We investigated whether the feasibility of left ventricular (LV) global longitudinal strain (GLS) in hypertrophic cardiomyopathy (HCM) varies according to the methodology .We retrospectively analyzed 111 consecutive patients with HCM  who underwent both transthoracic echocardiography (TTE) and cardiac magnetic resonance imaging . TTE-whole myocardial and TTE-endocardial GLS were measured and compared in terms of association with late gadolinium enhancement (LGE) extent and discrimination performance for extensive LGE (>15% of the LV myocardium).difference = 0.610). However, among patients with higher LV mass index (>70 g/m2), only TTE-whole myocardial GLS correlated with LGE extent and was independently associated with extensive LGE , while TTE-endocardial GLS did not. Additionally, TTE-whole myocardial GLS had better discrimination performance for extensive LGE than TTE-endocardial GLS .Although TTE-whole myocardial and TTE-endocardial GLS were significantly correlated, absolute TTE-endocardial GLS values (19.3 [16.2–21.9] %) were higher than TTE-whole myocardial GLS values . Both TTE-derived GLS parameters were significantly correlated with the LGE extent and independently associated with extensive LGE . Discrimination performance for extensive LGE was comparable between TTE-whole myocardial and TTE-endocardial GLS (area under the curve [AUC], 0.747 and 0.754, respectively, pTTE-derived GLS using either the endocardial or whole myocardial tracking technique is feasible in patients with HCM. However, in those with severe hypertrophy, TTE-whole myocardial GLS is better than TTE-endocardial GLS. Left ventricular (LV) global longitudinal strain (GLS) analysis is widely utilized to assess myocardial deformation . The LV While advancements in speckle tracking echocardiography have enabled the wide availability of strain assessment in clinical practice , competiTherefore, in the present study, we measured both LV endocardial and whole myocardial GLS on transthoracic echocardiography (TTE) in patients with HCM, and compared these parameters against GLS on cardiac magnetic resonance imaging (CMR) in terms of the association with the extent of myocardial fibrosis. In addition, we evaluated the discrimination performance of these GLS parameters for the detection of extensive late gadolinium enhancement (LGE), and determined whether the performance differed according to a high LV mass index (LVMI).We retrospectively screened 306 consecutive patients with HCM who underwent CMR, including both cine- and LGE-CMR, at Seoul National University Bundang Hospital, between 2010 and 2019. The clinical diagnosis of HCM was established by confirming a maximal end-diastolic wall thickness of ≥15 mm at any site of the LV, with an absence of other causes of hypertrophy [Echocardiography images were obtained using a standard ultrasound machine  at a frame rate ranging from 50 to 80 Hertz with a 2.5-MHz probe. Following guideline recommendations, 2-dimensional, M-mode, and Doppler images were acquired using a standard protocol , 11. LV For TTE-endocardial GLS assessment, the LV endocardial border was delineated semi-automatically at the end-systolic frame on apical view images using TomTec software. Subsequently, the software automatically tracked echo-speckles along the endocardial border over a single cardiac cycle. Peak longitudinal systolic strain in each myocardial segment was captured as the peak negative value during the cardiac cycle. Subsequently, the longitudinal strain of the selected image plane was calculated as the average of the strain values of the myocardial segments. The final LV endocardial GLS was calculated as the mean value of the longitudinal strain derived from apical 4-, 3-, and 2-chamber view images.For TTE-whole myocardial GLS assessment, the region of interest was determined automatically and manually adjusted to fit the thickness of the myocardium using EchoPac software. As with the TTE-endocardial GLS, the longitudinal strain was calculated in each standard apical view over a single cardiac cycle, based on the peak strain values of the myocardial segments. The calculated longitudinal strain values were then averaged to derive the LV whole myocardial GLS.Since LV GLS represents longitudinal shortening of myocardial fibers, it is expressed as negative values; thus, negative LV GLS values were converted to absolute |x| values for simpler interpretation. LV GLS values closer to 0 indicate more impaired LV systolic function.Details regarding CMR LV strain the LGE extent analysis are provided in the 2) was used as the cutoff value.Baseline clinical characteristics are presented as the median with interquartile range for continuous variables and as the number with percentage for categorical variables. Differences between GLS parameters derived from different software algorithms and imaging modalities were evaluated using the paired sample t-test. Spearman’s correlation analysis was used to investigate correlations between TTE-derived GLS values and CMR parameters, including CMR-TT GLS, LVMI, and LGE extent. Agreement between TTE-derived GLS and CMR-TT GLS values were assessed using the Bland-Altman plot. Discrimination performance for the presence of extensive LGE was assessed and compared between different GLS parameters using the area under the receiver operating characteristic (AUROC) curve, with 95% confidence intervals (CIs). In addition, logistic regression modelling was used to estimated odds ratios (ORs) of clinical and TTE parameters for extensive LGE. ORs of TTE-derived GLS values were also estimated with adjustment for age, sex, systolic blood pressure, and echocardiographic parameters . Additionally, we performed a subgroup analysis to evaluate whether the discrimination performance for, and association with, extensive LGE differed in patients with high LVMI; the median LVMI value . Two-sided p-values <0.05 were considered statistically significant.2. LGE was found in 110 (99.1%) patients, with extensive LGE (>15% of the LV mass) in 16 (14.5%) patients. The median interval between CMR and TTE acquisition was 15 (5–31) days.Clinical characteristics of the 111 patients  are summarized in The assessment of LV GLS on TTE and CMR images using different software algorithms in a representative case is shown in Results of the correlational analyses of each GLS parameter with LVMI and LGE extent are shown in difference = 0.610), although both parameters showed lower performance than CMR-TT GLS  (difference = 0.006)  and TTE-endocardial GLS  . However= 0.006) .On univariate logistic regression analysis for extensive LGE, e’ velocity , TTE-whole myocardial GLS , and TTE-endocardial GLS  were significantly associated with extensive LGE . On multdifference = 0.001); similarly, the addition of TTE-endocardial GLS to the model improved the predictive value  (difference = 0.001); however, the addition of TTE-endocardial GLS to the model did not improve the predictive value   and TTE parameters  in the entire study population, the addition of TTE-whole myocardial GLS to the model improved the predictive value  . However= 0.133) .To evaluate discrepancies between TTE-whole myocardial and TTE-endocardial GLS, the optimal cutoff values for discriminating extensive LGE were utilized to categorize patients with HCM. Notably, the optimal cutoff value for TTE-whole myocardial GLS (13.0%) was comparable to that for CMR-TT GLS (11.0%), but was much smaller than that for TTE-endocardial GLS (20.5%). Patients were categorized into Group 1 , with concordantly preserved GLS; Group 2 , with impaired TTE-whole myocardial GLS, but preserved TTE-endocardial GLS; Group 3 , with preserved TTE-whole myocardial GLS, but impaired TTE-endocardial GLS; and Group 4 , with concordantly impaired GLS . ExtensiIn the current study, we assessed the feasibility of LV GLS derived from different myocardial layers–the endocardial border and the whole myocardium–in patients with HCM. In the entire study population, both TTE-whole myocardial and TTE-endocardial GLS were significantly correlated with CMR-derived GLS, LVMI, and LGE extent, and were independently associated with extensive LGE over clinical and echocardiographic features. However, among patients with HCM with a high LVMI, TTE-whole myocardial GLS, but not TTE-endocardial GLS, was significantly correlated with the LGE extent and was independently associated with extensive LGE. The discrimination performance for extensive LGE was comparable for TTE-derived GLS parameters in the entire study population, but was significantly better with TTE-whole myocardial GLS than with TTE-endocardial GLS in patients with high LVMI. In cases with discrepancy between TTE-whole myocardial and TTE-endocardial GLS, impaired TTE-whole myocardial GLS better reflected the presence of extensive LGE and greater LGE extent.Although most prototypical vendors are currently evolving towards a comprehensive layer-specific strain analysis, these have been mainly applied for research purposes. Therefore, we considered comparisons in the feasibility and performance of the two most commonly used GLS algorithms as clinically relevant, especially in patients with HCM with thickened myocardium. In the present study, despite significant correlations between TTE-based GLS parameters, the absolute values of TTE-endocardial GLS were significantly higher than those of TTE-whole myocardial GLS. Given a transmural gradient in GLS  that exists in the healthy population , 16, thiAlthough the association between TTE-derived GLS and myocardial fibrosis has been repeatedly demonstrated –21. veryAlthough previous studies support endocardial GLS as a sensitive indicator of myocardial ischemia and infarction, which primarily affect the endomyocardium –8, thereThe strength of the present study arises from its unique design, which evaluated three different GLS parameters at the patient level, derived from TTE and CMR images. Additionally, the present study findings provide practical value in the evaluation of patients with HCM via GLS assessment. However, the results should be interpreted under consideration of the following limitations. First, the study cohort was not population-based and comprised a relatively small number of patients from a single tertiary medical center. Although we screened consecutive patients with HCM, the inclusion criteria of TTE and CMR within 6 months inevitably limited the study population. Therefore, caution must be taken when applying the current preliminary results to the general population. In addition, our findings may have insufficient power to support firm conclusions regarding the discrepancy between TTE-whole myocardial and TTE-endocardial GLS. Especially for patients with high LVMI, the multivariate model could be exposed at risk of overfitting due to the relatively small number of cases. Secondly, the median time interval between CMR and TTE examinations was only 15 days; although small, this time gap might have differentially affected the correlations between GLS parameters and LGE extent, considering differences in examination conditions. Finally, the discrimination performance of TTE-derived GLS parameters for extensive LGE may vary in patients with HCM with higher LVMI , 25 or lGLS analysis on strain echocardiography using either the endocardial or whole myocardial tracking technique is feasible in patients with HCM. However, in those with increased LV mass, whole myocardial GLS is better than endocardial GLS for the assessment of global LV function and myocardial fibrosis.S1 Appendix(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S1 Graphical abstract(TIF)Click here for additional data file.


