Soft tissue sarcoma is an umbrella term for a group of rare cancers that are difficult to treat. In addition to surgery, neoadjuvant chemotherapy has shown the potential to downstage tumors and prevent micrometastases. However, finding effective therapeutic targets remains a research challenge. Here, a previously developed computational approach called mechanistic models of signaling pathways has been employed to unravel the impact of observed changes at the gene expression level on the ultimate functional behavior of cells. In the context of such a mechanistic model, RNA-Seq counts sourced from the Recount3 resource, from The Cancer Genome Atlas (TCGA) Sarcoma project, and non-diseased sarcomagenic tissues from the Genotype-Tissue Expression (GTEx) project were utilized to investigate signal transduction activity through signaling pathways. This approach provides a precise view of the relationship between sarcoma patient survival and the signaling landscape in tumors and their environment. Despite the distinct regulatory alterations observed in each sarcoma subtype, this study identified 13 signaling circuits, or elementary sub-pathways triggering specific cell functions, present across all subtypes, belonging to eight signaling pathways, which served as predictors for patient survival. Additionally, nine signaling circuits from five signaling pathways that highlighted the modifications tumor samples underwent in comparison to normal tissues were found. These results describe the protective role of the immune system, suggesting an anti-tumorigenic effect in the tumor microenvironment, in the process of tumor cell detachment and migration, or the dysregulation of ion homeostasis. Also, the analysis of signaling circuit intermediary proteins suggests multiple strategies for therapy. Sarcomas are a group of rare heterogeneous malignancies that arise from cells of the mesenchymal lineage, including connective tissue, muscle, fat, bone, and cartilage . These vAt a molecular level, sarcomagenesis is driven by genetic and epigenetic alterations, including mutations, copy number changes, and gene expression alterations, which result in the activation of oncogenes or the loss-of-function of tumor suppressors, leading to uncontrolled cell proliferation. Mutations in sarcomas can occur in a variety of genes, including TP53, CDKN2A, CTNNB1, RB1, PTEN, and members of the RAS/RAF/MAPK pathway . SomaticTo understand the complex landscape of functional strategies that cells use to initiate their malignization and further progress to different sarcoma subtypes, a strategy based on mathematical modeling of the molecular cancer mechanisms has been used. Specifically, mathematical mechanistic modeling of cell signaling pathways provides a causal link between variation occurring at the level of gene activity  or integrity  and the observed phenotype diversity  . ActuallThis study elucidates the impact of general and specific processes in soft tissue sarcoma samples gathered from the Cancer Genome Atlas (TCGA) project , which cSelected data from GTEx tissues and the SARC project were downloaded. Samples from GTEx on putative sarcomagenic tissues of mesenchymal origin were: 1293 adipose, 520 fibroblasts, 881 skeletal muscle, 553 esophagus muscularis, 384 stomach, 159 uterus, and 946 vessels. From TCGA, the 206 sarcoma samples include 7 subtypes: 50 dedifferentiated liposarcomas (DDLPS), 80 leiomyosarcomas (divided into 53 soft tissue STLMS and 27 uterine ULMS), 5 malignant peripheral nerve sheath tumors (MPNST), 17 myxofibrosarcomas (MFS), 10 synovial sarcomas (SS), and 44 undifferentiated pleomorphic sarcoma (UPS). Downloaded gene read counts from these samples were preprocessed together with TMM normalization and used for the estimation of activity values with HiPathia. Data exploration by dimensionality reduction with t-SNE and clustering showed a strong correlation of samples by the project . On sarcA common assumption in genome-scale modeling methodologies is considering RNA-Seq counts as trustable proxies for protein abundance and, consequently, protein activity . This enExploration of sample clustering with either t-SNE or a heatmap did not reveal significant differences compared to sample distances using expression values see , indicatRB1, see PTK2) acts as an effector, exhibit a high hazard ratio. However, signaling pathways may have multiple roles in tumorigenesis, which will be further discussed.The activation of signaling pathways is closely related to cell fate and may have a causal influence on patient survival. Here, the association of the modeled circuit activity values with sarcoma patients’ overall survival was explored in detail. To achieve this, Cox proportional hazard models and Kaplan–Meier plots  were empSPI1 (a.k.a. PU.1) is the TF whose targets are present in most of the circuits. In particular, it has 41 targets within the signaling circuits whose expression is dysregulated, including PIK3CG and AKT2, central components of the PI3K/Akt signaling network present in various pathways, as well as components of the MAPK signaling network such as IKBKB, MAPK10, or RAC2. IKZF1 (Ikaros protein) targets participate in the protective circuits of the Fc epsilon signaling pathway, platelet activation, and HIF-1 signaling pathway. Among Ikaros targets, Lyn kinase and PRKCB  were found to be upregulated. Lyn participates in the activation of PI3K/Akt networks within the indicated pathways, while it activates PKC by downstream calcium signaling.Given the low expression levels exhibited by transcription factors (TF), the observed gene expression values in transcriptomic experiments may not represent their appropriate activity in the cell. A workaround to indirectly estimate the activity of a TF is to assess the activity of its target genes using any enrichment method. Here, the TFTEA  method wTo assess the altered cellular functions of sarcoma, the signaling activities of each sarcoma subtype were contrasted with those of their putative sarcomagenic normal tissues. In addition to some common deregulated signaling circuits, each sarcoma subtype also exhibited its own unique set of deregulated circuits. Functional annotation of the circuits that are dysregulated by each sarcoma subtype primarily highlights DNA replication, apoptotic processes, responses to cytokines, and Fc-receptor signaling . OverallDifferentially activated signaling circuits were associated with hallmarks of cancer using CHAT. With this classification, the abundance of such hallmarks on the dysregulated circuits for each sarcoma subtype was assessed. The most frequently dysregulated processes are related to cellular energetics, invasion/metastasis, and the avoidance of the immune system. At the same time, less frequently, they are involved in enabling replicative immortality, evasion of growth suppressors, and genomic instability and mutation . In consRaw gene counts for tumor and normal tissue samples were obtained from the recount3 R package . RecountLow-quality and non-primary tumor samples from the sarcoma project were filtered out. The subtype classification was modified according to previous expert pathology reviews . We keptvoom function provided in the limma package version 3.46.0 [The downloaded raw counts for all samples were normalized using the Trimmed Mean of M-values (TMM) method , as impln 3.46.0 , which aSn) for the current node (n) is determined by its normalized gene expression value (nv). This determination is influenced by the sets of activation signals (as) from the set of activation edges (A) and inhibitory signals (is) from the set of inhibition edges (I), as detailed in [The mechanistic model replicates the dynamics of the signaling circuits defined within pathways. These circuits are depicted as directed graphs, establishing connections from receptor to effector proteins through a series of activations and inhibitions mediated by intermediate proteins. Ultimately, effector proteins at the end of these circuits initiate distinct cellular functions. Malfunctions of these circuits can trigger cancer hallmarks in neoplastic cells. The mechanistic model emulates signal transduction along these circuits by considering protein activity levels. In this context, gene expression values serve as proxies for the presence of their corresponding active proteins ,43,44,45ailed in . This monormalize_data from the package HiPathia 2.6.0 [hipathia function. The function normalize_paths was used to account for the length of the circuits in the activity calculation.Prior to pathway activity computation, normalized values were rescaled to the range 0–1 using the function ia 2.6.0 . HiPathiia 2.6.0 ,26. HumaVisualization of normalized counts and activity values was performed using the t-distributed stochastic neighbor embedding method (t-SNE) for visualization of the structure of high-dimensional data  using thcoxph function. The p-value of each model was adjusted for multiple tests with the Benjamini and Hochberg method  [cox.zph function of the survival package. Kaplan–Meier survival curves were constructed with the Survminer package version 0.4.9 [Survival analysis was performed on sarcoma samples using the survival R package  version ry rate) . In addion 0.4.9  from selAdditional survival analysis of circuit-integrating genes was achieved on the web application Kaplan–Meier plotter , where gFor differential comparisons between sarcoma subtypes and normal tissues of both normalized expression and activity values, limma  was usedThe results of differential expression analysis were used for indirect estimation of transcription factor activity by enrichment analysis of their corresponding target genes using the Transcription Factor Target Enrichment Analysis (TFTEA)  tool. TFGene ontology (GO) enrichment analysis was performed with the package enrichR version 3.0 . Gene efThe activity of signaling pathways on sarcoma samples has been analyzed to, on the one hand, find how the degree of signaling pathway activity affects overall patient survival and, on the other hand, discover the alterations in sarcoma samples in contrast to sarcomagenic non-diseased tissues. In the present work, 13 circuits from 8 pathways associated with sarcoma patient survival, nine differentially activated circuits belonging to five pathways, and a set of eight enriched transcription factors have been reported.Implementations of mechanistic models, such as HiPathia, provide a sophisticated and detailed interface for closely studying the functional state of cells using RNA-Seq data with high sensitivity ,13. In tSPI1 could be used as a predictor together with FCER1G of the immune infiltration, suggesting a protective role for this TF [AKT1–3, JNK—MAPK8–10, and p38—MAPK11–14) from a common upstream receptor. Remarkably, these proteins activate