miRNA-7145-cuedc2 axis controls hematopoiesis through JAK1/STAT3 signaling pathway.

Hematopoiesis ensures tissue oxygenation, and remodeling as well as immune protection in vertebrates. During embryogenesis, hemangioblasts are the source of all blood cells. Gata1a and pu.1 are co-expressed in hemangioblasts before hemangioblasts are differentiated into blood cells. However, the genes that determine the differentiation of hemangioblasts into myeloid or erythroid cell lineages have not been fully uncovered. Here we showed that miRNA-7145, a miRNA with previously unknown function, was enriched in erythrocytes at the definitive wave, but not expressed in myeloid cells. Overexpression and loss-of-function analysis of miRNA-7145 revealed that miRNA-7145 functions as a strong inhibitor for myeloid progenitor cell differentiation while driving erythropoiesis during the primitive wave. Furthermore, we confirmed that cuedc2 is one of miRNA-7145 targeted-genes. Overexpression or knock-down of cuedc2 partially rescues the phenotype caused by miRNA-7145 overexpression or loss-of-function. As well, overexpression and loss-of-function analysis of cuedc2 showed that cuedc2 is required for myelopoiesis at the expense of erythropoiesis. Finally, we found that overexpression of zebrafish cuedc2 in 293 T cell inhibits the JAK1/STAT3 signaling pathway. Collectively, our results uncover a previously unknown miRNA-7145-cuedc2 axis, which regulate hematopoiesis through inhibiting the JAK1/STAT3 signaling pathway.

Association Between the Red Blood Cell Distribution Width and 30-Day Mortality in Intensive Care Patients Undergoing Cardiac Surgery: A Retrospective Observational Study Based on the Medical Information Mart for Intensive Care-IV Database.

Background: Millions of patients undergo cardiac surgery each year. The red blood cell distribution width (RDW) could help predict the prognosis of patients who undergo percutaneous coronary intervention or coronary artery bypass surgery. We investigated whether the RDW has robust predictive value for the 30-day mortality among patients in an intensive care unit (ICU) after undergoing cardiac surgery. Methods: Using the Medical Information Mart for Intensive Care-IV Database, we retrieved data for 11,634 patients who underwent cardiac surgery in an ICU. We performed multivariate Cox regression analysis to model the association between the RDW and 30-day mortality and plotted Kaplan-Meier curves. Subgroup analyses were stratified using relevant covariates. Receiver operating characteristic (ROC) curves were used to determine the predictive value of the RDWs. Results: The total 30-day mortality rate was 4.2% (485/11,502). The elevated-RDW group had a higher 30-day mortality rate than the normal-RDW group (P<0.001). The robustness of our data analysis was confirmed by performing subgroup analyses. Each unit increase in the RDW was associated with a 17% increase in 30-day mortality when the RDW was used as a continuous variable (adjusted hazard ratio=1.17, 95% confidence interval, 1.10-1.25). Our ROC results showed the predictive value of the RDW. Conclusions: An elevated RDW was associated with a higher 30-day mortality in patients after undergoing cardiac surgery in an ICU setting. The RDW can serve as an efficient and accessible method for predicting the mortality of patients in ICUs following cardiac surgery.

Development of a stemness-related prognostic index to provide therapeutic strategies for bladder cancer.

Bladder cancer (BC) is a heterogeneous disease with varying clinical outcomes. Recent evidence suggests that cancer progression involves the acquisition of stem-like signatures, and assessing stemness indices help uncover patterns of intra-tumor molecular heterogeneity. We used the one-class logistic regression algorithm to compute the mRNAsi for each sample in BLCA cohort. We subsequently classified BC patients into two subtypes based on 189 mRNAsi-related genes, using the unsupervised consensus clustering. Then, we identified nine hub genes to construct a stemness-related prognostic index (SRPI) using Cox regression, LASSO regression and Random Forest methods. We further validated SRPI using two independent datasets. Afterwards, we examined the molecular and immune characterized of SRPI. Finally, we conducted multiply drug screening and experimental approaches to identify and confirm the most proper agents for patients with high SRPI. Based on the mRNAsi-related genes, BC patients were classified into two stemness subtypes with distinct prognosis, functional annotations, genomic variations and immune profiles. Using the SRPI, we identified a specific subgroup of BC patients with high SRPI, who had a poor response to immunotherapy, and were less sensitive to commonly used chemotherapeutic agents, FGFR inhibitors, and EGFR inhibitors. We further identified that dasatinib was the most promising therapeutic agent for this subgroup of patients. This study provides further insights into the stemness classification of BC, and demonstrates that SRPI is a promising tool for predicting prognosis and therapeutic opportunities for BC patients.