These results demonstrate the critical role of continued Tax and IL-10 expression for the survival of primary ATL cells, highlighting their relevance as therapeutic targets.Adult T cell leukemia/lymphoma (ATL) is an aggressive malignancy secondary to chronic infection with human T cell leukemia virus type 1 (HTLV-1). The viral oncoprotein Tax initiates T cell transformation through activation of critical cellular pathways, including NF-κB. Unexpectedly, Tax protein is not detectable in most ATL cells, in contrast to the HTLV-1 HBZ protein which antagonizes Tax effects. Here, we demonstrate that primary ATL cells from patients with acute or chronic ATL express very low levels of Tax mRNA and protein. Critically, survival of these primary ATL cells is dependent on continued Tax expression. Mechanistically, Tax extinction results in reversal of NF-κB activation, P53/PML activation and apoptosis. Tax drives interleukin-10 (IL-10) expression and recombinant IL-10 rescues the survival of  Adult T cell leukemia/lymphoma (ATL) is a rare blood malignancy carrying a dismal prognosis , which iATL development is preceded by oligoclonal expansion of some HTLV-1 infected cells, driven by expression of the viral oncoprotein Tax . Tax is Tax oncogenic capacity is well documented, as its sole expression transforms T cells in vitro, and induces leukemia in transgenic mice or flies –23. WhilNevertheless, some indications suggest a role of Tax in the maintenance of the leukemic phenotype in vivo. Transient bursts of Tax expression occur in small fractions of HTLV-1 infected cells or the ATL-derived cell line MT-1 , 31. Anttax-transgenic flies rescues Tax-induced cellular transformation , the strong immunogenicity of Tax protein ultimately leading to the rapid elimination of Tax-expressing cells by the immune system –58, and We show that, only a small percentage of uncultured primary ATL cells barely express very low levels of Tax protein. Upon short-term culture, most ATL cells express Tax protein, albeit also at very low levels. These results are in agreement with previous reports in HTLV-1 asymptomatic carriers, showing that the HTLV-1 sense-strand transcription, encoding Tax and viral structural proteins, is usually silent at a given time in each cell, but gets reactivated upon cellular stress, enabling the transmission of the virus to a new host cell . Indeed,Reversal of Tax-induced NF-κB activation likely contributes to inhibition of proliferation, since NF-κB inhibitors induced apoptosis and selectively decreased HTLV-1-infected cells in the peripheral blood of virus carriers . That TaATL patients exhibit high levels of IL-10, that promote proliferation of HTLV-1-infected cells through STAT3 and IRF4 , which cIn conclusion, our results pinpoint that ATL is not only a virus-initiated leukemia, but also a virus-addicted malignancy, providing a strong rationale for the clinical development of Tax- targeting therapy . PotentiReproducibility checklistSupplemental material


Non-small cell lung cancer (NSCLC) is the prevailing lung cancer type, accounting for ~85% of all lung cancer cases. Despite their initial promise, current chemotherapeutic protocols are reaching their limits. This necessitates the prompt discovery of new molecular drivers and the development of novel regimens for advanced NSCLC. Herein, we pose that there is a need to systematically profile the human kinome activity of NSCLC. Using available state-of-the-art technologies, a wide gamut of kinase activities can be simultaneously mapped and quantified specifically in the primary or metastatic cancer states, with oncogenic kinase functions being likely linked to mutation signatures and malignant features of NSCLC. New chemical compound libraries can then be screened for kinase inhibitory properties in preclinical model systems, with presumptive induction of programmed cell-death subroutines and signaling-disintegration routes serving as major outputs of novel inhibitor tumor-suppressor potentials. Lung cancer has been recently reported as the second most frequent human malignancy and the major cause of cancer-related mortality worldwide Kirsten RAt Sarcoma (KRAS) and Epidermal Growth Factor Receptor (EGFR) genetic loci are frequently detected in NSCLC cases. Early-stage lung adenocarcinoma is associated with 29.1% and 14.2% of KRAS and EGFR genomic alterations, respectively, while metastatic adenocarcinoma is related to 29.9% and 30.3% of the respective oncogenic aberrations Anaplastic Lymphoma Kinase (ALK) gene rearrangements , together with co-occurring pathogenic drivers that include, among others, the mutated Fibroblast Growth Factor Receptors 1/2 (FGFRs1/2), neuroblastoma Ras (N-Ras), REarranged during Transfection (RET) and ROS proto-oncogene 1 (ROS1) cell signaling protein components Oncogenic mutations in KRAS mutations are notably common in lung adenocarcinoma, with KRASG12C number exceeding any other number of targetable mutations including EGFR and ALKKRASG12C oncogenic activity Targeted, as opposed to cytotoxic , chemotherapy (and immunotherapy) is considered as a relatively selective and rather safe approach, capable of markedly improving NSCLC patient prognosis. Next-generation inhibitors targeting oncogenic activities of EGFR or ALK molecular drivers in advanced NSCLCs harboring gain-of-function respective mutations, have shown beneficial effects in median survival KRASG12C inhibition efficacy can be overcome by the synergistic action of PhosphoInositide 3-Kinase (PI3K) inhibitors Signaling adaptation-triggered limitations of Figure 1).Accordingly, the NSCLC-specific activities  of multiple protein kinases should be profiled, in comparison to healthy tissues, both in the primary and metastatic areas. Utilizing validated high-throughput protein kinase activity profiling technologies, serine/threonine and tyrosine protein kinase species displaying the most important functional differences between primary tumor versus healthy samples and primary versus metastatic tumor specimens can be assembled in signal transduction networks, via sophisticated bioinformatic platforms. In that vein, the implementation of Artificial Intelligence (AI) approaches in high-throughput anatomopathological and histological analyses, including immunohistochemical studies, is becoming more and more relevant. Consequently, a new multiple active-kinase (active-kinome) signature can be developed, likely opening new therapeutic avenues for the disease (in vivo with the most efficient anti-oncogenic kinase inhibitors (single- or multiple-compound regimens), and tumor size and growth rate be analyzed using both conventional biometrics and luciferase-based in vivo imaging tools.To verify this active-kinome chart, NSCLC cell lines obtained from either primary or metastatic (lymph nodes) tumors, together with normal lung epithelial cells (serving as control), can be targeted with commercially available or chemically-synthesized specific kinase inhibitors, in mono-therapy and cocktail-therapy schemes. Their apoptotic, autophagic and necroptotic/necrophagic cell-death responses can thus be studied, via employment of 3--2,5 diphenyl tetrazolium bromide (MTT), Western immunoblotting, Reverse Transcription-quantitative real-time Polymerase Chain Reaction (RT-qPCR), Fluorescence-Activated Cell Sorting (FACS) and ImmunoFluorescence (IF) assays. Additionally, Severe Combined Immune Deficient (SCID) mice-tumor xenografts can be treated This druggable map of NSCLC-specific active kinome offers a valuable toolbox to proficiently combat and overcome the molecular diversity and acquired resistance frequently observed during therapy of advanced and metastatic NSCLC. The application of AI tools will be of outmost importance in the analysis of this kinome network, allowing prediction of drug resistance mechanisms and identification of potential synthetic lethalities.Given the cellular heterogeneity, heavy mutation load and frequent resistance to therapy of NSCLC, new “omics” strategies must be promptly developed to successfully cope with knotty and tangly molecular networks that govern NSCLC pathobiology. Novel mutation signatures, mechanistic/functional biomarkers and targeted drug schemes must be discovered in order to significantly reduce the mortality of advanced, metastatic and chemoresistant NSCLC. To this end, mutation burden, active-kinome profile and kinase inhibition are expected to serve as a powerful and precious toolbox for the successful management of NSCLCs in the clinical setting. This new prospect of kinome activity-dependent tumor drugging and therapeutics will likely result in multiple benefits, including lower cost of health-care system, reduced hospitalization and sick-leave time, shorter recovery time after chemotherapy, lower systemic toxicity and decreased regimen price. NSCLC subtype-dependent mutation maps, together with tumor-specific kinome activities and their targeted drugging with new generations of chemically-synthesized inhibitors, will most probably improve the prognosis of advanced, metastatic and chemoresistant NSCLC patients.In summary, the aforementioned treatment plan holds strong promise for the efficient, safe and economically abating therapy of NSCLC patients.