transcription factors within mast cells and are ultimately involved in the production and release of cytokines and arachidonic acid [COX-2 responds to TNF and p38/NF-κB activation and has been found to be protective against breast cancer [COX isoforms were observed, but also for the activation of the COX-2 activator NF-κB through the “MAPK signaling pathway”.Overall, a protective role of an enhanced immune system was observed, supported by low hazard ratios in immune-related pathways such as the Fc epsilon RI signaling pathway with four significant circuits . One of  this TF . The othnic acid ,65,66, wnic acid ,68,69. Anic acid . Anothernic acid . The indt cancer . In sarcWAS/WASL genes (encoding WASP and N-WASP nucleation-promoting factors). The heptameric Arp2/3 complex nucleates actin fibers into branched filaments that, in macrophages, participate in FcγR-mediated phagocytosis, integrin-dependent responses, and motility [An increased activation of the immune-related “Fc gamma R-mediated phagocytosis” pathway towards the activation of the Arp2/3 complex (Actin-related protein 2/3 complex) was also observed, as well as the previous node with motility ,74. Fullmotility . In sarcmotility . A higher hazard ratio was found on a short circuit of the “Rap1 signaling pathway: calcium cation.” The circuit consists of TCR (T-cell receptor), the adaptor protein linker of activated T cells (LAT), and phospholipase Cγ1 (PLCG1). It is involved in the activation of T cells, resulting in the mobilization of calcium cations. In disease situations, overactive unstimulated TCRs induce changes in T cells to become unresponsive as a mechanism of immune evasion . FurtherSarcoma samples appear to have the energy metabolism wired towards gluconeogenesis, which, linked with the previous section on the TME, is relevant to maintaining anti-tumor immune conditions. Cellular energetics is also modulated by the NF-κΒ pathway, where the alternative NF-κΒ (RelB/p52 dimers) is activated by the IKK alpha dimer  to promoPPARGC1A), activates the gluconeogenic enzyme fructose 1,6-bisphosphatase (FBP), a putative inhibitor of sarcoma growth [ALDOA, ALDOB, and ALDOC). Previous research revealed a positive association of ALDOB expression with the survival of patients with gastric cancer [ALDOC as a target for positive regulation by the retinoblastoma (RB) tumor suppressor protein [ALDOA correlates positively with the immune infiltration of macrophages, neutrophils, and T cells [Protective roles are predicted for glucose metabolism mediators on the insulin and HIF-1 signaling pathways through the PI3K/Akt network. Activation of the “insulin signaling pathway” results in the inhibition of FOXO1 by phosphorylation. FOXO1 is a well-known TF that, in association with the coactivator PGC-1α  receive signaling from cell attachment to neighbor cells. When detachment occurs, it triggers a specific type of apoptosis, called anoikis [EFNB) by the EPH receptors triggers a signaling cascade that increases FAK catalytic activity. This cascade causes the cells to modify their morphology into a round shape by remodeling the actin cytoskeleton and leading to cell repulsion [The preparation of tumor cells for migration includes cell-cell detachment accompanied by a change in cell morphology. In the first step, integrins and the focal adhesion kinase ( anoikis . For preepulsion . In TCGAepulsion .CDKN2A (a.k.a. INK4A/ARF locus), which encodes the proteins p16 and ARF that control the Rb and p53 pathways, respectively, with p16 being an inhibitor of the kinases that phosphorylate Rb and leading to growth arrest [MDM2), a negative regulator of p53/Rb proteins inhibited by ARF [MDM2 is reported as an oncogene and driver alteration of liposarcoma [Tumor cells usually acquire mechanisms to prevent cell death and enhance proliferation. Thus, it is no surprise that the “cell cycle pathway: RB1” (retinoblastoma protein as effector) signaling circuit  was founh arrest . The secd by ARF ,89. MDM2PI3K a druggable target for anticancer therapy due to its role in the stimulation of cell growth and proliferation. Bcl-2 is a relevant driver of synovial sarcoma (SS) [IPTR genes) and over-activation of TWIK-related acid-sensitive potassium channels (TASK by KCNK genes) mediated by the pathway “Aldosterone Synthesis and Secretion”. Excessive calcium release from IP3Rs disrupts mitochondrial membrane integrity and leads to apoptosis, a process suppressed by the anti-apoptotic Bcl-2 protein [On the other hand, the anti-apoptotic protein Bcl-2 appears to be overactivated by the “PI3K-Akt signaling pathway.” As seen before, this pathway is key in carcinogenesis, making oma (SS) , being o protein . Meanwhi protein ,93; overBesides common pathways that are generally dysregulated in sarcoma, there are also pathways specifically dysregulated in each sarcoma subtype . Here, wThe specifically dysregulated circuits observed in DDLPS are closely related to metabolic alterations. Thus, the activation of the hedgehog signaling pathway, previously reported in sarcoma patients, would promote a dedifferentiated morphology of adipocytes, decreased lipid accumulation, and insulin resistance ,95. IncrABCC8  is associated with a poor prognosis [Specific responses observed in MFS are varied, including metabolic-, motility-, apoptosis-, and immune-related circuits. Increased uptake of glucose by tumor cells would raise the ATP/ADP ratio, leading to the inhibition of ATP-sensitive potassium channels and triggering a cascade to secrete insulin. In glioma cells, lower expression of rognosis . Inhibitrognosis . RYR1, leading to the secretion of oxytocin. In both cases, the prolactin and oxytocin pathways activate the MAPK signaling pathway to enhance cell proliferation through cyclin D1. These hormones induce tumor cell viability, and targeting their receptors may pose a treatment [In MPNST, significant differential activation in circuits related to the protective effect triggered by the TME on the FceRI signaling pathway, previously described, as well as a putative melanotic feature and neuron response to the hormones prolactin and oxytocin, was found. Different receptors activate Phospholipase C, triggering, in one case, melamin synthesis, previously observed in malignant melanotic nerve sheath tumors , and, inreatment ,102.In SS, the down-activation of circuits with anticancer properties was mostly found. SS is related to MPNST, having a neural origin . As suchIn STLMS, reduced signaling from tumor necrosis factor (TNF) in the adipocytokine signaling pathway leads to elevated activation of IRS, involved in insulin sensitivity, whose expression has been related to malignant sarcoma progression. This protein was also found to be constitutively activated in leiomyosarcoma . UnexpecBAD and CASP9 and decreased COX-2 activation. The production of VEGF promotes a mechanism to evade the immune system by inhibiting the migration of T cells after modification of gene expression with a reduction in adherent factors in endothelial cells [Dysregulated signaling in the VEGF signaling pathway in ULMS, putatively produced from the reduced expression of the VEGFA ligand, leads to increased anti-apoptotic al cells .2+ release-activated Ca2+) channel ORAI1, a potential contributor to cancer progression. On the other hand, under-activation of the antiproliferative GnRH signaling pathway via JNK/c-jun signaling was observed. Therapeutic anticancer treatments over these pathways include complement-dependent cytotoxicity with antibodies and the utilization of GnRH agonists to counteract the signaling of growth-factor receptors or antagonists to induce apoptosis [UPS overactivates circuits involved in cell proliferation, such as the JAK/STAT signaling pathway in response to chemokine, with the production of complement anaphylatoxins. These anaphylatoxins play a role in regulating inflammation, enhancing cell stemness, and activating the CRAC  for additional support. Although the results provided by KMplot and UALCAN are obtained from different cancers, Chemotherapy and targeted therapy are both potent methods to treat cancer. However, due to their different forms of action, the side effects vary. While chemotherapeutics affect cells in the body with rapid proliferation, they affect not only tumor cells but also cells in the bone marrow and hair. On the other hand, targeted therapy agents, usually small molecules or antibodies directed towards specific proteins, have reduced side effects that commonly include diarrhea and skin reactions. The study of signaling pathways allows for the detection of putative targets on the pathways, either on specific nodes or in adjacent nodes, that may contribute to the programmed therapeutic perturbation. Many circuits share alterations in the PI3K/Akt/mTOR and Ras/MAPK networks. Both signaling networks may be activated by multiple RTKs, for which numerous tyrosine kinase inhibitors (TKIs) are sarcoma-approved drugs like pazopanib, sunitinib, sorafenib, or erlotinib . Besides signaling pathway modulation, immunotherapy is a promising field for the treatment of soft tissue sarcoma patients. As previously discussed, our results indicate that an enhanced TME is usually a sign of a good patient prognosis. A challenge is to promote an anti-tumor TME and reduce resistance to therapy. The adoptive cell therapy of T-cells consists of manipulating the TCR to recognize tumor-specific antigens and promote an anti-tumor response in the patient . AnotherSarcoma tumors are difficult to treat, leading to poor overall survival for patients. In the present work, the activation of pathways that have a significant association with patient survival and those that are dysregulated in tumor samples compared to normal tissues was assessed. Traditional neoadjuvant therapies usually target known oncogenic circuits, while newer therapies are focused on improving the immune anticancer response. In that respect, the protective role of signaling and metabolic pathways that promote the infiltration of anti-tumorigenic immune cells into the sarcoma TME has been discussed. On the other hand, different tumorigenic functions, such as cytoskeleton remodeling and the promotion of tumor and pre-metastatic cell survival, were found to negatively affect patient outcomes. Interestingly, the same elements may be indicative of a good or bad prognosis depending on the specific pathway and cell type they participate in. A systematic review has been conducted, taking into consideration the high variability of cellular processes existent in different types of sarcoma. A relevant outcome was the identification of common circuits that potentially influence patient survival and tumor progression in soft tissue sarcoma. The findings presented here provide hints for new therapeutic interventions in sarcoma.