LASS2 enhances chemosensitivity to cisplatin by inhibiting PP2A-mediated ß-catenin dephosphorylation in a subset of stem-like bladder cancer cells.

BACKGROUND: The benefits of first-line, cisplatin-based chemotherapy for muscle-invasive bladder cancer are limited due to intrinsic or acquired resistance to cisplatin. Increasing evidence has revealed the implication of cancer stem cells in the development of chemoresistance. However, the underlying molecular mechanisms remain to be elucidated. This study investigates the role of LASS2, a ceramide synthase, in regulating Wnt/ß-catenin signaling in a subset of stem-like bladder cancer cells and explores strategies to sensitize bladder cancer to cisplatin treatment. METHODS: Data from cohorts of our center and published datasets were used to evaluate the clinical characteristics of LASS2. Flow cytometry was used to sort and analyze bladder cancer stem cells (BCSCs). Tumor sphere formation, soft agar colony formation assay, EdU assay, apoptosis analysis, cell viability, and cisplatin sensitivity assay were used to investigate the functional roles of LASS2. Immunofluorescence, immunoblotting, coimmunoprecipitation, LC-MS, PCR array, luciferase reporter assays, pathway reporter array, chromatin immunoprecipitation, gain-of-function, and loss-of-function approaches were used to investigate the underlying mechanisms. Cell- and patient-derived xenograft models were used to investigate the effect of LASS2 overexpression and a combination of XAV939 on cisplatin sensitization and tumor growth. RESULTS: Patients with low expression of LASS2 have a poorer response to cisplatin-based chemotherapy. Loss of LASS2 confers a stem-like phenotype and contributes to cisplatin resistance. Overexpression of LASS2 results in inhibition of self-renewal ability of BCSCs and increased their sensitivity to cisplatin. Mechanistically, LASS2 inhibits PP2A activity and dissociates PP2A from ß-catenin, preventing the dephosphorylation of ß-catenin and leading to the accumulation of cytosolic phospho-ß-catenin, which decreases the transcription of the downstream genes ABCC2 and CD44 in BCSCs. Overexpression of LASS2 combined with a tankyrase inhibitor (XAV939) synergistically inhibits tumor growth and restores cisplatin sensitivity. CONCLUSIONS: Targeting the LASS2 and ß-catenin pathways may be an effective strategy to overcome cisplatin resistance and inhibit tumor growth in bladder cancer patients.

Vitamin D levels and the risk of overactive bladder: a systematic review and meta-analysis.

CONTEXT: Overactive bladder is treated mainly with behavioral and drug therapy, and symptoms of urinary frequency and incontinence are challenging to eliminate. There is thus a continuous unmet need for new drugs with a substitution effect mechanism. OBJECTIVE: It not known whether vitamin D deficiency can lead to overactive bladder or urinary incontinence or whether vitamin D supplementation alleviates bladder symptoms. This comprehensive systematic review with meta-analysis was conducted to determine whether overactive bladder is associated with vitamin D deficiency. DATA SOURCES: The PubMed and Cochrane Library databases were searched systematically up to July 3, 2022. DATA EXTRACTION: Initially, 706 articles were identified in the literature search, of which 13 were included in the systematic review: 4 randomized controlled trials, 3 cohort studies, 3 cross-sectional studies, and 3 case-control studies. DATA ANALYSIS: An increased risk of overactive bladder and urinary incontinence was observed with vitamin D deficiency (odds ratio [OR] = 4.46; 95%CI, 1.03-19.33; P = 0.046 and OR = 1.30; 95%CI, 1.01-1.66; P = 0.036, respectively). Vitamin D levels were relatively low in patients with overactive bladder or urinary incontinence (SMD = -0.33; 95%CI, -0.61 to -0.06, P = 0.019). On the basis of existing data, the risk of urinary incontinence was reduced by 66% after vitamin D supplementation (OR = 0.34; 95%CI, 0.18-0.66; P = 0.001). Egger test was conducted to assess publication bias, and the results were tested for robustness using a sensitivity analysis. CONCLUSIONS: Vitamin D deficiency increases the risk of overactive bladder and urinary incontinence, and vitamin D supplementation reduces the risk of urinary incontinence. The development of new strategies to prevent or alleviate bladder symptoms is crucial. Vitamin D supplementation may be gaining recognition as an effective strategy for prevention or alleviation of bladder symptoms such as overactive bladder and incontinence. SYSTEMATIC REVIEW REGISTRATION: PROSPERO registration no. CRD42022351443.