Dear Editor,PIGA)-mutated cells that PNH-G populations originate from, because their percentages usually remain low for a long period of time  Gs) are often detected in the peripheral blood (PB) of patients with acquired aplastic anemia (AA) and are thought to represent immune pathophysiology of bone marrow failure –3. Among of time . In addiIGA gene . Conversgoclonal , 6. HowePIGA-mutated committed progenitor cells rather than from hematopoietic stem and progenitor cells (HSPCs) with PIGA mutations  years) and eight cord blood (CB) samples who had been judged to be negative (0%–0.002%) for small-PNH-G populations by a high-sensitivity flow cytometry [highFLAER- dots that formed a tight cluster, in 24 (80%) of the 30 HIs  Gs of six out of 24 HIs. PIGA-AmpliSeq revealed 1–3 different PIGA mutations in four of the six subjects  Gs obtained from one male CB sample (CB 1) showed a sole PIGA mutation  Gs in the three HIs prompted us to study 30 HIs  PNH-G populations of AA patients arose from a few hematopoietic stem cells (HSCs) [PIGA-mutated HSCs by immune mechanisms, such as GPI-specific T cells [Several studies demonstrated that unlike GPI(-) Gs from patients with florid PNH, small-PNH-G populations detected in AA patients consisted of cells with multiple ytic PNH , 5. Alths (HSCs) . These f T cells , may occPIGA mutations at frequencies up to 0.005% in most HIs, which became undetectable two months later, except for one clone in a HI, which persisted for up to 164 days [PIGA-mutated committed myeloid progenitor cells rather than HSPCs. Hu et al. also concluded that PNH-G populations in HIs are all derived from committed progenitor cells by demonstrating that PIGA-mutated sequences in myeloid cells generated from proaerolysin-resistant colony-forming cells were highly diverse [PIGA-mutated sequences detected in the minor PNH-G populations persisted for more than ten months. Therefore, the minor GPI(-) G populations possessed by HIs are thought to be clonal populations derived from a limited number of PIGA-mutated HSPCs. However, given that the persistence of the PIGA-mutated sequence was demonstrated only in one HI and the rest of the mutated sequences in the two miniscule-PNH-G samples obtained over 6 months apart from the same HI varied greatly, it is possible that PIGA mutation was sometimes in a bona fide stem cell and sometimes in a more downstream progenitor cells. As several X-linked genes, such as GPA and XK, share similarities with PIGA in that mutant phenotypes result in loss of specific proteins, which can be detected by flowcytometry [According to Dingli’s hypothesis, granulocytes derived from committed progenitor cells persist for up to 120 days . A previ164 days , suggest diverse . Howeverytometry , 15, anaPIGA mutation in miniscule-PNH-G populations of one CB sample. Spencer et al. analyzed HSPCs in healthy fetuses using whole genome sequencing and revealed that HSPCs acquired around 40 somatic mutations by 18 weeks after conception [PIGA mutations during fetal development and that they minimally contribute to hematopoiesis.This study also identified miniscule-PNH-G populations in CB samples that were negative for small-PNH-G populations and single nception . Wong etnception . Our finPIGA mutations were determined using a small amount of DNA extracted from isolated GPI(-) Gs, false-positive mutations might have occurred due to replication errors during amplification of the PIGA gene. Although a minimum cut-off value of 2% was used for variant allele calling in order to avoid false-positive mutation calling, we could not completely exclude false-positive results. Second, in contrast with false-positive mutations, some PIGA mutations with low AFs might be misclassified as negative and the number of PIGA mutations in miniscule-PNH-G populations could be underestimated. Nevertheless, our results clearly demonstrate that GPI(-) Gs of HIs have limited PIGA mutations with high AFs and some of GPI(-) Gs with the same PIGA mutations were continuously detected at different time points.This study has several limitations that need to be considered. First, as PIGA-mutated HSPCs, not from committed myeloid progenitor cells, suggesting that the selection of PIGA-mutated HSPCs by immune mechanisms may occur at the time of early onset of AA, not in the transition from AA to PNH. Very small numbers of clonal GPI(-) Gs are present more frequently in HIs than previously thought and might also be derived from a few HSPCs with somatic PIGA mutations that occur during the fetal stage.In conclusion, minor GPI(-) Gs detectable in AA patients and HIs are derived from a few Supplemental data


DNA methylation is the most stable epigenetic modification. In mammals, it usually occurs at the cytosine of CpG dinucleotides. DNA methylation is essential for many physiological and pathological processes. Aberrant DNA methylation has been observed in human diseases, particularly cancer. Notably, conventional DNA methylation profiling technologies require a large amount of DNA, often from a heterogeneous cell population, and provide an average methylation level of many cells. It is often not realistic to collect sufficient numbers of cells, such as rare cells and circulating tumor cells in peripheral blood, for bulk sequencing assays. It is therefore essential to develop sequencing technologies that can accurately profile DNA methylation using small numbers of cells or even single cells. Excitingly, many single-cell DNA methylation sequencing and single-cell omics sequencing technologies have been developed, and applications of these methods have greatly expanded our understanding of the molecular mechanism of DNA methylation. Here, we summaries single-cell DNA methylation and multi-omics sequencing methods, delineate their applications in biomedical sciences, discuss technical challenges, and present our perspective on future research directions. DNA methylation refers to the phenomenon in which a methyl group CH3) from S-adenosylmethionine is transferred to the C-5 position of cytosine by DNA methyltransferases (DNMTs)  from S-aDNA methylation plays a critical role at the molecular, biological, and pathological levels De novo methylation is catalyzed by DNMT3A, 3B, and 3L, while the methylation profiles of dividing cells are maintained by DNMT1 -hydroxymethylcytosine (5hmC), then to 5-formylcytosine (5fC), and finally to 5-carboxylcytosine (5caC) under the catalysis of ten-eleven translocation enzymes  The biochemical processes of methylation and demethylation are beginning to be elucidated. DNA methylation analysis is essential to dissect its role in the development and human diseases, such as cancer. Methods for DNA methylation detection, including the principles and applications, have been described in the literature In this review, we summarize single-cell sequencing methods for the assessment of DNA methylation alone or in combination with other omics, outline the applications, and present with our perspective on these technologies.Table 1Figure ). Micromanipulators utilize automated pipetting under microscopic observation, allowing operators to isolate single cells efficiently and accurately . Laser capture microdissection (LCM) also allows targeted cell collection under microscopic visualization. The difference is that LCM focuses on isolating single cells from stained tissue and is, therefore, able to collect single cells with specific histological characteristics .Isolating intact individual cells is crucial for single-cell sequencing, and various methods have been documented and summarized in Figure ), and one of its main advantages is the throughput, which enables the isolation of hundreds of single cells within 30 minutes Figure ), the BD Rhapsody™ Single-Cell Analysis System , and the 10× Chromium Single Cell Gene Expression Solution  Many single-cell isolation platforms use flow cytometry or microfluidic devices to automatically sort single cells with high throughput. The most common platform is the fluorescence-activated cell sorting (FACS) system .The advent of single-cell isolation technologies and the optimization of methylation sequencing technologies are accelerating the development of single-cell DNA methylation sequencing. Most, if not all, single-cell sequencing methods are based on the corresponding bulk-based assays. Here we focus mainly on the most common types of single-cell DNA methylation sequencing technologies based on either restriction digestion (including methylation-insensitive and methylation-sensitive restriction endonucleases) or post-bisulfite adapter tagging (PBAT)  to uracil and leaves methylated C unchanged. Therefore, unmethylated and methylated Cs can be accurately inferred from sequencing analysis RRBS is the first NGS-based method for DNA methylation profiling Figure ). After two rounds of PCR enrichment, the amplified scRRBS libraries were pair-ended sequenced and the sequencing data indicated that scRRBS was capable of covering up to 1.5 million CpGs To generate single-cell RRBS (scRRBS) libraries, Guo and colleagues minimized the library DNA loss by performing five consecutive reactions from cell lysis, MspI digestion, end-repair, A-tailing to adapter ligation and the bisulfite conversion in one tube (Extended-representation bisulfite sequencing (XRBS) deliberately uses Illumina adapters with unphosphorylated bottom strands. After the sodium bisulfite treatment, the converted DNA fragments have only a 5'-terminal adapter, and the 3'-terminal adapter sequences are introduced using random hexamer-tagged PCR primers. As a result, XRBS captures more CpG sites within two MspI cleavage sites MSREs are a group of restriction endonucleases that cannot cleave DNA if their recognition sites contain methylated cytosines. Interestingly, some have isoschizomers with identical recognition sequences but are insensitive to methylation. MSRE-based assays can use multiple enzymes to extend genomic coverage, and the missed CpG sites in the enzyme binding sites are methylated and inferred from the sequencing analysis Figure ), can only detect a limited number of CpGs The first MSRE-based single-cell DNA methylation assay was described in 2011, and the method, restriction enzyme-based single-cell methylation assay (RSMA)  is another MSRE-based sequencing technology in vitro transcription and PCR application of transcribed RNAs. Thus, scMspJI-seq specifically enriches methylated sites and has been used to study the dynamics of DNA demethylation in early development In contrast to MSRE-based assays, which generally detect symmetric DNA methylation on both the plus and minus strands, single-cell MspJⅠ-dependent sequencing (scMspJI-seq) is designed to assess strand-specific 5mC Table 3.In conventional bisulfite-based sequencing methods, fragmented DNA is typically tagged by methylated adapters prior to bisulfite conversion. PBAT implements an initial bisulfite treatment protocol and then uses random primers to amplify bisulfite-converted DNA fragments, allowing more DNA fragments to be subsequently amplified and sequenced Figure ). Two critical steps, direct bisulfite treatment of the single-cell lysate and amplification of converted DNA before the purification of synthesized first-strand DNA, minimize DNA loss. In addition, the use of modified random hexamers eliminates the need to trim artificial bases introduced during conventional library preparation. On average, scBS-seq can detect 3.4 million CpGs per single cell. However, the method often fails to detect methylation differences for some alleles due to allele dropout caused by bisulfite conversion and enrichment-induced bias Single-cell bisulfite sequencing (scBS-seq) is the first PBAT-based genome-wide methylation sequencing method in which bisulfite-treated DNA is subjected to two cycles of random primer extension The scWGBS method developed in the Bock laboratory primarily uses a commercial product, the EpiGnome™ Methyl-Seq Kit , to generate sequencing libraries Single-cell PBAT (scPBAT) uses a pair of primers, the Bio-PEA2-W4N4 primer (5'-biotin-ACACTCTTTCCCTACACGACGCTCTTCCGATCTWWWWNNNN-3') and the PE-index-W4N4 primer (5'-CAAGCAGAAGACGGCATACGAGATXXXXXXXXXGTAAAACGACGGCCAGCAGGAAACAGCTATGACWWWWNNNN-3') for the first- and second-strand DNA synthesis sequentially. It is noteworthy that scPBAT does not undergo PCR-based amplification and is tailored for methylation analysis of repetitive regions Some single-cell methylation libraries start from single-cell lysates, while others utilize single-cell nuclei instead, such as single-cell combinatorial indexing for methylation analysis (sci-MET), sciMETv2 linear amplification (sciMETv2.LA), sciMETv2 splint ligation (sciMETv2.SL), and single-nucleus methylcytosine sequencing (snmC-seq) Both snmC-seq and snmC-seq2 rely on barcoded random primers to amplify bisulfite-converted DNA and on the adaptase (Swift Biosciences) to tag a short oligo tail at the 3'-terminal of synthesized DNA. Sequencing libraries are generated by PCR using a pair of custom indexing primers containing Illumina P7 and P5 sequences, respectively. Several modifications, including the use of a different degenerate random primer  and the deactivation of free random primers and dNTP, dramatically improve the library qualities of snmC-seq2 compared to snmC-seq, such as better mapping rates (64.7±2.6% vs. 52.4±4% for the mouse genome), fewer artifactual reads (6.1±5.2%) and improved library complexity (30.8±7.5% vs. 22.2±5.7) Table 4). Applications of these technologies have greatly improved our understanding of cellular and molecular heterogeneities and the internal correlations within multi-omics in development and human disease.The emergence of single-cell genomic, epigenomic, and transcriptomic sequencing methods motivates scientists to explore technologies for parallel single-cell multi-omics profiling. Remarkably, single-cell multi-omics sequencing technologies have been reported and are summarized  or accessible chromatin regions often overlap with transcriptional regulatory elements. Methods capable of simultaneously assessing the chromosomal accessibility and DNA methylation include single-cell nucleosome occupancy and methylation (scNOMe-seq) The single-cell multiple omics assay for genotype, gene expression, and methylation profiling (sc-GEM) combines the single-cell restriction analysis of