Bioceramics are calcium-phosphate-based materials used in medical and dental implants for replacing or repairing damaged bone tissues; however, the effect of bioceramic sintering on the intracellular signaling pathways remains unknown. In order to address this, we analyzed the impact of sintering on the cell signaling pathways of osteoblast cells using sintered and non-sintered hydroxyapatite (HA) and beta-tricalcium phosphate (β-TCP). X-ray diffraction indicated that only the morphology of HA was affected by sintering; however, the sintered bioceramics were found to have elevated the calcium concentrations in relation to the non-sintered variants. Both bioceramics inhibited the JNK signaling pathway; the sintered HA exhibited half the value of the non-sintered variant, while the sintered β-TCP rarely expressed a p-JNK value. The total Src and Raptor protein concentrations were unaffected by the sintering, while the p-Src concentrations were decreased. The p-EGFR signaling pathway was regulated by the non-sintered bioceramics, while the p-p38 concentrations were reduced by both the sintered β-TCP and HA. All of the bioceramics attenuated the total AKT concentrations, particularly the non-sintered HA, and the AKT phosphorylation concentration, except for the non-sintered β-TCP. Thus, the sintering of bioceramics affects several intracellular signaling pathways. These findings may elucidate the bioceramic function and expand their application scope as novel substrates in clinical applications. Bone regeneration after fracture is a time-consuming process. Human bone is composed of 60–70% calcium phosphate, which has a chemical composition similar to hydroxyapatite (HA) . AlthougBioceramics, such as HA and β-TCP, are mainly used as substitutes for hard tissues, such as teeth and bone. However, the mechanical properties of these materials can be further improved through sintering . SinteriSintered HA bioceramics exhibit different biomechanical strengths and physical properties depending on the sintering temperature . SinteriVarious studies have been conducted to examine the effect of bioceramic surfaces on cell adhesion, spreading, and proliferation; however, the effect of sintering on the intracellular signaling pathways and calcium concentration has not been widely explored thus far. To address this gap in knowledge, we conducted a study investigating the hypothesis that sintering affects the intracellular signal transduction and calcium concentration in osteoblasts. To achieve this, HA and β-TCP were selected as bioceramic substrates, and their effect on these parameters before and after sintering were studied. In the experiments, five signaling pathways , which are involved in cell death and proliferation, were analyzed.2 at 37 °C in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum and 1× penicillin–streptomycin (Gibco).MG63 (osteoblast) cells were purchased from Seoul National University . The cells were maintained in an atmosphere of 5% CONon-resorbable HA and resorbable β-TCP were selected as the bioceramic substrates. Both were prepared as sintered and non-sintered powders. First, non-sintered HA (HA-n) was synthesized using octacalcium phosphate (OCP) as the starting material. The synthesis was carried out via OCP hydrolysis, as previously reported ,27,28:CaNext, sintered HA (HA-s) was prepared by heat-treating reagent-grade HA powder  at 1200 °C for 2 h, followed by mechanical crushing to afford a fine powder .Reagent-grade TCP powder  was used for the non-sintered samples (β-TCP-n). Commercially available β-TCP powder  was used for the sintered samples (β-TCP-s).To confirm that the prepared powders were suitable for the study, phase analysis was performed using XRD  with a Cu-Kα radiation source. Scans were conducted at a rate of 2.0°/min and a step size of 0.022°. The XRD analysis results were compared with the publicly available inorganic crystal structure (ICSD) data.The surface morphology of the powder samples was examined by FE-SEM  at magnifications of 1000× and 5000×.Elemental analysis of the bioceramic powders was carried out using a Kα XPS spectrometer . Each sample surface was sputter-cleaned with argon ions to remove contaminants before performing the XPS measurements . The ele2 for 24 h. After incubation, the dishes were used for the cellular studies at 37 °C and 5% CO2.Solutions of the bioceramics (10 mg/mL in DMEM) were prepared in 100 pi cell-culture plate dishes  and incubated at 37 °C and 5% COThe signaling proteins JNK/p-JNK, EGFR/p-EGFR, Src/p-Src, Raptor/MTORC1, p38/p-p38, and AKT/p-AKT were examined in the signaling pathway experiments. The proteins were obtained via cell lysis after the cells were incubated in the bioceramic-loaded plates.g and 4 °C for 30 min. After centrifugation, the supernatant was removed, and the protein concentration was measured. The proteins were then eluted for 15 min at 70 °C using a 4× lithium dodecyl sulfate sample buffer (diluted in radio-immunoprecipitation assay (RIPA) buffer) supplemented with β-mercaptoethanol. The samples were then separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the mini-Protean Tetra Handcast System . The isolated proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes, which were subsequently blocked with 5% non-fat dry milk in PBS-Tween 20 (PBS-T).First, the cell solutions were centrifuged at 15,000× After blocking, the protein-loaded membranes were incubated overnight at 4 °C with the complementary primary antibodies. Subsequently, the membranes were washed five times with PBS-T before further incubation with complementary secondary antibodies. To detect the protein bands, a SuperSignal™ West Femto Maximum Sensitivity Substrate  was used. The primary antibodies used were anti-EGFR , anti-phospho-EGFR , anti-SAPK/JNK , anti-phospho-SAPK/JNK , anti-Src , anti-phospho-Src , anti-p38 , anti-phospho-p38 , anti-AKT , and anti-phospho-AKT (Ser473) . The HRP conjugated secondary antibodies used for western blot were anti-mouse IgG and anti-rabbit IgG (used at 1:4000 dilution). All of the antibodies were purchased from Cell Signaling Technology (USA.). Anti-Raptor antibodies  were purchased from Santa Cruz Biotechnology, Inc. .SP600125 , wortmannin , and SB203580  were used in this study.2 or CaCl2 for each condition. The Fura-2-loaded cells were then transferred to a perfusion chamber on an optical microscope .Poly-L-lysine-coated 12 mm coverslips were used for the attachment of the MG63 cells before and after exposure to the sintered and non-sintered bioceramics. After deposition, the cells were loaded with Fura-2 AM (5 μM) and 0.01% pluronic F-127 in a normal Tyrode (NT) solution at 37 °C for 30 min. The NT solution was composed of 140 mM NaCl, 5 mM KCl, 10 mM HEPES, 10 mM glucose, and 1 mM of MgCl2+/ionomycin . For the 340 and 380 nm excitation wavelengths, a high-speed wavelength-switching device  was used. The MetaFluor image analysis software  was used to measure the fluorescence intensity ratio (340:380 nm) of several regions across the Fura-2-loaded cell samples.To confirm the basal calcium concentration in each sample, the MG63 cells were treated with 10 mM EGTA  and 10 mM Cap-value of <0.05 was defined as a significant difference, and all of the statistical analyses were represented as the mean ± the standard error of the mean (SEM.).All of the protein concentration analyses were performed using ImageJ . The results were compared using one-way analysis of variance (ANOVA). A The obtained XRD patterns for the sintered and non-sintered bioceramics are shown in The particle morphologies of the bioceramics were examined by FE-SEM at 1 k and 5 k magnifications B. Here, To investigate the Ca/P molar ratio in each bioceramic, energy dispersive spectroscopy (EDS) was conducted by coupling the EDS system with the FE-SEM instrument. The ratios obtained for HA-n and HA-s were 1.29 and 1.55, respectively, thus indicating a disparity in the Ca/P ratio after sintering. Conversely, β-TCP-n and β-TCP-s showed similar ratios of 1.23 and 1.24, respectively. These results were further corroborated by the XPS measurements, as shown in Intracellular calcium is important for regulating the cell signaling pathways; thus, we examined the calcium concentration from the cells after exposure to the sintered and non-sintered bioceramics via fluorescence measurements. It was found that the HA-n exhibited a smaller intracellular calcium concentration than that of the HA-s. The β-TCP powders showed similar results B. This iFor the signaling pathway studies, the cells were incubated in solutions of the sintered and non-sintered bioceramics and lysed to obtain the intracellular signaling proteins for western blotting analysis. First, the JNK pathway was examined A. The HAAll of the bioceramics were found to inhibit the p-Src pathway considerably. The HA-s and both of the β-TCP variants suppressed the p-Src protein concentration to approximately zero, whereas the exposure to HA-n resulted in comparatively higher p-Src activity B.With sintering, the HA and β-TCP did not exhibit a difference in the p-EGFR protein concentration in relation to the control sample; however, the non-sintered bioceramics attenuated the p-EGFR signaling pathway considerably. The total amount of EGFR protein did not change with or without sintering C. This iWith and without sintering, both ceramics exhibited negligible differences in the Raptor protein concentration in relation to the control sample; thus, these bioceramics do not influence the total Raptor protein concentration D.The effect of sintering on the p38 signaling pathway was different depending on the type of bioceramic used; for HA, there was no observable difference in the p38 concentration between the sintered and non-sintered variants, whereas β-TCP-n significantly enhanced the p38 phosphorylation, by approximately 50%, in relation to that of β-TCP-s. The total p38 protein concentrations with HA and β-TCP were similar to that of the control group A.The AKT signaling pathway was individually affected by the two types of bioceramics. While all of the bioceramics decreased the total AKT protein concentration in relation to the control sample, p-AKT (S473) exhibited a higher AKT activity with β-TCP-n in relation to that of β-TCP-s. Conversely, both HA-n and HA-s significantly decreased the AKT protein concentration, but did not exhibit a notable difference in the p-AKT protein concentration between the sintered and non-sintered variants. However, HA-n did exhibit a considerable decrease in the total AKT protein concentration, whereas the other bioceramics gave a similar reduction compared to the control sample B.The sintering process uses high-temperature heat treatment to modify both the physical and chemical properties of bioceramics. Previous studies have mainly focused on examining the macroscopic changes in these parameters such as the porosity, strength, and brittleness ,17,22,23Through XRD and FE-SEM measurements, it was established that HA alters its phase composition after sintering, whereas β-TCP is unaffected ; HA-s alCalcium acts as an intracellular messenger that controls numerous cellular processes, including proliferation, mitosis, neurotransmission, gene transcription, and apoptosis. In previous studies, it has been reported that when appropriate mechanical stimulation is given to osteoblasts, the calcium channels in the cell membrane are activated, resulting in an influx of extracellular calcium into the cells. This increases the intracellular calcium concentration and promotes a corresponding increase in bone production . Our resFive major intracellular signaling pathways, JNK, Src, EGFR, p38, and AKT, were analyzed in this study. The MAPK signaling cascade pathway, an important signaling pathway known to be involved in the regulation of osteoblast physiology, plays a major role in several processes, including cell proliferation, death, and differentiation . This siSrc activity has been reported to maintain bone homeostasis and regulate cell invasion and growth ,37. Its EGFR is characteristic of multiple carcinomas and significantly affects the cell signaling pathway . In osteWe also examined the effect of the total Raptor protein concentration on cell proliferation; however, this was not impacted by the bioceramics, regardless of sintering D.p38 is a member of the MAPK family and is associated with accelerating cell differentiation and proliferation . The treβ-TCP-n does not influence AKT phosphorylation, whereas β-TCP-s slightly decreases the p-AKT concentration. HA also decreases the p-AKT concentration, regardless of sintering B. All AKThrough this study, it was demonstrated that the sintering process alters various physicochemical factors in the bioceramics, which can affect the intracellular calcium concentration and signal transduction pathways in osteoblasts. Recently, it has been reported that exacerbated intracellular signaling results in cleft lip, palate, and alveolus (CLPA) defects in children and adolescents . BioceraIn this study, several bioceramics were shown to differently regulate the intracellular signaling pathways and intracellular calcium concentration based on whether the substrates were sintered. Bioceramics may play a significant role in improving the future quality of these treatments and promoting bone regeneration. We investigated the effects of bioceramics on cells and how to improve the utilization of calcium phosphate-based bioceramics. Our findings suggest that the employed bioceramics exhibit individual effects on the regulated intracellular signaling pathways. The results of this study enhance the understanding of the intracellular response to bioceramics used in combination with various biomaterials to promote bone regeneration and can be used as basic data for the development of biomaterials containing bioceramics in the future. Bioceramics may play a significant role in improving the future quality of these treatments and promote wound healing. It is expected to help to improve the quality of clinical research. In future studies, it is necessary to examine the specific physicochemical changes induced by bioceramic sintering for an in-depth understanding of the effect on the intracellular calcium concentration and signal transduction pathway of osteoblast cells.