Unraveling the parameters and biological mechanisms of CO2 laser therapy for acute pain relief.

Acute pain-related pathology is a significant challenge in clinical practice, and the limitations of traditional pain-relief drugs have made it necessary to explore alternative approaches. Photobiomodulation (PBM) therapy using CO2 laser has emerged as a promising option. In this study, we aimed to identify the optimal parameters of CO2 laser irradiation for acute pain relief through in vivo and in vitro experiments. First, we validated the laser intensity used in this study through bone marrow mesenchymal stem cells (BMSCs) experiments to ensure it will not adversely affect stem cell viability and morphology. Then we conducted a detailed evaluation of the duty cycle and frequency of CO2 laser by the hot plate and formalin test. Results showed a duty cycle of 3% and a frequency of 25 kHz produced the best outcomes. Additionally, we investigated the potential mechanisms underlying the effects of CO2 laser by immunohistochemical staining, and found evidence to suggest that the opioid receptor may be involved in its analgesic effect. In conclusion, this study provides insights into the optimal parameters and underlying mechanisms of CO2 laser therapy for effective pain relief, thereby paving the way for future clinical applications.

The spatiotemporal dynamics of spatially variable genes in developing mouse brain revealed by a novel computational scheme.

To understand how brain regions form and work, it is important to explore the spatially variable genes (SVGs) enriched in specific brain regions during development. Spatial transcriptomics techniques provide opportunity to select SVGs in the high-throughput way. However, previous methods neglected the ranking order and combinatorial effect of SVGs, making them difficult to automatically select the high-priority SVGs from spatial transcriptomics data. Here, we proposed a novel computational pipeline, called SVGbit, to rank the individual and combinatorial SVGs for marker selection in various brain regions, which was tested in different kinds of public datasets for both human and mouse brains. We then generated the spatial transcriptomics and immunohistochemistry data from mouse brain at critical embryonic and neonatal stages. The results show that our ranking and clustering scheme captures the key SVGs which coincide with known anatomic regions in the developing mouse brain. More importantly, SVGbit can facilitate the identification of multiple gene combination sets in different brain regions. We identified three dynamical sub-regions which can be segregated by the staining of Sox2 and Calb2 in thalamus, and we also found that Nr4a2 expression gradually segregates the neocortex and hippocampus during the development. In summary, our work not only reveals the spatiotemporal dynamics of individual and combinatorial SVGs in developing mouse brain, but also provides a novel computational pipeline to facilitate the selection of marker genes from spatial transcriptomics data.

Targeting the PRC2-dependent epigenetic program alleviates urinary tract infections.

Urinary tract infection (UTI) is a pervasive health problem worldwide. Patients with a history of UTIs suffer increased risk of recurrent infections, a major risk of antibiotic resistance. Here, we show that bladder infections induce expression of Ezh2 in bladder urothelial cells. Ezh2 is the methyltransferase of polycomb repressor complex 2 (PRC2)-a potent epigenetic regulator. Urothelium-specific inactivation of PRC2 results in reduced urine bacterial burden, muted inflammatory response, and decreased activity of the NF-κB signaling pathway. PRC2 inactivation also facilitates proper regeneration after urothelial damage from UTIs, by attenuating basal cell hyperplasia and increasing urothelial differentiation. In addition, treatment with Ezh2-specific small-molecule inhibitors improves outcomes of the chronic and severe bladder infections in mice. These findings collectively suggest that the PRC2-dependent epigenetic reprograming controls the amplitude of inflammation and severity of UTIs and that Ezh2 inhibitors may be a viable non-antibiotic strategy to manage chronic and severe UTIs.

Generation of Vgll4-DreER transgenic mouse for visualizing and manipulating VGLL4-expressing cells in vivo.