methylation (SCARM) technique with NGS-based single-cell genotyping. Targeted-gene transcripts are assessed by qPCR. Most of the experimental procedures are performed on the Fluidigm C1 single-cell auto-prep system Single-cell nucleosome, methylation, and transcription sequencing (scNMT-seq) can concurrently evaluate chromatin accessibility, DNA methylation, and gene transcription by applying M.CviPI to label the open chromatin regions. The method also uses oligo-dT-coated magnetic beads to precipitate mRNAs for RNA-seq library construction, leaving M.CviPI-treated DNA in the lysate for methylation analysis Figure 5).Conventional sequencing approaches require thousands to millions of cells and provide average changes at the genetic, epigenetic, and transcriptional levels. However, bulk sequencing technologies cannot reveal what is happening in rare cells or subpopulations of cells. Single-cell sequencing technologies provide tools to precisely profile DNA methylation and other omics for individual cells. Applications of single-cell DNA methylation and single-cell multi-omics sequencing are primarily focused on the development and human disease, particularly cancer Mammalian life begins at fertilization, where both paternal and maternal genomes undergo global demethylation, reaching its lowest level at the blastocyst stage Mouse is the most commonly used model animal to study early mammalian development. Single-cell DNA methylation analysis of the paternal and maternal genomes in mouse zygotes shows that the demethylation process of the genic region is faster than that of the intergenic regions de novo methylation of the genome After blastocyst implantation, DNMT3A and 3B catalyze Extensive studies show that epigenetic abnormalities are closely associated with the development and evolution of cancer Cellular heterogeneity is closely associated with cancer development, evolution, and response to treatment. Many studies have used single-cell DNA methylation sequencing to investigate cellular heterogeneity in cancers, such as colorectal cancer, breast cancer, liver cancer, and chronic lymphocytic lymphoma (CLL) Understanding tumor heterogeneities and clonal evolutionary trajectories could help scientists elucidate the underlying mechanisms and develop specific targeted drugs. Using scTrio-seq, Hou et al. reported two subpopulations based on the CNV, methylation, and transcriptional profiles of 25 single cells isolated from the liver tissue of one patient with hepatocellular carcinoma Single-cell multi-omics sequencing technology is able to identify differentially expressed and differentially methylated genes in colorectal cancer, which can be used as biomarkers to guide targeted therapy for patients Circulating tumor cells (CTCs) are cancer cells shed from primary or metastatic tumors into the peripheral blood. CTCs are rare, and often fewer than ten cells can be isolated from 10 ml of peripheral blood Applications of single-cell DNA methylation sequencing and single-cell multi-omics sequencing technologies have also been extended to other research areas, such as neuroscience and aging. DNA methylation in neurons exhibits a unique feature, with a significant amount of methylated cytosine at CpH sites (H=A/T/C) in post-mitotic human and mouse neurons A hallmark change of aging is genome-wide DNA hypomethylation Over the last two decades, DNA methylation profiling technologies have changed dramatically from Sanger sequencing-based low-throughput to NGS-based high-throughput, from bulk DNA/RNA inputs to requiring only single cells Conversely, TET-assisted pyridine borane sequencing (TAPS) is based on TET oxidation of 5mC and 5hmC to 5caC, followed by pyridine borane reduction of 5caC to dihydrouracil. Enzyme-based bisulfite conversion is milder and generally does not cause DNA damage. TAPS can effectively identify modified cytosines with better mapping rates and uniform coverage Sequencing costs have fallen dramatically over the last two decades, but profiling genetic and epigenetic changes at the single-cell level remains a challenge for many academic laboratories. One critical reason is that single-cell-based assays typically require sequencing hundreds or even thousands of single cells to obtain a comprehensive population-level picture Single-cell DNA methylation sequencing has been widely used to profile rare cells and investigate cellular heterogeneity. CTCs preserve tumor genetic and epigenetic information well and are excellent candidates for cancer prognosis and diagnosis Finally, the role of DNA methylation in gene regulation is complex


After induction, 59 (46%) patients were mutation-negative (0.0024 VAF detection limit) and 69 (54%) had ≥1 persisting NGS-detectable mutation. Compared with NGS-negative patients, NGS-positive patients had shorter overall survival  and a shorter time to relapse . Among 95 patients with a complete morphologic remission (CR), 43 (45%) were MRD-positive by NGS and 52 (55%) were MRD-negative. These MRD-positive CR patients had a shorter overall survival  than did the MRD-negative CR patients. Post-treatment persisting MRD positivity, defined by the same NGS-based test used at diagnosis, is thus a more sensitive biomarker for low-level leukemic clones compared to traditional non-molecular methods and is prognostic of subsequent relapse and death.Treated AML patients often have measurable residual disease (MRD) due to persisting low-level clones. This study assessed whether residual post-treatment somatic mutations, detected by NGS, were significantly prognostic for subsequent clinical outcomes. AML patients ( Acute myeloid leukemia (AML) is an aggressive clonal hematopoietic stem cell malignancy with a five-year overall survival of ~30%. Although most patients with AML achieve a morphologic complete remission (CR) after intensive induction chemotherapy, approximately half later relapse, with a poor subsequent prognosis. This high rate of relapse implies the post-treatment low-level persistence of residual leukemic clone(s) that were not detected by routine disease monitoring methods. The detection of this post-treatment measurable residual disease (MRD), by a number of different laboratory methods in AML-focused research labs, has been shown to be both a potent prognostic biomarker for long-term disease outcomes (relapse and death) and a shorter-term predictor of early relapse –4.NPM1, typically require less technical sophistication but are only applicable to a minority of AML patients, thus leaving most patients without a molecular approach to monitor MRD. Multi-gene NGS-based assays for AML, however, are becoming routinely available in clinical diagnostic labs, for informing targeted therapy and/or risk stratification in pre-treatment AML diagnostic samples.Although MRD is a proven prognostic biomarker in AML, its real-world mass implementation by more oncologists and laboratories is likely curtailed by the requirement for specialized MRD-specific laboratory methods, equipment, and expertize that are difficult to validate, standardize, and implement in a routine clinical diagnostic lab. Flow cytometric assays for AML MRD, for example, are technically complex and difficult to quality control , 5. SingWe hypothesize that the same sensitive clinical laboratory-validated NGS assay, initially applied at the time of the AML diagnosis to define the somatic mutation profile, can also be applied to define molecular MRD after treatment. Utilization of the same clinical diagnostic lab NGS assay both before and after treatment would likely lead to MRD monitoring being applied to more AML patients – by reducing the practical and/or economic barriers associated with the routine clinical diagnostic lab having to send the post-treatment sample to an outside referral lab with appropriate expertise. This local lab in-house serial testing approach could also assist in confirming more NGS-defined somatic mutations as clinically useful MRD markers, and thus further enhance the applicability of these assays to more patients. To assess this hypothesis in a real-world diagnostic lab environment, we utilized the same sensitive clinically validated NGS assay to both define the somatic mutation profile of pre-treatment AML patients and subsequently measure post-treatment MRD – and confirm its prognostic capabilities for long-term clinical outcomes.This was a retrospective, single-center study of 128 patients with a diagnosis of AML as per consensus WHO diagnostic criteria (excluding acute promyelocytic leukemia) . We inclPatient response to treatment was evaluated by routine morphologic, cytogenetic, and/or flow cytometric analyses of post-treatment bone marrow samples, as performed in the diagnostic clinical laboratories at OHSU, with previously described methods . CompletTM-based multiplex PCR methodology was used to generate the amplicon-based sequencing libraries that were sequenced on a PGM sequencer (Life Technologies), as previously described ) age at diagnosis was 58 (44–65) years. Of these 128 subjects, 23 (18%) had secondary (versus de novo) AML, and 45 (35%) had adverse disease prognostic characteristics as per the ELN classification system . Additio28 AML paFLT3 (33% of subjects), NPM1 (32%), NRAS (30%), and DNMT3A (25%) , RUNX1 (12%), and IDH1 (10%)  Fig. .Fig. 1FrDNMT3A, TET2, and/or ASXL1 (DTA); mutations in these genes are commonly found in benign age-related clonal hematopoiesis, often persist after therapy without prognostic significance, and have been recommended by consensus experts to be excluded as reliable molecular MRD markers  ]  and death   had a significantly shorter overall survival (median 17 months) compared to the 59 subjects who cleared each of their known non-DTA pre-treatment somatic mutations  Fig. . After c1)] Fig. . To asseP = 0.47) ] , 43 (45%) subsequently died. In comparison, of the 33 subjects with treatment-refractory disease (by the same methods), 17 (52%) subsequently died (supplementary Table 47) Fig. . The hig9)] Fig. .Fig. 4SuIn this retrospective study of 128 AML patients who were tested by the same sensitive 42-gene targeted NGS panel both at diagnosis and after their initial induction chemotherapy, the post-treatment persistence of a detectable somatic mutation (MRD-positivity) was shown to be independently predictive of a significantly inferior clinical outcome (earlier relapse and death). The assessment of post-induction MRD by an NGS-based method that was universally applied to all pre-and post-treatment AML samples in a routine diagnostic pathology lab was also shown to be more analytically sensitive than traditional non-molecular MRD-detection methodologies such as morphology, flow cytometry, and cytogenetics/FISH. In particular, only 26% of the post-treatment AML subjects were MRD-positive by these traditional diagnostic methods , compared to 54% who were MRD-positive by NGS. Furthermore, this significant increase in MRD diagnostic yield afforded by NGS was not just indicative of an analytically superior laboratory detection methodology for post-treatment persisting clonal disease, but was also shown to be a clinically relevant prognostic biomarker for predicting long-term disease outcomes, as confirmed by the significantly shorter overall survival among the 45% of CR subjects who were NGS-positive (as compared to those CR subjects with no persisting mutations). Conversely, the attainment of a traditional non-molecular CR was not prognostic of an improved overall survival, likely due to the persistence of low-level post-treatment AML clones detectable only by NGS. The detection of post-treatment MRD by this more sensitive NGS method (in more than double the number of subjects detectable by non-molecular methods) thus defines a routinely available laboratory test to identify more AML patients with low-level residual disease who are most likely to relapse and die. Identifying these high-risk patients is a necessary first step toward applying more intensive induction and/or consolidation-based therapies towards the ultimate therapeutic goal of disease eradication (to impart the best long-term prognosis). Perhaps equally important, however, is the clinical utility of this same sensitive NGS MRD method to identify the ~50% of AML patients who are at lower risk after their initial therapy and may not require additional (often toxic) treatments to achieve improved clinical outcomes, beyond standard of care consolidation (with or without stem cell transplant).The NGS-based assay approach that was used in this study was novel in that the same clinically validated method targeting 42 gene targets was routinely applied to all pre- and post-treatment samples [extending 40 months and 6 samples per subject (median) after diagnosis] within a conventional diagnostic clinical pathology laboratory. Although this “all time point” universal NGS assay did require a manual inspection of NGS reads for low-level trackable mutations (after treatment), and an initial extensive validation phase to meet stringent US government regulatory requirements for a clinical diagnostic test, there was no need for additional dedicated post-treatment MRD-specific methods, requiring specialized reagents, equipment, and/or bioinformatics. This practical approach to MRD assay validation may allow analogous molecular MRD assays to become more universally available in routine diagnostic clinical labs, thus improving patient access to these proven life-extending biomarkers, beyond the more restricted and specialized research lab settings where such technologies have been traditionally available.Our NGS-based MRD assay that could be practically and equivalently applied to all pre- and post-treatment AML samples was not only more analytically sensitive and prognostically relevant than the non-molecular assays traditionally used to assess disease burdens, but it was also more clinically applicable. In particular, 85% of our AML patients had at least one somatic non-DTA mutation that could be monitored by post-treatment clinical-grade NGS (relative to research-based exome sequencing) . The percentage of mutation-negative AML cases not eligible for NGS-based disease monitoring will, of course, be dependent on the number of genes being targeted by the NGS assay. However, when designing NGS assays, most clinical laboratories must appropriately balance the