Opuntia humifusa extract (MA-OHE). The MA-OHE was loaded onto zinc-aminoclay (ZnAC) and encapsulated in a Pickering emulsion system (MA-OHE/ZnAC PE) to evaluate its therapeutic potential against acne-inducing microbes. Dynamic light scattering and scanning electron microscopy were used to characterize MA-OHE/ZnAC PE with a mean particle diameter of 353.97 nm and a PDI of 0.629. The antimicrobial effect of MA-OHE/ZnAC was evaluated against Staphylococcus aureus (S. aureus) and Cutibacterium acnes (C. acnes), which contribute to acne inflammation. The antibacterial activity of MA-OHE/ZnAC was 0.1 and 0.025 mg/mL to S. aureus and C. acnes, respectively, which were close to naturally derived antibiotics. Additionally, the cytotoxicity of MA-OHE, ZnAC, and MA-OHE/ZnAC was tested, and the results showed that they had no cytotoxic effects on cultured human keratinocytes in a range of 10–100 μg/mL. Thus, MA-OHE/ZnAC is suggested to be a promising antimicrobial agent for treating acne-inducing microbes, while MA-OHE/ZnAC PE is a potentially advantageous dermal delivery system.Acne is a common skin condition caused by the growth of certain bacteria. Many plant extracts have been investigated for their potential to combat acne-inducing microbes, and one such plant extract is microwave-assisted  Cutibacterium acnes (C. acnes) and Staphylococcus aureus (S. aureus), which are associated with inflammatory acne. Various studies have attempted to treat acne-inducing microbes using the antibiotic properties of antioxidant plant extracts (Opuntia humifusa (O. humifusa) extract (MA-OHE) has been found to have antimicrobial activity and is useful for treating acne-inducing microbes [O. humifusa extract exhibits beneficial effects, especially in preventing cancer, diabetes, and cardiovascular disease in humans, through its various bioactive compounds [Human skin provides a fertile environment for specific Gram-positive microbes, including extracts . In partmicrobes ,2,3. In ompounds ,5,6. Theompounds ,8,9. Theompounds .Combining the advantages of microwave-assisted extraction and Zinc-aminoclay (ZnAC), this approach is expected to show promising results in combating acne-inducing microbes. ZnAC, also known as 3-aminopropyl zinc phyllosilicate, is an aminopropyl functionalized 2D layered material formed by the sol-gel transition of 3-aminopropyltriethoxysilane (APTES) and cationic zinc ions . During Because ZnAC nanoparticles were dispersed in water, a solidified encapsulation system is essential for stability . An emul−1 region, C-O tertiary alcohol at the 1124–1205 cm−1 region, O-H carboxylic acid at the 1395–1440 cm−1 region, and C=C α,β-unsaturated ketone at the 1620–1610 cm−1 region were identified in MA-OHE/ZnAC. Peak shifts of MA-OHE/ZnAC were noted at 650, 900, 965, 1040, 1410, and 1490 cm−1, respectively. In addition, the N-H and OH stretching bands for the MA-OHE/ZnAC overlapped and displayed a combined vibrational band in the wavelength range 3500–3000 cm−1. Similar to other 2D nanomaterials, peak shifting and overlapping were attributed to the bioactive compound loaded on the ZnAC surface and occurred mainly due to covalent bonding [FT-IR spectra showing information related to the functional groups and peak alteration of MA-OHE, ZnAC, and MA-OHE/ZnAC are shown . The C-H bonding ,33. In tSEM images revealed the morphological structure of MA-OHE/ZnAC PE A,B. Homo2 value of the standard curve was >0.99 . The total phenolic amount of the MA-OHE/ZnAC was 39.40 ± 1.06%, as stated earlier, and the Rs >0.999 . The diss >0.999 . A total−9 m2/s diffusion coefficient and −4.17 × 10−9 flux  and ethanol were purchased from Daejung Chemicals & Metals Co. Ltd. . APTES was purchased from Tokyo Chemical Industry . Folin-Denis reagent, sodium carbonate (Na2CO3), glycerol, and cetyl palmitate were purchased from Sigma-Aldrich . Phosphate buffered saline (PBS) was purchased from Thermo Fisher Scientific Inc. . A Milli-Q Millipore filter system  was used to obtain distilled (DI) water. Potassium bromide (KBr) was procured from PIKE Technologies .Fresh O. humifusa [O. humifusa cladodes were washed with DI water and dried in ambient air. The dried cladodes were then mixed with an equal weight of DI water and blended using a domestic blender to obtain the O. humifusa mixture. Next, 30 g of the O. humifusa mixture was irradiated using a domestic microwave oven  at 1000 W for 8 min. The irradiated O. humifusa mixture was ground using a pestle, to which 30 mL of DI water was added, and the diluted mixture was passed through a 0.22 µm pore-sized hydrophilic filter  to remove impurities and debris. Finally, the filtrate was freeze-dried under conditions of −45 °C and 10 Pa to obtain MA-OHE.The microwave-assisted extraction method was used to extract humifusa . Briefly2 was dispersed in 200 mL of ethanol and stirred gently for 10 min. Next, 10 mL of APTES was added and stirred gently for 16 h. The mixture was then centrifuged at 4000 rpm for 20 min, and the supernatant was discarded. The precipitate was dried in a dry oven at 60 °C, and the dried ZnAC was ground using a pestle to form ZnAC powder.ZnAC was synthesized using a sol-gel transition process previously reported . Briefly−1, and H2O and CO2 signals were removed after data acquisition.For the aqueous phase, 40 mg of ZnAC and the same weight of MA-OHE powder were mixed with 20 mL of DI water and gently stirred for 6 h at ambient conditions. Bioactive compound-loaded ZnAC (MA-OHE/ZnAC) was obtained by centrifugation at 4000 rpm for 20 min and further analyzed. Fourier transform-infrared (FT-IR) spectroscopy  was performed to determine the functional groups of MA-OHE, ZnAC, and MA-OHE/ZnAC. The KBr pellet was measured as a baseline and scanned from 4000 to 400 cmThe water-in-oil formulation of MA-OHE/ZnAC PE was prepared using the anti-solvent method . BrieflyScanning electron microscopy (SEM) and dynamic light scattering (DLS) were used to demonstrate the morphology of MA-OHE/ZnAC PE. SEM was used to determine the morphological structure of MA-OHE/ZnAC PE  and a DLS instrument  was used to measure its particle size (Z-average) distribution and polydispersity index (PDI). Adequate MA-OHE/ZnAC PE was dispersed in DI water and carried into a quartz cell for measurement.w/v) of Na2CO3 solution was added to each well. The plate was incubated for 1 h in the dark at 22 °C, and absorbance was measured at 750 nm using a BioTek Synergy H1 hybrid multi-mode reader .The bioactive compound entrapment efficiencies of MA-OHE/ZnAC and MA-OHE/ZnAC PE were measured using total phenolic assay with slight modifications . This meTotal indicates the total phenolic content of MA-OHE added for MA-OHE/ZnAC or MA-OHE/ZNAC PE, and MA-OHEFree indicates the total phenolic content of free MA-OHE amount in MA-OHE/ZnAC or MA-OHE/ZnAC PE.Entrapment efficiency was calculated using the following formula:Bioactive compound dissolution kinetics of MA-OHE/ZnAC was determined using the dialysis method . BrieflyThe in vitro drug permeation kinetics were determined using a transdermal diffusion system  and artificial human epidermis tissue  . BrieflyS. aureus (ATCC 23235) was purchased from the American Type Culture Collection , and C. acnes (KCCM 42791) was purchased from the Korean Culture Center of Microorganisms . Tryptone was purchased from Condalab . Bacto Yeast Extract was purchased from Thermo Fisher Scientific Inc. Sodium chloride was purchased from Sigma-Aldrich. Luria-Bertani (LB) media was prepared using 5 g of tryptone, 2.5 g of yeast extract, and 5 g of sodium chloride. For making LB-agar plates, 10 g of agar powder  was added to the medium. Reinforced clostridial medium (RCM) and AnaeroBag were purchased from KisanBio .S. aureus was cultured in LB medium at 37 °C, while C. acnes was cultured in RCM at 37 °C under anaerobic conditions achieved using an AnaeroBag. For downstream experiments, 1.5 × 108 CFU/mL (0.5 Mcfarland) of bacteria in the growth phase was used.C. acnes or S. aureus in growth phase was seeded into a 96-well plate, followed by the addition of 100 μL of the different test samples to the wells. The plate was then incubated for 24 h, after which 100 μL of C. acnes from relevant wells was seeded on RCM agar media and incubated for 74 h. Likewise, 100 μL S. aureus from relevant wells was seeded on LB agar media and incubated for 24 h. The MIC values were determined by comparing the colony amount to the control.MIC values were determined using the broth micro-dilution method . BrieflyDulbecco’s modified eagle medium (DMEM), fetal bovine Serum (FBS), and penicillin/streptomycin (pen/strep) were purchased from Thermo Fisher Scientific Inc. Immortalized human skin keratinocyte cell line (HaCaT) was purchased from Cell Lines Service . Water-soluble tetrazolium (WST) solution was purchased from DoGenBio .2 [HaCaT cells were cultured in DMEM supplemented with 10% FBS and 100 U/mL penicillin/streptomycin in an incubator set at 37 °C and 5% CO2 . The cul4 cells/well) and incubated at 37 °C and 5% CO2 for 24 h. Next, 10 μL of MA-OHE, ZnAC, or MA-OHE/ZnAC of various concentrations  was added to the cells and incubated for 24 h. After incubation, the medium was rinsed three times. Next, 10 μL of WST solution was added into each well, and cells were further incubated for 2 h. Finally, cell viability was measured using a plate reader at 450 nm .WST assay was performed to evaluate cell viability when HaCaT cells were cultured in the presence of MA-OHE, ZnAC, or MA-OHE/ZnAC . Briefly2) were calculated using SPSS ver.25.0. The graphs were generated using Origin software version 2022b.Analysis of variance was performed, and the coefficient of determination values (RC. acnes was greater compared to the use of ZnAC alone due to the extended-release characteristic of MA-OHE/ZnAC. The antimicrobial activity of MA-OHE/ZnAC against S. aureus was also strong enough to be comparable to antibiotics. The entrapment efficiencies of MA-OHE/ZnAC and MA-OHE/ZnAC PE were measured, and MA-OHE/ZnAC PE was found to have a high bioactive compound entrapment efficiency. Furthermore, rapid permeation of MA-OHE/ZnAC PE was observed under in vitro conditions. Remarkably, no lag time was observed during the in vitro drug release kinetics assay. It seems that the major advantages of the MA-OHE/ZnAC PE formula are rapid permeation and extended release with high entrapment efficiency. Thus, ZnAC possibly acts as a bioactive compound carrier and an antimicrobial agent. Hence, MA-OHE/ZnAC is a potential novel antimicrobial agent that can be used for treating acne-inducing microbes. Moreover, our study highlighted the advantages of using Pickering emulsion for fabricating the MA-OHE/ZnAC delivery system. With emerging interest in lipid nanoparticles, the current research reinforces their advantages.In this study, a Pickering emulsion system was formulated by loading MA-OHE onto ZnAC for the treatment of acne-inducing microbes. Morphological structures of MA-OHE/ZnAC PE were obtained using DLS and SEM images. MA-OHE/ZnAC exhibited strong antimicrobial activity without exerting any cytotoxic effects on HaCaT cells. Specifically, the antimicrobial activity of MA-OHE/ZnAC against


Sexual dimorphism exists in the epidemiology of cardiovascular disease (CVD), which indicates the involvement of sexual hormones in the pathophysiology of CVD. In particular, ample evidence has demonstrated estrogen's protective effect on the cardiovascular system. While estrogen receptors, bound to estrogen, act as a transcription factor which regulates gene expressions by binding to the specific DNA sequence, a subpopulation of estrogen receptors localized at the plasma membrane induces activation of intracellular signaling, called “non-nuclear signaling” or “membrane-initiated steroid signaling of estrogen”. Although the precise molecular mechanism of non-nuclear signaling as well as its physiological impact was unclear for a long time, recent development of genetically modified animal models and pathway-selective estrogen receptor stimulant bring new insights into this pathway. We review the published experimental studies on non-nuclear signaling of estrogen, and summarize its role in cardiovascular system, especially focusing on: (1) the molecular mechanism of non-nuclear signaling; (2) the design of genetically modified animals and pathway-selective stimulant of estrogen receptor. SexuaEstrogen receptors (ERs) regulate gene expression as transcription factors in the nucleus, known as nuclear signaling. However, a subpopulation of ERs is present at the plasma membrane and initiates intracellular signaling, referred to as “non-nuclear signaling” or “membrane-initiated steroid signaling”. Despite their relatively small numbers compared to nuclear ERs , 12, an In this article, we first describe the characteristics of ERs and the molecular mechanism of non-nuclear signaling. Next, the role of non-nuclear signaling in cardiovascular systems is discussed through studies using genetically modified animals and pathway-selective stimulators. A concise review of GPER is also provided.2.α and ERβ, belong to the nuclear hormone receptor superfamily and share common structural characteristics . Two subtypes of ERs, EReristics –17. ER ceristics . The N-tmologous , while tmologous . In addiunctions .Upon binding to their ligands, ERs undergo a conformational change and form a stable dimer –23, whicEstradiol (E2) is the most potent endogenous estrogen in pre-menopause women, whereas estrone (E1) plays a larger role after menopause, and estriol (E3) shows a greater importance during pregnancy . Estetroα-mediated breast and ovarian cancer cell proliferation and prevents hypoxia-induced cardiomyocyte apoptosis  inhibits E2-induced nitric oxide synthase expression and re-endothelialization of murine carotid artery . In contpoptosis .3.3.1.α and ERβ  concentration in the rat uterus within minutes . Followi and ERβ , 49, 50, and ERβ –54. This and ERβ , 56.α and ERβ is responsible for palmitoylation . In consequence, activated cGMP-dependent protein kinase (PKG)-I stimulates cystathionine γ-lyase in endothelial cells, resulting the rapid release of hydrogen sulfide, which acts as a vasodilator   , 77. Pronine 260 .α on the plasma membrane binds to the p85α subunit of PI3K and rapidly stimulates eNOS via the PI3K-Akt pathway   of human ERstriatin . In a huKI mice) .αKI/KITie2Cre mice was established. In this mouse model, ERα non-nuclear signaling is inactivated by disrupting its binding to the p85α subunit of PI3K through an arginine 263 to alanine mutation in a tissue-specific manner under the Cre-loxP system  have demonstrated preserved E2 acceleration of carotid artery re-endothelialization after electric injury , an underlying mechanism of neointimal hyperplasia (α and striatin and protein phosphatase 2A (PP2A), leading to subsequent kinase inactivation  is exclusively localized in the cytoplasm. This altered localization is assumed to be caused by enhanced NES function, which is partially restored by leptomycin B, a nuclear export inhibitor. H2 + NES ERα exhibits impaired transcriptional activity but maintains a non-nuclear response of ERK1/2 phosphorylation after E2 administration (α (H2NESKI) exhibits an interesting cardiovascular phenotype where the degree of carotid artery re-endothelialization after electric injury is similar to that of E2-treated wild-type female mice, even in the absence of estrogen by ovariectomy. This observation suggests an intrinsically enhanced non-nuclear ERα signaling in H2NESKI mice  or low-density lipoprotein receptor-deficient (Ldlr−/−)  –118. Whi451A-ERα  and R264 , estrogen-dendrimer conjugate (EDC), and pathway-preferential estrogens (PaPEs) have been developed as selective stimulator of non-nuclear signaling and widely used in various studies, including cardiovascular research.5.1.β-estradiol conjugated to bovine serum albumin (E2-BSA) is membrane impermeable and therefore selectively stimulates cell-surface ERs. The exposure to E2-BSA leads to an increase in intracellular calcium and NO release in human artery endothelial cells, which is presumed to be mediated by non-nuclear signaling  E2-BSA solution contains some free E2 by cleaving from BSA, which may activate nuclear signaling; (2) the pattern of E2-BSA binding to ERs is influenced by the BSA linking site, which leads to different biological responses , 135.5.2.A novel conjugate of estrogen and a polyamidoamine dendrimer was developed, in which the estrogens were linked to the dendrimer through a hydrolytically stable bond . This coIn bovine artery endothelial cells, EDC activates eNOS and promotes cell proliferation and migration. Moreover, EDC accelerates re-endothelialization of carotid artery after electric injury , which iIn an ischemia/reperfusion model, E2 decreases cardiomyocyte apoptosis and infarct size , 139. Prin vivo, as evidenced by the bioluminescence assay using ERE-luciferase reporter mouse and real-time PCR  are synthesized from estradiol by altering its steroid structure and adding modifications. PaPE-1 binds to ERc injury .Ldlr−/− female mice fed a hypercholesterolemic diet  of estrogen, identified in 1997 . GPER inα –150.Interestingly, GPER is not localized only at plasma membrane but also in intracellular compartments including endoplasmic reticulum and Golgi apparatus –99. The GPER mediates multiple physiological effects of estrogen on various organs, including the cardiovascular system. In contrast, it has been reported that GPER is dispensable for estrogenic effects in the reproductive system . Here, w6.1.G-1, a selective agonist of GPER , inducesα non-nuclear signaling in atherosclerosis (GPER also mediates vasculo-protective effects of estrogen against atherosclerosis. In ovary-intact female mice fed on atherogenic diet, GPER deletion reduces vascular NO bioavailability and aggravates aortic inflammation and atherosclerosis , which cclerosis , 98. G-1clerosis , which cclerosis .6.2.GPER stimulation also confers cardiac protection in a PI3K-dependent mechanism. The pretreatment with G-1 attenuates contractile dysfunction and reduces infract size following ischemia/reperfusion injury , which iG-1 inhibits angiotensin II-induced cardiomyocyte hypertrophy . Studies6.3.GPER-deficient female mice lack estrogenic response to insulin, exhibiting hyperglycemia and glucose intolerance . Another7.The series of studies focusing on non-nuclear signaling brought a new perspective on intracellular signaling and expanded our understanding of estrogen function. Further research advance would lead to a therapeutic approach that effectively distinguish cardiovascular protective effects from unfavorable ones such as cancer-progression and thrombosis.