Vestigial like family member 4 (VGLL4), a member of the Hippo pathway, is a transcriptional cofactor involved in many biological processes, such as tumor progression, postnatal heart growth, and muscle regeneration. However, the VGLL4 expression pattern in vivo remains unclear. To detect and trace Vgll4-expressing cells and their progeny, we generated and characterized a new tamoxifen-inducible Dre knock-in mouse line, Vgll4-DreER. This mouse line expressed DreER (Dre recombinase fused to the estrogen receptor) under the control of the endogenous Vgll4 promoter. After crossing the Vgll4-DreER mouse line with the Dre-responsive reporter H11-rRFP, Dre-mediated recombination in the tissue was monitored on the basis of red fluorescent protein (RFP) signals, which indicated the distribution of VGLL4-positive cells in vivo. Our data revealed that VGLL4 is widely expressed in various cell types at embryonic and neonatal stages. After comparison with our previously reported Vgll4-GFP mouse, we found that the RFP signal profile was wider than the green fluorescent protein (GFP) pattern, indicating that Vgll4-DreER is more sensitive for labeling VGLL4-expressing cells. We next used a dual-recombination system to simultaneously label VGLL4- and keratin 5 (KRT5)-positive cell populations, and no crosstalk was observed in the Krt5-CreER;Vgll4-DreER;R26-rGlR mice. Taken together, the Vgll4-DreER mouse line is a valuable new tool for examining the precise VGLL4 expression profile and conditional manipulating of VGLL4-expressing cells and their progeny.

Heparanase-2 protein and peptides have a protective effect on experimental glomerulonephritis and diabetic nephropathy.

Introduction: The endothelial glycocalyx degrading enzyme heparanase-1 (HPSE1) is a major contributor to kidney diseases, such as glomerulonephritis and diabetic nephropathy. Therefore, inhibition of HPSE1 could be an interesting therapeutic strategy to treat glomerular diseases. A possible HPSE1 inhibitor is heparanase-2 (HPSE2) because HPSE2 is a structural homolog of HPSE1 without enzymatic activity. The importance of HPSE2 has been recently demonstrated in HPSE2-deficient mice, since these mice developed albuminuria and died within a few months after birth. We postulate that inhibition of HPSE1 activity by HPSE2 is a promising therapeutic strategy to target albuminuria and resulting renal failure. Methods: First, we evaluated the regulation of HPSE2 expression in anti-GBM and LPS-induced glomerulonephritis, streptozotocin-induced diabetic nephropathy, and adriamycin nephropathy by qPCR and ELISA. Second, we measured the HPSE1 inhibiting capacity of HPSE2 protein and 30 different HPSE2 peptides and assessed their therapeutic potential in both experimental glomerulonephritis and diabetic nephropathy using kidney function and cortical mRNA expression of HPSE1 and cytokines as outcome parameters. Results: HPSE2 expression was downregulated under inflammatory and diabetic conditions, whereas this effect on HPSE2 expression was absent with HPSE1 inhibition and in HPSE1-deficient mice. Both HPSE2 protein and a mixture of the three most potent HPSE1 inhibitory HPSE2 peptides could prevent LPS and streptozotocin induced kidney injury. Discussion: Taken together, our data suggest a protective effect of HPSE2 in (experimental) glomerular diseases and support the therapeutic potential of HPSE2 as HPSE1 inhibitor in glomerular diseases.

A deep learning model for drug screening and evaluation in bladder cancer organoids.

Three-dimensional cell tissue culture, which produces biological structures termed organoids, has rapidly promoted the progress of biological research, including basic research, drug discovery, and regenerative medicine. However, due to the lack of algorithms and software, analysis of organoid growth is labor intensive and time-consuming. Currently it requires individual measurements using software such as ImageJ, leading to low screening efficiency when used for a high throughput screen. To solve this problem, we developed a bladder cancer organoid culture system, generated microscopic images, and developed a novel automatic image segmentation model, AU2Net (Attention and Cross U2Net). Using a dataset of two hundred images from growing organoids (day1 to day 7) and organoids with or without drug treatment, our model applies deep learning technology for image segmentation. To further improve the accuracy of model prediction, a variety of methods are integrated to improve the model's specificity, including adding Grouping Cross Merge (GCM) modules at the model's jump joints to strengthen the model's feature information. After feature information acquisition, a residual attentional gate (RAG) is added to suppress unnecessary feature propagation and improve the precision of organoids segmentation by establishing rich context-dependent models for local features. Experimental results show that each optimization scheme can significantly improve model performance. The sensitivity, specificity, and F1-Score of the ACU2Net model reached 94.81%, 88.50%, and 91.54% respectively, which exceed those of U-Net, Attention U-Net, and other available network models. Together, this novel ACU2Net model can provide more accurate segmentation results from organoid images and can improve the efficiency of drug screening evaluation using organoids.