inherent desire to target more genes with the ensuing inevitable disadvantage of requiring more overall sequence depth to achieve a sufficiently low mutant allele detection limit - with a consequent increase in cost and turnaround time. The 42-gene NGS panel used in this study was large enough to detect an NGS-trackable mutation in 85% of AML patients, but small enough to achieve sufficiently deep sequencing coverage to confer a low-level detection limit below 0.003 VAF.The poor prognosis imparted by persisting post-treatment NGS-detectable MRD has also been reported by other investigators, at varying AML post-treatment time points, including after initial induction chemotherapy , 16, befNPM1 C-terminal insertion, have been well documented to impart a significant subsequent relapse risk when they persist after AML therapy [DNMT3A, TET2, and ASXL1 (DTA) genes, which typically originate in the early preleukemic and/or clonal hematopoiesis stage of disease [The complexity and non-standardization of MRD assessments in AML stems from not only the choice of methodology  but also the choice of gene/protein targets. For NGS-based MRD assays, which typically target dozens of myeloid malignancy-related genes, there is abundant evidence confirming that not all genes and/or mutations are equivalently useful for MRD prognosis. For example, some mutations, most notably the common  therapy , 15. Alt disease , 20, 21.Relapsed AML is often clonally evolved compared to the pre-treatment precursor clone(s), frequently with the addition of new or expanding mutations, beyond those initially seen at diagnosis. These newly evolved or expanding mutations will not be detectable at low clonal burdens (<0.02 VAF) before frank relapse using our “trackable mutation” strategy for MRD monitoring, a specific limitation of this study. The median clonal mutation burden after treatment, however (0.064 VAF) will be easily detectable by NGS, without any pre-test knowledge of its prior presence. Other limitations of this study include its relatively small size, restriction of MRD monitoring to a single post-treatment time point, and the single-site retrospective patient cohort. Potential selection bias in the AML study subjects, always a concern in retrospective studies, has been minimized by including all AML patients at our institution with serial NGS monitoring who consented to participate in the larger Beat AML study, which has done extensive genomic and drug susceptibility characterizations of this disease , 22. TheThe vast majority of patients in studies assessing the prognostic value of MRD, including ours, were treated with intensive chemotherapy modalities, typically including an initial induction with anthracyclines and cytarabine (the standard “7 + 3” therapy), a therapeutic approach that has not substantively changed in decades. In elderly patients and/or those deemed unfit to tolerate this traditional intensive chemotherapy regimen, an exciting new treatment option is the BCL-2 inhibitor venetoclax, which has shown good tolerability, high response rates, and prolonged overall survival when combined with hypomethylating agents or low dose cytarabine , 24. An In summary, we have shown that in newly diagnosed AML subjects initially treated with intensive induction chemotherapy, the attainment of an NGS-defined absence of detectable molecular MRD at the end of the induction phase is a significant and independent prognostic marker for subsequent relapse and death. The increasing availability of these sensitive MRD assays in a growing number of clinical diagnostic labs should allow the identification of the significant fraction of patients (45% in our cohort) with low-level persisting disease (and its associated poorer outcomes), even after achieving a complete remission by less sensitive laboratory methods. Given this valuable prognostic information, future clinical trials should assess whether more intensive therapies directed specifically at MRD-positive patients can improve long-term outcomes without excessive toxicity. For example, in AML patients with an initial favorable-risk genetic profile who remain MRD-positive after intensive induction chemotherapy, would subsequent stem cell transplantation ameliorate the relapse risk? MRD analyses are also now being used in clinical trials of novel induction and/or consolidation therapies as a surrogate endpoint for longer-term relapse and death – to hopefully accelerate the current suboptimal decade-long drug development timeline. These highly sensitive NGS assays will be a crucial tool for defining the ultimate goal for patients undergoing treatment for this deadly malignancy, namely disease eradication.Supplementary Tables (3) with Legend


Single-cell sequencing (scRNA-seq) technology provides higher resolution of cellular differences than bulk RNA sequencing and reveals the heterogeneity in biological research. The analysis of scRNA-seq datasets is premised on the subpopulation assignment. When an appropriate reference is not available, such as specific marker genes and single-cell reference atlas, unsupervised clustering approaches become the predominant option. However, the inherent sparsity and high-dimensionality of scRNA-seq datasets pose specific analytical challenges to traditional clustering methods. Therefore, a various deep learning-based methods have been proposed to address these challenges. As each method improves partially, a comprehensive method needs to be proposed. In this article, we propose a novel scRNA-seq data clustering method named AttentionAE-sc (Attention fusion AutoEncoder for single-cell). Two different scRNA-seq clustering strategies are combined through an attention mechanism, that include zero-inflated negative binomial (ZINB)-based methods dealing with the impact of dropout events and graph autoencoder (GAE)-based methods relying on information from neighbors to guide the dimension reduction. Based on an iterative fusion between denoising and topological embeddings, AttentionAE-sc can easily acquire clustering-friendly cell representations that similar cells are closer in the hidden embedding. Compared with several state-of-art baseline methods, AttentionAE-sc demonstrated excellent clustering performance on 16 real scRNA-seq datasets without the need to specify the number of groups. Additionally, AttentionAE-sc learned improved cell representations and exhibited enhanced stability and robustness. Furthermore, AttentionAE-sc achieved remarkable identification in a breast cancer single-cell atlas dataset and provided valuable insights into the heterogeneity among different cell subtypes. Single-cell sequencing (scRNA-seq) has been widely used in numerous biological studies to reveal heterogeneity at the cellular level. Accurate cell type identification serves as the foundation for scRNA-seq data analysis, and unsupervised cluster analysis is commonly employed when an appropriate reference is not available. However, the inherent sparsity and high-dimensionality of scRNA-seq datasets pose specific analytical challenges to traditional clustering methods. To address this, we propose a novel scRNA-seq data clustering method named AttentionAE-sc (Attention fusion AutoEncoder for single-cell). By integrating denoising representation learning and cluster-friendly representation learning through an attention mechanism, AttentionAE-sc demonstrated outstanding performance in the evaluation phase. Firstly, when compared to benchmark methods on the real scRNA-seq datasets, AttentionAE-sc consistently achieved superior external and internal clustering evaluation metrics. Secondly, AttentionAE-sc exhibited robustness and stability across various experimental conditions. Lastly, AttentionAE-sc not only delivered excellent clustering results but also unveiled potential biological insights on a breast cancer single-cell atlas dataset. Single Cell RNA sequencing (scRNA-seq) techniques facilitate exploring the heterogeneity and diversity of cells at a cellular level, providing a more detailed mechanism for a variety of biological researches , 2. AccuIn the last few years, various clustering methods involved in other fields have been applied to scRNA-seq datasets. For example, RaceID  applied To efficiently integrate single-cell clustering methods with dimension reduction, data denoising and batch effects removal processes, various deep learning-based tools have been proposed. DCA  raised aDifferent from the previous methods, GNN-based methods need to manually construct the topologic information between cells in the absence of prior knowledge. Two types of graphs are mainly included: cell-gene graph and cell-cell graph. scDeepSort  proposedMotivated by the researches above, we propose an attention mechanism-based scRNA-seq clustering method named AttentionAE-sc (Attention fusion AutoEncoder for single-cell). As shown in scanpy package . Th. ThBCAS3In this work, we propose AttentionAE-sc, a novel deep learning-based scRNA-seq clustering method, relying on an attention-based information fusion block, which combines the denoising autoencoder with the graph autoencoder to learn a clustering-friendly cell representation. It can not only deal with sparsity and dropout events of scRNA-seq datasets by ZINB-based autoencoder, but also directly considers the relationship among cells to guide the dimension reduction and get a clustering-friendly representation. Therefore, AttentionAE-sc can easily extract denoising features from the sparse scRNA-seq data, and the ideal clustering partition is obtained without specifying the number of groups.AttentionAE-sc acquired an excellent performance on 16 scRNA-seq datasets, with an average of 60% ARI score and 18% NMI score higher than 9 baseline methods. Compared with these state-of-art methods, AttentionAE-sc achieved an exceptional performance, including a high internal metric silhouette score that reflects the quality of cell representations. Furthermore, we explored the relationship between the outstanding clustering results and the clustering-friendly cell representation obtained during the optimizing clustering stage. We also analyzed the stability and robustness of AttentionAE-sc and examined the contribution of each module component. Moreover, experiments conducted on BRCA datasets demonstrated the excellent performance of AttentionAE-sc in clustering analysis and its capability to unveil the biological significance within scRNA-seq datasets.However, AttentionAE-sc as well as some other methods , 11 aimsS1 FigDifferent embeddings from DAE and GAE are combined by a multi-head attention layer and residual connection is adopted to promote the training efficiency of DAE module.(TIF)Click here for additional data file.S2 Figa. NMI scores of AttentionAE-sc and 9 baseline methods. b. Silhouette scores of 6 community detection-based methods (including AttentionAE-sc). c. Davies-Bouldin score of 6 community detection-based methods (including AttentionAE-sc). Arithmetic mean is taken as results of each dataset after running each method five times under different random seeds. Methods that need to specify the number of clusters are marked with an asterisk (*).(TIF)Click here for additional data file.S3 FigThe 5 subfigures in each row are the cell projections obtained from the 2-dimentional UMAP features, which are calculated from the cell representation obtained by the different methods. Each dataset consists of 2 rows of subfigures that adopt the ground truth or predicted labels by different method respectively (different color clusters in the subfigure). In summary, AttentionAE-sc obtained the larger distinction between each group and the degree of internal tightness and the cell annotation is closer to the ground truth.(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Figa. The ARI score of clustering results with random manual dropout rate of gene expression values set as 10%, 20%, 30% and 50% in the scRNA-seq datasets. All experiments run 5 times with different random seeds. Base (the pink box) is the performance of AttentionAE-sc without manual dropout. Compared AttentionAE-sc  with ordinary DAE , AttentionAE-sc is less influenced. b. The ARI score of clustering results with random and stratified input down-sampling rate of cells set as 20%, 40%, 60% and 80% respectively in the scRNA-seq datasets. All experiments run 5 times with different random seeds. Base (the pink box) is the performance of AttentionAE-sc with the whole scRNA-seq datasets as input. The average score is displayed below the x label on each box.(TIF)Click here for additional data file.S7 Figa), the resolution parameters in the Leiden algorithm (b), and the number of highly variable genes (c).Three hyperparameters significantly influence the clustering performance of AttentionAE-sc: the number of attention heads in the information fusion blocks ((TIF)Click here for additional data file.S8 FigNMI, silhouette score (ASW) and Davies-Bouldin score (DB) are used as metrics. Four conditions were tested, including the absence of the information fusion block (wo attn), the absence of the ZINB loss function (wo zinb), the absence of residual connections (wo res), and the absence of GAE (wo gnn).(TIF)Click here for additional data file.S9 Figa. Venn diagram comparing the difference of highly variable genes after the preprocessing process between original clustering stage and sub-clustering stage. b. The results of KEGG enrichment analysis in the cluster 6 and cluster 31 based on the acquired DEGs in the (TIF)Click here for additional data file.S10 FigTo assess the capacity for recognizing rare cell types, we opted to visualize cell types with a limited number of cells in the datasets. The results demonstrate that AttentionAE-sc possesses the capability to predict clusters with a small number of cells and distinguish rare cell types.(TIF)Click here for additional data file.S1 TableOverview contains code source and operational environment for experiment in the real scRNA-seq datasets.(DOCX)Click here for additional data file.S2 TableFor each clustering, the threshold for considering the presence of differential expression is 0.05 .(DOCX)Click here for additional data file.S3 TableCompare with another GNN-based methods in the 14 real scRNA-seq datasets. Nvidia RTX 3070Ti (8 GB RAM) was used in the gpu test.(DOCX)Click here for additional data file.