Diaphragmatic dysfunction is prevalent among intensive care unit patients. The use of Fu’s subcutaneous needling (FSN) for respiratory problems is a new issue and few study has been conducted so far.Despite conventional treatments, the patient continued using noninvasive ventilation after discharge from the intensive care unit due to diaphragmatic dysfunction.Diaphragmatic dysfunction.After the myofascial trigger points were confirmed in the neck, chest, and abdomen area, FSN therapy was performed using disposable FSN needles. FSN needles were penetrated into the subcutaneous layer.The patient dyspnea and tachypnea improved, and noninvasive ventilation time dropped significantly. The patient was successfully weaned from the ventilator after 3 sessions of FSN therapy, and there was an increase in diaphragmatic excursion and tidal fraction of the diaphragm via the ultrasound imaging. We found no evidence of relapse 12 months after treatment.FSN therapy has potential as an alternative strategy for patients with diaphragmatic dysfunction and severe neurologic disabilities who do not respond well to conventional therapies, but further research is still required to establish the effects of FSN on diaphragmatic function. This precludes respiratory muscle rehabilitation for patients with significant neurological illness or cognitive impairment.Enhancing clinical awareness of the effects of critical illness on respiratory muscles and diaphragmatic function, coupled with prioritizing preventative measures are essential to improving patient outcomes. Studies have shown that respiratory muscle rehabilitation can improve diaphragmatic strength and aid in weaning critically ill patients from ventilation.–9 From the perspective of FSN, the limb muscles can impact neighboring tissues and organs, including the respiratory system smooth muscles, leading to clinical manifestations such as chronic cough, asthma, shortness of breath, and breathing difficulties. The use of FSN for respiratory problems is a new issue and few studies have been conducted so far. Therefore, we report the first case report of FSN as an adjunctive therapy for treating diaphragmatic dysfunction in a critically ill patient with a severe neurologic disability.Fu’s subcutaneous needling (FSN), originating from Chinese acupuncture and dry needling has emerged as a popular treatment technique, but studies are largely limited to the treatment of myofascial trigger point (MTrP)-induced musculoskeletal disorders.An 18-year-old male patient with a history of hypoxic-ischemic brain injury, resulting in severe motor and language dysfunction as well as mild cognitive impairment, was admitted to a rehabilitation ward. He subsequently developed a high fever, recurrent cough, and dyspnea, which later worsened. He was transferred to the ICU for 16 days due to breathing problems. Upon stabilization, the patient was returned to the rehabilitation ward with persistent symptoms of dyspnea and tachypnea (30–40 breaths per minute). This necessitated continuous post-extubating noninvasive ventilation, which limited the ability to receive aggressive rehabilitation therapy. He was subjected to 24 hours of noninvasive ventilation for the first 4 days, which was reduced to 12 hours a day for the next 3 days. Ultrasound, performed after suspecting a complication of prolonged mechanical ventilation, revealed diaphragm weakness.,11 Results from touch-based physical examination revealed the presence of myofascial trigger points (MTrPs) around his neck, chest, and abdomen . Besides, we observed increases both in diaphragmatic excursion (DE) and thickening fraction of the diaphragm (TFdi) after the second treatment and after the last treatment. Just after the second treatment, the DE increased from 1.36 cm to 1.52 cm immediately, and after the last treatment, the DE was 1.87 cm (Fig. To the best of our knowledge, this is the first report describing efficacy of FSN as an adjunctive therapeutic approach for the management of diaphragmatic dysfunction. Notably, the patient was successfully extubated after 3 FSN treatments, and significant improvement was observed in DE and TFdi.–9,11,12 recent studies,14 reveal that stretching subcutaneously can influence adjacent VA and FSN may enhance gastrointestinal motility and decrease gastric drainage volume. Unlike traditional Chinese acupuncture methods that may involve deep needle insertion, FSN entails the shallow insertion of a disposable needle into the subcutaneous area near the affected muscle, referred to as the “tightened muscle” with one or more MTrPs. The needle is then manipulated using a horizontal swaying movement, rather than the deeper stimulation techniques used in traditional acupuncture.The patient in the present case study was subjected to FSN therapy, a method that combines traditional Chinese acupuncture and dry needling. Studies have reported that FSN is efficacious against various soft tissue injuries and medical issues, Therefore, the development and implementation of a comprehensive treatment plan for ICU survivors throughout their rehabilitation journey are imperative to effective management, and clinicians have recommended inspiratory muscle training for patients with weak inspiratory muscles or trouble weaning from ventilation. In this case study, however, a major barrier to effective inspiratory muscle rehabilitation is that the patient must have adequate cognitive status and actively participate in the exercise. Thus, inspiratory muscle training could not be applied to the patient in the present case.The current therapeutic options for the management of diaphragmatic dysfunction are limited; conventional treatment strategies that target only 1 mechanism ineffective. Although early ICU rehabilitation programs have benefits, they are limited by various factors key among them patients complex conditions, lack of awareness among staff, and low staffing. These challenges significantly hinder rehabilitation outcomes across the ICU setting. Results from a recent randomized pragmatic trial revealed that the addition of acupuncture treatment to routine care was associated with improved disease-specific and health-related quality of life in patients with allergic asthma. Another study demonstrated that a single session of dry needling, applied over active MTrPs in the scalene muscles, effectively improved inspiratory vital capacity in individuals with mechanical neck pain. Consistently, results from another study revealed that the addition of dry needling over regions related to respiration to usual treatment was associated with a reduction in symptoms and an increase in exercise capacity in chronic obstructive pulmonary disease patients of medium severity. Collectively, these findings suggest that acupuncture and dry needling have the potential as adjunctive treatments for the management of respiratory diseases.The efficacy of dry needling and acupuncture as adjunctive treatment therapies for respiratory diseases, including chronic obstructive pulmonary disease and asthma, has been extensively studied. Results indicate that these treatments are not only well-tolerated but also provide additional benefits beyond routine care. Acupuncture could improve the symptoms of a patient with severe COVID-19, including breath rate, SpO2, and the heart rate. Similarly, the dry needling technique also has the ability to reduce muscle stiffness and improve various parameters of neuromuscular function, such as tone, relaxation, and creep. Recently, FSN therapy was found to improve the morphological structure and function of mitochondria within tightened muscles, as evidenced by the upregulation of mitochondrial citrate synthase and Complex II as well as elevated muscle energy metabolism.FSN therapy combines the advantages of traditional acupuncture and dry needling and is particularly effective in reducing muscle stiffness. Studies have shown that FSN therapy can suppress musculoskeletal pain by not only eliminating MTrPs but also reducing muscle tissue hardness.,24 Abdominal muscles play a crucial role in respiration and contribute to effective coughing and secretion clearance during forceful expiratory efforts. Results from a recent study revealed that patients with impaired abdominal muscle function are at risk of failing to be liberated from mechanical ventilation. Another study demonstrated that the application of functional electrical stimulation over the abdominal muscles could improve cough in people with spinal cord injury. Fascia, which connects the rectus abdominis and intercostal muscle to the diaphragm, transfers the effects of FSN to the painful area. Studies have also shown that inactivating MTrPs in the abdomen and chest muscles can result in the disappearance of associated MTrPs in the diaphragm.In the present case, FSN treatment aims to restore and enhance the function of respiratory muscles by eliminating MTrPs and alleviating muscle tightness and cramps.The patient received multiple other rehabilitation therapies, including neuromuscular electrical stimulation, manual hyperinflation, and thorax vibration, in accordance with the current standard of primary care practice. It is important to note that the magnitude of improvement observed in this case report may be influenced by the background of usual care received by the patient. Despite these limitations, the results of this case report are encouraging. Within the context of integrative therapies explored in this paper, FSN was found to supplement rather than supplant the impact of usual care. During the treatment course, however, he was not administered drugs with potential favorable effects on respiratory muscle function, such as theophylline or antioxidants. Collectively, these results suggest that FSN is an efficacious and safe adjunctive therapy for the treatment of diaphragmatic dysfunction in critically ill patients. However, this efficacy needs to be validated by further studies.Our study suggests that FSN, a type of dry needling, is efficacious for early active rehabilitation of patients with diaphragmatic dysfunction and severe neurologic disabilities. Despite being limited to a single case report, it is clear that the ease of administration and feasibility of FSN makes it a promising addition to the repertoire of respiratory rehabilitation strategies, especially considering the unsatisfactory outcomes observed with conventional treatments. In conclusion, FSN is a promising therapeutic strategy for improving recovery outcomes in critically ill patients with diaphragmatic dysfunction-associated weaning difficulties, but further research is still required to establish the effects of FSN on diaphragmatic function.Conceptualization: Hu Li.Investigation: Tianyu Bai.Methodology: Cong Cong Yang.Project administration: Hu Li, Tianyu Bai.Supervision: Jian Sun, Zhonghua Fu.Validation: Hu Li, Tianyu Bai.Visualization: Hu Li, Tianyu Bai.Writing – original draft: Hu Li.Writing – review & editing: Zhonghua Fu, Li-Wei Chou.