WITHDRAWN: The Analgesic Effect of Regional Blocks for Modified Radical Mastectomy: A Network Meta-analysis.

Ahead of Print article withdrawn by publisher.

Association between blood urea nitrogen to serum albumin ratio and in-hospital mortality of patients with sepsis in intensive care: A retrospective analysis of the fourth-generation Medical Information Mart for Intensive Care database.

Background: This study aimed to investigate the relationship between the blood urea nitrogen to serum albumin ratio (BAR) and in-hospital mortality in patients with sepsis. Materials and methods: This is a retrospective cohort study. All septic patient data for the study were obtained from the intensive care unit of Beth Israel Deaconess Medical Center. Adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated using multivariable Cox regression analyses. Survival curves were plotted and subgroup analyses were stratified by relevant covariates. Results: Among 23,901 patients, 13,464 with sepsis were included. The overall in-hospital mortality rate was 18.9% (2550/13464). After adjustment for confounding factors, patients in the highest BAR quartile had an increased risk of sepsis death than those in the lowest BAR quartile (HR: 1.42, 95% CI: 1.3-1.55), using BAR as a categorical variable. When BAR was presented as a continuous variable, the prevalence of in-hospital sepsis-related death increased by 8% (adjusted HR: 1.08, 95% CI: 1.07-1.1, P < 0.001) for each 5-unit increase in BAR, irrespective of confounders. Stratified analyses indicated age interactions (P < 0.001), and the correlation between BAR and the probability of dying due to sepsis was stable. Conclusion: BAR was significantly associated with in-hospital mortality in intensive care patients with sepsis. A higher BAR in patients with sepsis is associated with a worse prognosis in the ICU in the USA. However, further research is required to confirm this finding.

Association between glucose-to-lymphocyte ratio and in-hospital mortality in intensive care patients with sepsis: A retrospective observational study based on Medical Information Mart for Intensive Care IV.

Background: This study aimed to evaluate the association between the glucose-to-lymphocyte ratio (GLR) and in-hospital mortality in intensive care unit (ICUs) patients with sepsis. Methods: This is a retrospective cohort study. Patients with sepsis from the Medical Information Mart for Intensive Care-IV (MIMIC-IV) database had their baseline data and in-hospital prognosis retrieved. Multivariable Cox regression analyses were applied to calculate adjusted hazard ratios (HR) with 95% confidence intervals (CI). Survival curves were plotted, and subgroup analyses were stratified by relevant covariates. To address the non-linearity relationship, curve fitting and a threshold effect analysis were performed. Results: Of the 23,901 patients, 10,118 patients with sepsis were included. The overall in-hospital mortality rate was 17.1% (1,726/10,118). Adjusted for confounding factors in the multivariable Cox regression analysis models, when GLR was used as a categorical variable, patients in the highest GLR quartile had increased in-hospital mortality compared to patients in the lowest GLR quartile (HR = 1.26, 95% CI: 1.15-1.38). When GLR was used as a continuous variable, each unit increase in GLR was associated with a 2% increase in the prevalence of in-hospital mortality (adjusted HR = 1.02, 95% CI: 1.01-1.03, p = 0.001). Stratified analyses indicated that the correlation between the GLR and in-hospital mortality was stable. The non-linear relationship between GLR and in-hospital mortality was explored in a dose-dependent manner. In-hospital mortality increased by 67% (aHR = 1.67, 95% CI: 1.45-1.92) for every unit GLR increase. When GLR was beyond 1.68, in-hospital mortality did not significantly change (aHR: 1.04, 95% CI: 0.92-1.18). Conclusion: There is a non-linear relationship between GLR and in-hospital mortality in intensive care patients with sepsis. A higher GLR in ICU patients is associated with in-hospital mortality in the United States. However, further research is needed to confirm the findings.

Arid1a regulates bladder urothelium formation and maintenance.