The results of human observational studies on the correlation between gut microbiota perturbations and polycystic ovary syndrome (PCOS) have been contradictory. This study aimed to perform the first systematic review and meta-analysis to evaluate the specificity of the gut microbiota in PCOS patients compared to healthy women.I2) for alpha diversity were calculated. Qualitative syntheses of beta-diversity and microbe alterations were performed.Literature through May 22, 2023, was searched on PubMed, Web of Science, Medline, Embase, Cochrane Library, and Wiley Online Library databases. Unreported data in diversity indices were filled by downloading and processing raw sequencing data. Systematic review inclusion: original studies were eligible if they applied an observational case-control design, performed gut microbiota analysis and reported diversity or abundance measures, sampled general pre-menopausal women with PCOS, and are longitudinal studies with baseline comparison between PCOS patients and healthy females. Systematic review exclusion: studies that conducted interventional or longitudinal comparisons in the absence of a control group. Two researchers made abstract, full-text, and data extraction decisions, independently. The Joanna Briggs Institute Critical Appraisal Checklist was used to assess the methodologic quality. Hedge’s g standardized mean difference (SMD), confidence intervals (CIs), and heterogeneity  that investigated gut microbiota by collecting stool samples were included, with 26 and 27 studies having provided alpha-diversity and beta-diversity results respectively. A significant decrease in microbial evenness and phylogenetic diversity was observed in PCOS patients when compared with control participants . We also found that reported beta-diversity was inconsistent between studies. Despite heterogeneity in bacterial relative abundance, we observed depletion of Lachnospira and Prevotella and enrichment of Bacteroides, Parabacteroides, Lactobacillus, Fusobacterium, and Escherichia/Shigella in PCOS. Gut dysbiosis in PCOS, which might be characterized by the reduction of short-chain fatty acid (SCFA)-producing and bile-acid-metabolizing bacteria, suggests a shift in balance to favor pro-inflammatory rather than anti-inflammatory bacteria.Twenty-eight studies (Gut dysbiosis in PCOS is associated with decreased diversity and alterations in bacteria involved in microbiota-host crosstalk.PROSPERO registration: CRD42021285206, May 22, 2023.The online version contains supplementary material available at 10.1186/s12916-023-02975-8. PCOS is one of the most common female reproductive disorders and affects 5–20% of reproductive-age women worldwide , 2. It iThe gut microbiota is associated with a wide range of diseases, including reproductive and gynecological diseases –6. ThereHowever, many studies that contain cohorts from a range of regions and ethnicities report inconsistent and, occasionally, contradictory results. Though this variance may be explained by limited sample size and different choices of sequencing platforms, the heterogeneity among studies should be addressed to identify disease-specific bacterial biomarkers across a population, which could deepen our understanding of disease pathogenesis and would enable the development of new and effective therapies. Therefore, there is a need to compare microbial perturbations and differentiate a set of homogeneous gut microbial features from different studies. Meta-analysis is an effective method for integrating existing knowledge to uncover commonalities across different research, and interestingly, there is currently a lack of published meta-analyses that explore bacterial characteristics in PCOS patients. The present meta-analysis fills this gap in the literature by evaluating alterations in the gut microbiota of patients with PCOS across multiple studies and aims to elucidate consistent gut microbiota profiles and features with therapeutic potential.https://www.crd.york.ac.uk/prospero/) and conducted and reported according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline . Supplemental Table 2.[Detailed characteristics of the included studies measured alpha diversity indexes]. Supplemental Table 3. [Stool sample processing methods in the included studies]. Supplemental Table 4. [Publish bias assessment by egger regression test in alpha diversity indexes]. Supplemental Table 5. [Subgroup and meta-regression analysis of α-diversity (Chao 1) difference between PCOS patients and healthy control]. Supplemental Table 6. [Subgroup and meta-regression analysis of α-diversity (Observed species) difference between PCOS patients and healthy control]. Supplemental Table 7. [Subgroup and meta-regression analysis of α-diversity (PD) difference between PCOS patients and healthy control]. Supplemental Table 8. [Sensitivity analyses of α-diversity indices difference between PCOS patients and healthy control]. Supplemental Table 9. [Summary of various measures of beta-diversity]. Supplemental Table 10. [Univariate logistic-regression analysis of factors potentially associated with Beta-diversity]. Supplemental Table 11. [Summaries of the bacterial function description from the included studies]. Supplemental Figure 1. [Funnel plot (A) and trim-and-fill funnel plot (B) assessing publication bias in themeta-analyses of Chao 1 index]. Supplemental Figure 2. [Forest plot by trim-and-fill analysis of Chao 1]. Supplemental Figure 3. .Additional file 2: SupplementalTable 12. [the clinical characteristics and sequencinginformation of the studies reported alpha diversity].


Obsessive-Compulsive Disorder (OCD) is a chronic disabling condition, with considerable lifetime prevalence. There are interindividual differences regarding personality dimensions and how they affect obsessive- compulsive (OC) symptomatology. Furthermore, there is a connection between OC symptoms and the use of maladaptive emotion regulation strategies (expressive suppression) instead of using more cognitive reappraisal.Explore the relationship between personality, emotion regulation strategies and OC symptoms by testing a path analytic model in a sample of healthy participants and in a sample of OCD patients.Two samples of participants were utilized. Sample 1 consists of 787 healthy participants from the general Portuguese population. Sample 2 is composed of 33 OCD patients and 32 Healthy Controls (HC). Participants completed different scales: Emotion Regulation Questionnaire (ERQ), Neuroticism-Extraversion-Openness Five-Factor Inventory (NEO-FFI) and Obsessive-Compulsive Inventory-Revised (OCI-R), through online surveys (sample 1) or a clinical interview (sample 2). These questionnaires were then analyzed with a path-analytic approach.In sample 2, we found significant differences between OCD patients and HC in every OCI-R subscale, except Hoarding and Neutralizing. In the NEO-FFI, OCD patients scored higher on Neuroticism and lower on Extraversion. No significant differences were found regarding the ERQ. Relatively to sample 1: path analysis results showed that 13,4% of the variance of OC symptoms was explained by the best-fitting model. Only Neuroticism and Extraversion were directly associated with higher OCI-R Total scores, whereas Agreeableness predicted less OC symptoms. The use of Expressive Suppression was associated with more OC symptomology, but no significant connection was found with Cognitive Reappraisal. Regarding sample 2, no model was found, showing no modifying effect of emotion regulation strategies on OC Symptoms.There is a deep-rooted interconnection between personality and emotion regulation regarding OC symptomatology in a sample of healthy participants but no effect of emotion regulation was seen regarding OCD patients.To sum up, promising results were obtained and it could be an important field for the OCD in terms of diagnostic, severity and treatment.None Declared


N-containing groups that are recognized by the proton-coupled organic cation antiporter might cross the BBB through CMT. PROTAC-antibody conjugates (PACs) might cross the BBB through RMT. Subsequently, such small molecule-based PROTACs released in the brain interstitial fluid would be transported into cells such as neurons through passive diffusion and then demonstrate arbitrary protein degradation. In this review, I introduce the potential and advantages of PROTAC delivery into the brain across the BBB through CMT or RMT using PACs in a non-invasive way.Drug development for neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease has challenging difficulties due to the pharmacokinetic impermeability based on the blood-brain barrier (BBB) as well as the blurriness of pharmacodynamic targets based on their unclarified pathogenesis and complicated progression mechanisms. Thus, in order to produce innovative central nervous system (CNS) agents for patients suffering from CNS diseases, effective, selective delivery of CNS agents into the brain across the BBB should be developed. Currently, proteolysis-targeting chimeras (PROTACs) attract rising attention as a new modality to degrade arbitrary intracellular proteins by the ubiquitin-proteasome system. The internalizations of peptide-based PROTACs by cell-penetrating peptides and that of small molecule-based PROTACs through passive diffusion lack cell selectivity. Therefore, these approaches may bring off-target side effects due to wrong distribution. Furthermore, efflux transporters such as multiple drug resistance 1 (MDR1) expressed at the BBB might interrupt the entry of small molecule-based PROTACs into the brain. Nonetheless, intelligent delivery using machinery systems to absorb the nutrition into the brain for homeostasis, such as carrier-mediated transport (CMT) or receptor-mediated transcytosis (RMT), can be established. PROTACs with  It is true that medical treatment has benefited humankind in terms of health and longevity but the development of new pharmaceutical agents has become more and more difficult due to lack of seeds such as druggable targets and potent compounds, huge cost, adverse side-effects, and technological problems such as poor stability, poor solubility, cell membrane impermeability, and wrong distribution. There remain many intractable diseases that distress patients extremely. Thus, novel therapeutic modalities are required to resolve this present situation. Among them, the proteolysis-targeting chimera (PROTAC)  1,2] is,2 is1,2]PROTAC is a novel therapeutic modality using the ubiquitin-proteasome system. PROTAC molecules degrade target proteins due to the endogenous decomposition system, different from the competitive inhibition or irreversible inhibition systems and slightly similar to the messenger RNA (mRNA) degradation system by short interfering RNAs (siRNA) called RNA interference. The ubiquitin-proteasome system is a crucial protein degradation system in all eukaryotes. Ubiquitin-activating enzymes (E1), ubiquitin conjugases (E2), ubiquitin ligases (E3), ubiquitin, and the 26S proteasome play essential roles in intracellular proteolysis  10,11].,11.10,11Canonical PROTACs are heterobifunctional molecules composed of a target protein ligand and an E3 ligase ligand via a suitable linker  12]. Th. Th12]. n = 115) were classified into a high-permeability class (n = 46), a moderate-permeability class (n = 44), and a low-permeability class (n = 25). CRBN-based PROTACs (n = 113) were classified into a high-permeability class (n = 59), a moderate-permeability class (n = 45), and a low-permeability class (n = 9). Surprisingly, it turned out that small molecule-based PROTACs showed high and moderate membrane permeability. However, it was uncertain whether the transport mechanisms were based on passive diffusion or carrier-mediated transportation . An. An21]. HD is a neurodegenerative disease caused by heterogeneous aggregation of mutated huntingtin proteins with normal huntingtin proteins . The patPROTACs can lower the intracellular tau and α-synuclein levels by degradation based on the ubiquitin-proteasome system. Nonetheless, how to deliver PROTACs selectively and effectively into the brain is an important problem. Carrier-mediated transport and receptor-mediated transcytosis are solutions.PROTACs for CNS diseases must enter the brain across the BBB. It was revealed that small molecule-based PROTACs showed high and moderate membrane permeability through passive diffusion ,16. ThusN-containing groups. Thus, some of