Hand hygiene is a cost-effective measure to reduce healthcare-associated infections (HAIs) in healthcare facilities. The impact of the coronavirus disease 2019 (COVID-19) pandemic on hand hygiene performance (HHP) provided evidence for targeted hand hygiene intervention measures.This study evaluated the HHP rate in a tertiary hospital before and after the COVID-19 outbreak. HHP was checked by infection control doctors or nurses every day, and they inputted the HHP rate to the full-time infection control staff every week. A random examination of HHP was conducted by a confidential worker every month. The HHP of healthcare workers (HCWs) was monitored in the outpatient department, inpatient ward, and operating room from January 2017 to October 2022. The influence of COVID-19 prevention and control strategies on HHP was elucidated by analyzing the results of HHP during the study period.P < 0.001). The HHP rate was the highest (93.01%) in September 2022 when the local epidemic occurred. Among the different occupation categories, medical technicians showed the highest HHP rate (89.10%). The HHP rate was the highest after contact with body fluids or blood of patients (94.47%).The average HHP rate of HCWs was 86.11% from January 2017 to October 2022. The HHP rate of HCWs after the COVID-19 pandemic was statistically significantly higher than that before the pandemic (The HHP rate of HCWs in our hospital showed an increasing trend in the recent 6 years, especially during the COVID-19 pandemic, and the increase was most obvious during the local epidemic. P < 0.0001) during the study period, and there was a weak but statistically significant negative correlation between the monthly incidence of HAIs and HH compliance. Therefore, hand hygiene is the simplest, the most effective, the most convenient, and the most economical means of infection control.Healthcare-associated infections (HAIs) are the most prevalent adverse events of hospital care, posing a substantial threat to patient safety, which have an association with increased financial burden and mortality , 2. HAIsHowever, the present situation of HH of HCWs at home and abroad is not satisfactory. Erasmus and Tjbrug  conducteIn a laboratory experiment, SARS-CoV-2 was found to be stable on the skin for up to 14 days at 4°C, 96 h at 22°C, and for at least 8 h at 37°C . With thThis study was carried out in a university teaching hospital in Jining, Shandong Province from January 2017 to October 2022. A new hospital area was added in 2021. Therefore, this hospital currently has 4,100 beds and 5,254 healthcare workers. With the updating and development of disciplines, the hospital currently has 82 clinical departments and 15 intensive care units, responsible for the healthcare of over 20 million people in the southwestern region of Shandong Province, China. In 2021, the hospital had 4 million outpatients, 199,000 discharged patients, 128,000 operations, and 6.6 days of hospitalization on average. This study analyzed the HHP of HCWs in the outpatient department, inpatient ward, and operating room. The study subjects were related personnel engaged in daily diagnosis and treatment, cleaning, and logistics activities in the hospital, including medical personnel, nursing personnel, medical technical personnel, and workers (cleaning and logistics support), including training personnel, standardized training personnel, and interns.This study was approved by the Ethics Committee of the Affiliated Hospital of Jining Medical University (2022-12-C018). The application for exemption of informed consent has been approved. The reasons for the exemption of informed consent are as follows: First, the Affiliated Hospital of Jining Medical College formulated and issued the HH management system (M-9900101-006) and implemented it. HCWs were informed through the system that they would be monitored for HH. Second, this study was based on the monitoring method of HHP in Appendix D of the Health Industry Standard of the People's Republic of China for Medical Personnel Hand Hygiene (WS/T-2019). According to our method, the direct observation method was adopted. The observation objects were randomly selected, and the timing and implementation of HH of HCWs were observed and recorded without informing them. If informed consent was obtained, it could not reflect the real situation of HH of HCWs. Finally, this study was retrospective. We collected the data of the study object, and the privacy of all participants was anonymous in our study.We issued a recruitment notice for HCWs to recruit 20 dispatched nurses as confidential observers for hand hygiene. First of all, we provided unified training for confidential observers, including the purpose, plan, content, and method of the investigation. Direct observers and confidential observers should master unified standards, use unified questionnaires, and ensure the accuracy of observation records. Second, an on-site examination would be conducted after the training, only those who passed the examination were allowed to take up their positions. We provided a certain economic subsidy to the confidential observers every year.We screened individuals who meet the inclusion criteria and exclusion criteria for observation. Inclusion criteria: healthcare workers with observation opportunities for hand hygiene, including doctors, nurses, external learning and further education personnel, cleaning personnel, logistics support personnel, and other third-party management personnel. Exclusion criteria: patient caregivers and personnel without hand hygiene opportunities. The investigation process is detailed in HHP was measured during healthcare work by direct observation by infection control doctors or nurses every day (self-examination), and they inputted the HHP results to the full-time infection control staff every week. A random examination of HHP was conducted anonymously by the confidential worker every month. Direct observation method or viewing surveillance video method was adopted by the confidential worker. Without informing the subjects, the confidential worker randomly selected the subjects and randomly observed them in daily professional activities. It was ensured that one healthcare worker was not observed for more than three hand hygiene times at a time and the duration of observation did not exceed 20 min. No <80 hand hygiene sessions per occupation per month were observed.One confidential worker was responsible for several departments. To avoid discovering the identity of the confidential worker, the departments investigated by the confidential worker were adjusted every month. Two groups of observers covered the clinical departments of the entire hospital every month. The observation parameters included the observation date, observation start and end time, observation place, observation personnel, occupation category of observed personnel , hand hygiene indicators, whether performing hand hygiene and hand hygiene methods , and the accuracy of hand hygiene methods. Monthly reports of hand hygiene observation were obtained through the infection surveillance information system.Training on HHP monitoring was conducted every year for infection control doctors or nurses and confidential workers, including hand hygiene observation requirements, hand hygiene indications, and the use of the infection monitoring information system. Hand hygiene observers were retrained if changed. The full-time infection control staff of the hospital analyzed the feedback on the monitoring situation of HHP of the entire hospital every quarter.According to the WHO guidelines on hand hygiene in healthcare , the fivAfter the COVID-19 outbreak, to improve HHP, hand hygiene management measures were actively adopted to strengthen the hand hygiene management of healthcare workers. The detailed methods are shown in p < 0.05, two-tailed).All the data were analyzed using SPSS 20.0 software . HHP rate = hand hygiene execution time number/hand hygiene execution time number × 100%. We compared the HHP rates of different survey times and methods  and also compared the HHP rates of five hand hygiene opportunities and different professions of HCWs during different survey times. All comparisons were statistically assessed using Pearson's chi-square test, with levels of significance being set at 5% . The HHP rates showed an increasing trend every year in the recent 6 years, and the difference was statistically significant . The average HHP rates of HCWs were 83.77% from 2017 to 2019 and 88.25% from 2020 to 2022. The difference in the HHP rate before and after the COVID-19 epidemic was statistically significant  and the highest in 2022 . The HHP rate checked by infection control doctors or nurses was significantly higher than that checked by confidential workers . The HHP rates of doctors, nurses, medical technicians, refresher/intern nurses, cleaning staff, and service workers showed an increasing trend in the recent 6 years , followed by nurses (87.37%), doctors (86.85%), workers (85.86%), doctors undergoing further study/internship/regular training (79.48%), and nurses in training/practice (77.97%). The lowest HHP rate was observed among the cleaning staff (72.11%). The HHP rates showed a gradually increasing trend in the recent 6 years, and the difference was statistically significant . The HHP rate of different hand hygiene opportunities showed an increasing trend every year in the recent 6 years, with the differences being statistically significant  after contact with body fluids or blood of patients and the lowest (77.79%) after contact with patients' surroundings. The HHP rate of HCWs at different hand hygiene opportunities had statistical differences , among which the month of September showed the highest HHP rate of 93.01% . The HHP rate was the lowest (88.47%) in January . A comparison of the HHP rate of healthcare workers from January to October 2022 revealed a statistically significant difference  in different hand hygiene opportunities and different occupations. It was shown that hand hygiene compliance was improved after the COVID-19 pandemic in our study.The average HHP rate of HCWs in the recent 6 years was 86.11%, and HHP rates increased from 81.99% in 2017 to 89.50% in 2022 in this study; the difference in the HHP rate in the recent 6 years was statistically significant. There was a downward trend in hand hygiene compliance in 2019 compared with that in 2018. However, an obvious upward trend was observed after the COVID-19 outbreak in 2020. The difference in the HHP rate before and after the COVID-19 outbreak was statistically significant. During the epidemic period, the HAI management departments improved their prevention and control strategies, and simultaneously, the hand hygiene awareness of HCWs was strengthened. Therefore, the HHP rate significantly increased after the COVID-19 outbreak. This is consistent with the results reported by Liu et al. (Self-examination and random examination were used to monitor HHP in this study, and the HHP rates were calculated as 87.43 and 73.72%, respectively; the HHP rate was significantly lower in the random examination than that in the self-examination. The choice of monitoring methods is important, and we found that the hand hygiene examination method had a great impact on the HHP. This is consistent with the results of a study on the hidden hand hygiene investigation and analysis of eight comprehensive hospitals in Beijing . HoweverIn this study, the HHP rate of HCWs in different occupations was different as well as the highest. The HHP rates of doctors undergoing further study/internship/regular training, nurses in training/practice, and cleaning staff were low, a finding similar to that was reported by other studies , 22. TheThe HHP rate of HCWs was the highest after contact with body fluids or blood of patients; meanwhile, it was the lowest after contact with patients' surrounding environment in our study, which was consistent with other studies –27. ThisHand hygiene has never been more important than in the COVID-19 pandemic . The resIn this study, there were several limitations. First, the direct observation method was used in the monitoring of HHP, which may be influenced by the Hawthorne effect. Second, this study had not been combined with the consumption of hand hygiene products to evaluate the HHP of HCWs. In future studies, the direct observation method can be combined with the consumption of hand hygiene products to evaluate the hand hygiene compliance of HCWs in a more objective manner.In brief, due to the COVID-19 epidemic, we developed a relatively perfect hand hygiene monitoring system, strengthened hand hygiene training, and increased the corresponding reward and punishment system; therefore, the HHP rate improved. Improving hand hygiene compliance is a continuous improvement effort, and we will develop some improvement strategies in the future based on the current survey results. A long-term hand hygiene mechanism should be established to maintain the best effect of hand hygiene compliance.The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.This study was approved by the Ethics Committee of Affiliated Hospital of Jining Medical University, China. Written informed consent from the participants was not required to participate in this study in accordance with the national legislation and the institutional requirements.PW, YM, LW, YX, NL, DZ, TZ, and LL assisted in data collection. JiW, YM, XY, JuW, and MZ conducted the experiments. XZ, SW, and TZ analyzed the results. XM and LK conceived the study. XZ and XM supervised the study and prepared the manuscript. All authors read and approved the final manuscript.


