctival incision from the 8 o’clock to the 4 o’clock position and identified the LR, SR, and MR muscles. We cut Tenon’s capsule around the MR muscle, including the inter-muscle membrane and check ligament. Since the MR muscle was extremely difficult to pull because of the contracture, pulled-in-two syndrome (MR muscle tear) occurred during the MR recession procedure. The MR muscle ruptured at the muscle belly, approximately 5 mm from the insertion , and their combination.,6 In our case, because the preoperative evaluation showed large-angle hypoesotropia of the right eye, we expected that recession–resection surgery would induce under-correction. Therefore, we decided to perform a combination of Yokoyama’s surgery and MR recession. In both recession–resection and in combination surgery, MR recession must be performed. Pulled-in-two syndrome occurred during MR recession in our patient. However, the lost MR muscle was easily detected in a subsequent second surgery, and MR advancement could be performed without difficulty, at 3 months after the first surgery. Thus, loss of the MR muscle during surgery should not cause undue stress.Contracture of the MR muscle occurs with long-standing myopic strabismus fixus. This causes marked limitation of abduction, with stretching of the MR muscle. Pulled-in-two syndrome occurs in 0.14% of adults undergoing strabismus surgery. The complication occurs mainly in the MR or IR muscle, and particularly in the MR muscle (50%). The risk factors are advanced age, previous extraocular muscle surgery, extraocular muscle palsy, thyroid disease, the presence of a scleral buckle, and cranial nerve palsy. In our patient, the MR muscle had contracture with fibrosis due to the long-standing myopic strabismus fixus, thereby weakening the muscle. Additionally, strong tension was needed to stretch the muscle during the recession procedure. This case emphasizes the need for particular care during the high-risk MR recession in myopic strabismus fixus.In 1990, Greenwald and Parks described a rare complication of intraocular surgery, which occurred at the muscle-tendon junction, named “pulled-in-two syndrome,” an intraoperative tear of a rectus muscle caused by excessive tension or pull on the muscle or weakness in the muscle tissue. However, our patient showed consequent exotropia of 45 prism diopters; therefore, MR muscle advancement was needed. Our experience highlights 2 crucial points: first, in myopic strabismus fixus, MR tenotomy might cause large overcorrection, and second, it is possible to find the pseudotendon with the slipped true tendon fixed to the sclera, even 3 months after the tenotomy. A previous case report recounted that a muscle lost intraoperatively was found 18 years after the first strabismus surgery. Since the necessity for additional surgery varies across individuals, decisions should be made based on postoperative evaluation after the first surgery.The best rescue of this complication is finding and reattaching the lost muscle to the globe during the surgery. However, the operator may decide not to reattach it, particularly in cases with large-angle strabismus. A previous study reported 2 cases of pulled-in-two syndrome occurring during strabismus surgery for myopic strabismus fixus, in which lost muscles could not be recovered, but an acceptable appearance was achieved; therefore, they required no additional treatment.Pulled-in-two syndrome is characterized by extraocular muscle rupture under strong tension. This intraoperative complication is rarely encountered in clinical practice, but its rescue is sometimes difficult during the surgery. The clinical manifestations of pulled-in-two syndrome are related to limited duction in the field of action of the lost muscle. Major risk factors for pulled-in-two syndrome are cranial nerve palsy, prior surgery, advanced age, and myopic strabismus fixus. Here, we reported our experience of a case of contracture-related pulled-in-two syndrome of the MR muscle that occurred during strabismus surgery for myopic strabismus fixus. Furthermore, we described a method for rescuing this complication when necessary after the initial surgery.Conceptualization: Shinya Nakao, Manabu Miyata.Project administration: Manabu Miyata.Writing – original draft: Shinya Nakao.Writing – review & editing: Manabu Miyata, Mariko Hirai, Shusaku Mizoguchi, Hirokazu Ohashi, Hirokazu Nishiwaki.