them might cross the BBB as substrates of the proton-coupled organic cation antiporter. If they are not the substrates, the introduction of N-containing groups such as the 2-(dimethylamino)ethyl group to PROTAC molecules might enable them to be the substrates. Prodrug approaches would be effective when the interaction of PROTACs to the corresponding intracellular receptors is interrupted due to steric hindrance based on N-containing groups. The permeability and degradation activity of designed PROTACs with N-containing groups can be tuned by repeated evaluation in in vitro tests reflecting the assay results.Many existing small molecule-based PROTACs have ® is a drug delivery system into the brain across the BBB using anti-transferrin receptor ADCs with drugs [®, was clinically approved in Japan on 23 March 2021 for the treatment of Hunter syndrome. Anti-transferrin receptor ADCs can deliver drugs into the brain across the BBB through receptor-mediated transcytosis by the same strategy of J-Brain Cargo®. After the endocytosis triggered by ligand-receptor binding on the cell surface, antibodies are liberated from ADC-transferrin receptor complexes in endosomes as acidification. pH in endosomes gradually reduces from the early endosome (pH approximately 6.5) to late endosome (pH approximately 5.5) and then lysosome (pH approximately 4.5) by vacuolar H+-ATPase proton pumps [Substance deliveries into the brain across the BBB were carried out through receptor-mediated transcytosis using a transferrin receptor. J-Brain Cargoth drugs . Idursulth drugs , an antion pumps . pH-sensPROTAC-antibody conjugate (PAC) is a type of ADC, replacing ADC drugs with PROTACs. PACs, also called degrader-antibody conjugates (DACs), have attracted attention ,45. (i)A trastuzumab-PROTAC conjugate with a BRD4 ligand and a VHL ligand  showed B(ii)Several antibody-PROTAC conjugates with an ER ligand and an E3 ligase ligand were designed and evaluated for their ER degradation activity. Among them, an anti-HER2 antibody-PROTAC conjugate with an ER ligand and a VHL ligand  demonstr(iii)5a (drug-to-antibody ratio (DAR) 6.0)   is a membrane protein overexpressed in cancer cells. Anti-STEAP1 antibody-PROTAC conjugates with a BRD4 ligand and a VHL ligand, particularly STEAP1-AR) 6.0) , affordeer cells . The DC5DAR 5.9) , affordeIntravenously administered anti-transferrin receptor PACs would be delivered into the brain across the BBB, similarly to the anti-transferrin receptor ADC . If PACsMoreover, PACs have a long half-life based on neonatal Fc receptor (FcRn)-mediated salvation from the lysosomal degradation. FcRn is expressed at the apical membrane of the endothelial cells. Under physiological pH 7.0–7.4), antibodies and FcRn do not bind in the systemic circulation. Endocytosed by-stander antibodies bind FcRn under weak acidic conditions in endosomes as acidification. Antibody-FcRn complexes in endosome were exposed to the systemic circulation through exocytosis in the secretory pathway. Antibody-FcRn complexes were liberated under physiological pH ,7. The h.0–7.4, aNanoparticles as carriers used for drug delivery systems are biodegradable, biocompatible, stable, and easy to modify. NanoPROTACs are PROTACs using nanoparticles and make it possible to improve the delivery and mechanism of action of PROTACs . PROTAC-N-containing groups are delivered into the brain across the BBB. Amine derivatives are positively charged in the systemic circulation under physiological conditions and cannot penetrate the membrane through passive diffusion. Amine transporters absorb drugs with N-containing groups. The proton-coupled organic cation antiporter is suggested to mediate the absorption of drugs with N-containing groups, although its amino acid sequence has not been identified yet. Transporter-consciously designed compounds with N-containing groups can be transported in the capillary endothelial at the BBB. (ii) Some receptors such as transferrin receptors and insulin receptors at the apical membrane in the capillary endothelial cells deliver substances into the brain through receptor-mediated transcytosis. In the case of transferrin receptors, holo-transferrin (apo-transferrin-Fe (III) complex) binding to transferrin receptors mediates endocytosis. Holo-transferrin is liberated from the complex receptor as endosomal acidification and subsequently is released to the brain ISF through exocytosis based on the fusion between endosomes and the basolateral membrane. Artificial ligands of transferrin receptors, such as antibodies, induce receptor-mediated transcytosis. (iii) Endosomes triggered by clathrin-dependent endocytosis are 85–150 nm in diameter, which is enough to contain nanoparticles. Drugs encapsulated in nanoparticles are protected from enzymatic metabolism, like RNAs in exosomes. Anti-receptor antibody-loaded nanoparticles are ligands to the corresponding receptors that mediate transcytosis at the BBB. The clustering of ligand-receptor complexes derived from transferrin receptors enhances endocytosis [Neurodegenerative diseases such as AD, PD, and HD still remain intractable, because methods of effective treatment have not been established due to unclarified pathogenesis and complicated progression mechanisms. Moreover, most drugs do not reach the brain due to the impermeability based on the BBB. Innovative CNS agents to cross the BBB and elicit their activity there should be developed. At present, PROTAC is attention-getting as a novel modality. PROTAC molecules are constituted of heterobifunctional units of a target protein ligand and a E3 ligase ligand via a linker and induce the proteasomal degradation of the target proteins by the ubiquitin-proteasome system . They arocytosis ,55. AntiN-containing groups that are recognized by the proton-coupled organic cation antiporter [Therefore, as a summary, the delivery methods of PROTACs into the brain across the BBB are presented as three approaches: (i) PROTACs with tiporter , (ii) PRInspired readers are expected to suggest better ideas . Substan


Oryza sativa L.) and brown planthoppers  are used as a model system to study the molecular mechanisms underlying plant-insect interactions. Small RNAs (sRNAs) regulate growth, development, immunity, and environmental responses in eukaryotic organisms, including plants and insects. Recent research suggests that sRNAs play significant roles in rice-BPH interactions by mediating post-transcriptional gene silencing. The focus of this review is to explore the roles of sRNAs in rice-BPH interactions and to highlight recent research progress in unraveling the mechanism of cross-kingdom RNA interference (ckRNAi) between host plants and insects and the application of ckRNAi in pest management of crops including rice. The research summarized here will aid in the development of safe and effective BPH control strategies.Interactions between rice plants ( Oryza sativa L.) is a globally-important staple food which is susceptible to damage from hundreds of insect herbivores throughout its lifecycle , which is responsible for severely reduced rice yields and substantial economic losses each year -triggered immunity (PTI) . PTI actsRNAs are eukaryotic non-coding RNA molecules, approximately 20-30 nucleotides (nt) in length, which regulate gene expression via RNA silencing . AccordiR gene-mediated resistance, phytohormone signaling, callose deposition, reactive oxygen species (ROS) production, and secondary metabolite biosynthesis  genes have been identified in both wild and cultivated rice (R genes confer BPH resistance (Bph15) and susceptible rice varieties before and after BPH infestation, respectively. The identified DEMs were primarily involved in basal defense and BPH-specific resistance. Similarly, an integrated miRNA and mRNA analysis identified 217 DEMs between Bph6-carrying transgenic rice lines and wild type plants after BPH infestation  signaling (Osa-miR156-silenced plants (MIM156) exhibited increased resistance to BPH via upregulated expression of OsMPK3 and OsMPK6 and downregulated expression of OsWRKY70, a transcription factor which positively regulates JA signaling. Furthermore, the expression of the JA biosynthesis gene OsHI-LOX and the contents of JA and bioactive jasmonoyl-isoleucine (JA-Ile) were significantly reduced in MIM156 plants. Altogether, it appears that Osa-miR156 regulates JA biosynthesis and BPH resistance via the MAPK cascade in rice. In addition, Osa-miR162a is strongly induced by BPH herbivory in rice seedlings –OsF3H–flavonoid module (growth-regulating factor 8 (OsGRF8), the target gene of OsmiR396, exhibit enhanced BPH resistance due to downregulation of OsmiR396. Overall, it appears that OsmiR396-OsGRF8 modulates BPH resistance by regulating the expression of the flavanone 3-hydroxylase (OsF3H) gene, which is involved in flavonoid biosynthesis  have been confirmed to interact with NlInR genes, which are the part of IIS signaling pathway . Both Nlu-miR-1000-1-3p (Nlu-mir-9a (Ultrabithorax (NlUbx), and both were found to be differentially expressed between LW and SW BPH. Finally, the NlInRs/Nlu-mir-9a/NlUbx regulatory cascade appears to control wing dimorphism by regulating the host’s nutritional status  were injected into BPH, reducing both chitin biosynthesis and molting success  expression . The researchers identified 26 DEMs between these two BPH populations, suggesting that these BPH miRNAs may regulate adaptability to resistant rice varieties. However, the precise functions of these miRNAs require further confirmation.Finally, miRNAs play important regulatory roles in environmental responses such as the adaptation to resistant rice varieties. Botrytis cinerea)-derived sRNAs were found to be able to control the Arabidopsis thaliana RNAi system by binding to AGO1, ultimately silencing genes involved in plant immunity  expression in BPH through the cross-kingdom RNAi mechanism -overexpressing transgenic rice lines consistently resulted in reduced egg production and hatching success. These results suggest that these miRNAs confer resistance to BPH in rice, and that Osa-miR162a may be a potential target for BPH control  appear to play an important role in rice-BPH interactions through cross-kingdom regulation of NlTOR and NlVg expression, both of which regulate fecundity in BPH (Both of these rice-derived miRNAs (y in BPH Table 1.Nicotiana benthamiana) leaves induced the expression of defense-related genes and callose deposition, suggesting that these two proteins function as elicitors  is a novel concept based on the cross-kingdom RNAi mechanism. HIGS involves overexpressing insect-targeted double-stranded RNAs (dsRNAs) or artificial miRNAs in host plants to specifically block the expression of feeding- and survival-related genes in target pests and pathogens  Figure. In thislicitors . Comparesurvival . Similare plants . Althouge plants . These lMagnaporthe oryzae) by spraying dsRNA targeting the fungal pathogenicity gene MoDES1 (Sogatella furcifera) under laboratory conditions ” has been developed . As the e MoDES1 . Recentlnditions . The res in WBPH . These pA growing body of research has revealed the involvement of sRNAs in the interaction between rice and BPH. The majority of these sRNAs have been predicted and/or identified through multi-omics analyses, and their targets have been predicted computationally. However, many of these results still require experimental validation. Moreover, the molecular mechanisms underlying sRNA-mediated rice-BPH interactions remain poorly understood. The pathways of sRNA transfer between rice and BPH should also be comprehensively evaluated. Our growing understanding of cross-kingdom RNAi has paved the way for the development of promising agricultural pest control strategies, including HIGS and SIGS. Nevertheless, HIGS and SIGS face several technical challenges. The stability and uptake efficiency of dsRNA and sRNA need to be strengthened and off-target activities must be avoided. We predict that the development and application of environmentally-friendly RNAi-based technology will become an agronomic research focus, and that the communication of cross-kingdom sRNAs will emerge as a hot research topic.SJ: Project administration, Writing – original draft, Writing – review & editing. JX: Writing – review & editing. HT: Writing – review & editing. PL: Writing – review & editing. BY: Writing – original draft, Writing – review & editing. QL: Writing – original draft, Writing – review & editing.