The origin and intrafamilial transmission of Human T-Lymphotropic Virus Type 1 (HTLV-1) in non-endemic populations such as China is still unknown. In this study, donors from blood banks/centers in China (including 28 provinces and Shenzhen city) during 2019 and 2021 were screened for HTLV-1/2 antibody, and all the reactive samples were tested using a line immunoassay (LIA) and quantitative polymerase chain reaction (qPCR). Samples that can be detected using qPCR were amplified and sequenced for the long terminal repeat (LTR) region. The positive donors were contacted to identify their relatives. As a result, 4,451,883 blood donors were totally tested, and 50 of them were confirmed to be HTLV-1/2 positive. Viral LTR sequences genotyped from 26 HTLV-1 carriers demonstrated that all had the HTLV-1a genotype, of which Transcontinental and Japanese subgroups accounted for half each. There were 17 family members of 11 index donors detected, and the HTLV-1 infection rate in the spouses of male index donors  was significantly higher than that in the husbands of female index donors . However, 7 children of HTLV-1 positive women were tested and found negative. Therefore, our findings indicated that HTLV-1 is spreading silently from high-endemic to low-endemic areas in China. To prevent further HTLV-1/2 transmission, an efficient HTLV-1/2 screening strategy and counseling of the virus carriers are essential. Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiological agent of two severe diseases: adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) , predominantly based on nucleotide sequence analysis of its long terminal repeat (LTR) region. The HTLV-1a subtype is distributed worldwide and can be further divided into five subgroups based on the geographical localization of HTLV-1 infection: A-Transcontinental, B-Japanese, C-West African, D-North African, E-Black Peruvian, and F-Senegalese  for confirmation. According to the confirmatory process , all theSamples with limited plasma volume that were not sufficient for all assays' tests were excluded from our study. Enrolled samples with whole blood available were further quantified by qPCR established in our laboratory. Samples that can be detected using qPCR were also amplified for the LTR region of HTLV-1 by nested PCR. In addition, blood donors with positive results were recommended to collect blood samples from their relatives and test them using the same detection process. The positive donors were also asked to fill out a questionnaire about their personal experiences of probably being infected with HTLV-1/2 and some of the HTLV-1-related syndromes they have suffered.Samples were initially screened for HTLV1/2 antibodies using one of the following three commercially available ELISA assays, including Wantai HTLV-1/2 antibody ELISA kits , Murex HTLV I+II , and Foresight HTLV-1/2 antibody ELISA kits . Reactive specimens underwent retests by the same assay, and any one of the two-round retests that were reactive would be defined as an initially anti-HTLV-1/2 reactive specimen and was sent to the NCCL for further confirmation.Two different serologic assays, namely, Elecsys HTLV-I/II  and Murex HTLV I+II , were performed on one sample simultaneously in NCCL. Any sample that reacted to one of the two assays was confirmed and discriminated by the line immunoassay .pol region. The RPPH1 gene as an internal control was also amplified simultaneously. The primers and probes we used are listed in DNA was extracted from the whole blood of each blood sample by the Tiangen Magnetic Blood Genomic DNA Kit . HTLV-1 proviral DNA was detected and quantified as previously described , was signed by them. If they agreed to allow their relatives to be tested, then the family members would be contacted and they would need to sign written informed consent.2 test. P < 0.05 was considered statistically significant. SPSS software version 17.0 was used for the statistical analyses.The difference between proportions was evaluated using the χA total of 4,451,883 blood samples were initially screened for anti-HTLV-1/2 antibodies, and 1,276 were repeatedly reactive. Except for 32 samples that were not sent to NCCL, the other 1,234 were further confirmed according to the process described above. A total of 754 samples were not reactive in two additional screening assays (Elecsys HTLV-I/II or Murex HTLV I+II), and 480 were reactive in at least one screening assay. After confirmation by LIA, 42 samples were finally identified as HTLV-1 positive, 7 were HTLV positive, 97 were indeterminate, and 334 were negative. Most LIA-indeterminate samples expressed a single gp21 I/II pattern , while the others displayed p19 I/II plus p24 I/II or plus a gp46 I/II pattern. In addition, 226 samples with available blood cells were tested by qPCR; these samples included 29 HTLV-1 positive samples, 5 HTLV positive samples, 59 indeterminate samples, and 133 negative samples. It was discovered that all of the LIA negative samples could not be detected by qPCR, but 93.1% (27/29) HTLV-1 samples, 40% (2/5) HTLV-untyped samples, and 1.7% (1/59) indeterminate samples could all be detected. Consequently, 50 donors were determined to be HTLV-1/2 carriers . The aveAmong the 50 confirmed HTLV1/2-infected individuals, 46 provided demographic information . A total of 50 individuals finally confirmed HTLV-1/2 infection in 18 provinces, of which 26 samples collected from 12 provinces were successfully sequenced. As displayed in p < 0.05). Seven of the index cases provided information about how many sexual partners they had had. Two of them (index 2649 and 2751) had previously had 3–4 sexual partners, and the others  had only one. As for the vertical transmission, seven kids' blood samples were tested, but none were positive for HTLV-1/2 antibodies. Interestingly, four children who had been breastfed for more than 10 months by their infected mothers were not infected. Additionally, index case 2,613 was obtained from the Shandong Blood Hematopoietic Stem Cell Bank, and her child's umbilical cord blood was LIA-HTLV-1 positive but could not be detected using qPCR. While her baby's blood sample was serologically negative.All families of HTLV-1/2 positive individuals were contacted, and 11 agreed to allow their relatives to be tested. In this study, 17 samples were collected from family members of those HTLV-1-infected blood donors. Five index cases were women, and the others were men. As illustrated in The devastating diseases induced by HTLV-1 have a greater impact on health than is commonly cited, indicating that virus infection should not be neglected. In China, in the early 1980s, HTLV-1/2-infected cases were found in three Japanese and two Taiwanese patients, in one ATL patient, and in two Chinese women whose husbands were positive Japanese and Taiwanese individuals  routes. Prospective studies have shown an average vertical transmission rate of 20.5% among children breastfed for ≥6 months, 8.3% among those breastfed for <6 months, and 2.4% among those fed by other routes (Percher et al., To the best of our knowledge, this is the first study to investigate the familiar transmission of HTLV-1 infection in the non-endemic areas of China. We evaluated 11 HTLV-1-infected cases and 17 family members. As presented in vice versa, and younger generations were less susceptible to infection. However, the general population is vulnerable to being infected without a prophylactic vaccine and effective treatment of HTLV-1/2-related diseases. Therefore, in addition to regular HTLV-1/2 antibody testing, counseling of the virus carriers is critical in preventing further HTLV-1/2 transmission.In summary, although HTLV-1/2 prevalence in non-endemic regions in China is as low as previously reported, an effective HTLV-1/2 screening strategy should be adopted to reduce the risk of transmission from the southern coastal areas of China as well as other highly endemic countries. From the molecular analysis, the Transcontinental subgroup may be the predominant HTLV-1 subgroup in the southeastern coastal areas of China, but not in the northeastern coastal areas or other regions. With the expansion of international trade and the increased population migration rate, the Japanese subtype may gradually become more prevalent in some regions. Moreover, a higher transmission risk was observed from male to female than The original contributions presented in the study are included in the article/The studies involving humans were approved by the Beijing Hospital Ethics Committee. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants' legal guardians/next of kin.HJ: Data curation, Investigation, Writing—original draft. LC: Investigation, Methodology, Writing—review and editing. YY: Methodology, Validation, Writing—review and editing. HS: Investigation, Methodology, Writing—original draft. YL: Software, Validation, Writing—review and editing. LW: Validation, Writing—review and editing.