Since the discovery of the LDL receptor in 1973 by Brown and Goldstein as a causative protein in hypercholesterolemia, tremendous amounts of effort have gone into finding ways to manage high LDL cholesterol in familial hypercholesterolemic (HoFH and HeFH) individuals with loss-of-function mutations in the LDL receptor (LDLR) gene. Statins proved to be the first blockbuster drug, helping both HoFH and HeFH individuals by inhibiting the cholesterol synthesis pathway rate-limiting enzyme HMG-CoA reductase and inducing the LDL receptor. However, statins could not achieve the therapeutic goal of LDL. Other therapies targeting LDLR include PCSK9, which lowers LDLR by promoting LDLR degradation. Inducible degrader of LDLR (IDOL) also controls the LDLR protein, but an IDOL-based therapy is yet to be developed. Among the LDLR-independent pathways, such as angiopoietin-like 3 (ANGPTL3), apolipoprotein (apo) B, apoC-III and CETP, only ANGPTL3 offers the advantage of treating both HoFH and HeFH patients and showing relatively better preclinical and clinical efficacy in animal models and hypercholesterolemic individuals, respectively. While loss-of-LDLR-function mutations have been known for decades, gain-of-LDLR-function mutations have recently been identified in some individuals. The new information on gain of LDLR function, together with CRISPR-Cas9 genome/base editing technology to target LDLR and ANGPTL3, offers promise to HoFH and HeFH individuals who are at a higher risk of developing atherosclerotic cardiovascular disease (ASCVD). Familial hypercholesterolemia (FH) is one of the prevalent and widely studied monogenic metabolic disorders caused by mutations in the low-density lipoprotein receptor (LDLR) gene ,2,3, witThe marketed agents known to lower LDL in hypercholesterolemic individuals include statins ,27,28, eLow levels of LDLR activity and high circulating LDL cholesterol were first demonstrated in vitro in the fibroblasts isolated from FH patients . LowerinAmong LDLR-independent pathways, suppressing apoB  productiANGPTL3 is a 460-amino-acid secretory glycoprotein expressed in the liver and implicated in the metabolism of lipids through modulation of LPL. ANGPTL3 inhibits lipoprotein lipase (LPL)  in tissuIn vivo proof-of-concept as well as pharmacology studies of therapeutic agents /new biological entities (NBEs)), are first evaluated in relevant animal models since testing agents may show species ,130,131 125I-labeled LDL from blood eight to ten times more rapidly than normal mice. The plasma concentrations of apoproteins B-100 and E, the two ligands for the LDL receptor, declined by more than 90 percent, but the concentration of another apoprotein, apoA-I, was unaffected. These studies demonstrated that overexpression of the LDL receptor can dramatically lower the concentration of LDL in vivo. To further confirm that, indeed, an association exists between LDL receptor activity and circulating LDL concentration, the same group of researchers [125I-VLDL and 125I-LDL were prolonged by 30-fold and 2.5-fold, respectively. Intravenous injection of a recombinant replication-defective adenovirus encoding the human LDL receptor restored expression of the LDL receptor protein in the liver and increased the clearance of 125I-VLDL, further confirming the inverse association of LDL receptor activity and LDL level and the role of the LDL receptor in reversing the hypercholesterolemic effects of LDL receptor deficiency. An LDLR deficient mouse model has been extensively employed to ask important biologic questions, including the mechanism of atherosclerosis progression by nuclear hormone receptors [In mice, a clear association between LDLR activity and circulating LDL levels was demonstrated by either overexpressing the LDL receptor or making LDL-receptor-deficient mice . Hofmannearchers  generateeceptors ,137,138,eceptors  and playeceptors .Both LDLR  as well +/− hamsters. Further validation of the heterozygous hamster (LDLR+/−) model for testing therapeutic agents was shown in a study where hyperlipidemic heterozygous hamsters were treated with the PCSK9 monoclonal antibody [The golden Syrian (GS) hamster has been used with increasing frequency to study lipoprotein metabolism ,148 and antibody . In thisFamilial hypercholesterolemia (FH) is caused primarily by loss-of-function mutations in the LDLR gene ,80, lead+/−) showed similar characteristics as human heterozygotes (LDLR+/−) and a similar response when treated with statins . Homozygous (LDLR−/−) pigs also showed human-like advanced plaques and responded to statin (pitavastatin) treatment [−/−) and heterozygotes (LDLR+/−) Yucatan miniature pigs, another class of drug candidate, bempedoic acid, which inhibits ATP citrate lyase, showed a reduction in LDL and lesion formation [Yucatan miniature pigs are well established as translational research models because of similarities to humans in physiology, anatomy, genetics and size of the atherosclerotic plaques. The pig LDL receptor gene was inactivated to drive hypercholesterolemia and atherosclerosis by two groups of researchers using separate technologies ,166. Thereatment . In the ormation . The ablPCSK9 gene in humans showed low levels of LDL [Since circulating LDL levels are associated with LDLR activity, any agents that disrupt the function of LDLR can result in the elevation of LDL . Therefos of LDL  and prots of LDL ,172. On s of LDL . More cos of LDL , consists of LDL .IDOL is an E3 ubiquitin ligase involved in LDLR degradation. Untreated FH patients had higher serum levels of IDOL and PCSK9 than the control, and serum IDOL levels decreased after statin therapy, suggesting statins’ role in decreasing serum IDOL . This wap < 0.001). Importantly, a strong correlation between enhanced insulin sensitivity and both plasma apoC-III and TG suppression was observed together with lower HbA1c. An increased apoC-III/HDL ratio was shown to be associated with CAD as a result of impaired cholesterol efflux capacity [Loss-of-function phenotypes of both apoC-III ,117,119 capacity , suggestcapacity ,187. −/−, CETP-Tg/LDLR−/−), treatment with ANGPTL3 antisense oligo caused HDL lowering but increased RCT, as evidenced by >two-fold increases in fecal cholesterol. Thus, ANGPTL3 inactivation increased RCT despite lowering of HDL and showed no correlation between HDL level and RCT, similar to other findings [ANGPTL3 is also involved in the hepatic secretion of TG-rich lipoproteins and clearance of TRLs via inhibition of LPL . Loss-offindings , demonstfindings . Studies from LDLR knockout (loss-of-function)  and LDLR−/−/apobec1−/− double knockout mice [Patients with refractory hypercholesterolemia (RH), often seen in hereditary metabolic disorders like HoFH and compound heterozygous familial hypercholesterolemia (HeFH), have significantly elevated LDL levels despite treatment with maximum tolerated doses of lipid-lowering therapies. These patients are at a very high risk for developing ASCVD ,196. HoFout mice ,208  ,223 or p−/− mice [−/− mice, with no off-target mutagenesis. The precision of base editing as well as lack of off-target mutagenesis were also demonstrated in PCSK9 humanized mice [The new generation of CRISPR-Cas9-mediated gene editing is called base editing, where undesired off-target gene modifications can be avoided by utilizing the positioning and cleavage capability of the Cas nuclease to yield precise single-base substitution without the need for DNA cleavage . Two cla−/− mice . More thzed mice . Liver-szed mice , showingzed mice . In a sized mice ,225. In zed mice   and treated with LDLR-modifying agents, a tight association between LDLR activity and LDL was observed. However, in certain pathophysiological conditions such as diabetes and oxidative stress, other factors may weaken LDL and ASCVD risk correlations. Preclinical and clinical results demonstrate that gain of function by gene therapy and genome/base editing of LDLR-based therapy may be better in terms of management of hypercholesterolemia and overall safety pharmacology. While targeting lipid synthesis in the liver has shown marked benefit in lowering blood LDL, the inhibition of hepatic lipid secretion, although it improves circulating LDL and TRL, is often associated with accumulation of lipids in the liver. LDL clearance pathways via directly targeting LDLR or via other players, such as PCSK9, have shown benefits to HoFH and HeFH patients, respectively. Two approaches utilizing CRISPR-based gene/base editing currently underway to enhance clearance of LDL and TRL by targeting LDLR and ANGPTL3, respectively, offer great promise to help hypercholesterolemic patients achieve the therapeutic goals of LDL. One caveat of the enhancing LDLR expression approach is that one needs at least one functional LDL allele to target. Thus, only HeFH patients, and possibly other hypercholesterolemic patients with an intact LDLR gene, are likely to benefit. Based on non-human primate results, ANGPTL3 inactivation using CRISPR base editing appears promising to HoFH patients, once the safety of this approach is demonstrated in clinical studies.