Epigenetic regulation of gene expression plays a central role in bladder urothelium development and maintenance. ATPase-dependent chromatin remodeling is a major epigenetic regulatory mechanism, but its role in the bladder has not been explored. Here, we show the functions of Arid1a, the largest subunit of the SWI/SNF or BAF chromatin remodeling ATPase complex, in embryonic and adult bladder urothelium. Knockout of Arid1a in urothelial progenitor cells significantly increases cell proliferation during bladder development. Deletion of Arid1a causes ectopic cell proliferation in the terminally differentiated superficial cells in adult mice. Consistently, gene-set enrichment analysis of differentially expressed genes demonstrates that the cell cycle-related pathways are significantly enriched in Arid1a knockouts. Gene-set of the polycomb repression complex 2 (PRC2) pathway is also enriched, suggesting that Arid1a antagonizes the PRC2-dependent epigenetic gene silencing program in the bladder. During acute cyclophosphamide-induced bladder injury, Arid1a knockouts develop hyperproliferative and hyperinflammatory phenotypes and exhibit a severe loss of urothelial cells. A Hallmark gene-set of the oxidative phosphorylation pathway is significantly reduced in Aria1a mutants before injury and is unexpectedly enriched during injury response. Together, this study uncovers functions of Arid1a in both bladder progenitor cells and the mature urothelium, suggesting its critical roles in urothelial development and regeneration.

A novel deep learning segmentation model for organoid-based drug screening.

Organoids are self-organized three-dimensional in vitro cell cultures derived from stem cells. They can recapitulate organ development, tissue regeneration, and disease progression and, hence, have broad applications in drug discovery. However, the lack of effective graphic algorithms for organoid growth analysis has slowed the development of organoid-based drug screening. In this study, we take advantage of a bladder cancer organoid system and develop a deep learning model, the res-double dynamic conv attention U-Net (RDAU-Net) model, to improve the efficiency and accuracy of organoid-based drug screenings. In this RDAU-Net model, the dynamic convolution and attention modules are integrated. The feature-extracting capability of the encoder and the utilization of multi-scale information are substantially enhanced, and the semantic gap caused by skip connections has been filled, which substantially improved its anti-interference ability. A total of 200 images of bladder cancer organoids on culture days 1, 3, 5, and 7, with or without drug treatment, were employed for training and testing. Compared with the other variations of the U-Net model, the segmentation indicators, such as Intersection over Union and dice similarity coefficient, in the RDAU-Net model have been improved. In addition, this algorithm effectively prevented false identification and missing identification, while maintaining a smooth edge contour of segmentation results. In summary, we proposed a novel method based on a deep learning model which could significantly improve the efficiency and accuracy of high-throughput drug screening and evaluation using organoids.

Posttranslational regulation of FOXA1 by Polycomb and BUB3/USP7 deubiquitin complex in prostate cancer.

Forkhead box protein A1 (FOXA1) is essential for androgen-dependent prostate cancer (PCa) growth. However, how FOXA1 levels are regulated remains elusive and its therapeutic targeting proven challenging. Here, we report FOXA1 as a nonhistone substrate of enhancer of zeste homolog 2 (EZH2), which methylates FOXA1 at lysine-295. This methylation is recognized by WD40 repeat protein BUB3, which subsequently recruits ubiquitin-specific protease 7 (USP7) to remove ubiquitination and enhance FOXA1 protein stability. They functionally converge in regulating cell cycle genes and promoting PCa growth. FOXA1 is a major therapeutic target of the inhibitors of EZH2 methyltransferase activities in PCa. FOXA1-driven PCa growth can be effectively mitigated by EZH2 enzymatic inhibitors, either alone or in combination with USP7 inhibitors. Together, our study reports EZH2-catalyzed methylation as a key mechanism to FOXA1 protein stability, which may be leveraged to enhance therapeutic targeting of PCa using enzymatic EZH2 inhibitors.

Using Elastographic Ultrasound to Assess the Effect of Cupping Size of Cupping Therapy on Stiffness of Triceps Muscle.

OBJECTIVE: Cupping therapy may reduce muscle stiffness for managing fatigue. However, there is no scientific evidence showing changes of muscle stiffness after cupping therapy. Furthermore, it is unclear whether the cup size of cupping therapy affects the change of muscle stiffness. The objective of this study was to compare the effect of cup size of cupping therapy on muscle stiffness. DESIGN: A repeated measures design with a counterbalanced design was used to test three cup sizes (45, 40, and 35 mm in inner diameter) in 12 healthy participants. Strain elastography was used to measure stiffness of the triceps before and after cupping therapy at 300 mm Hg for 5 mins. Strain elastogram was converted to the grayscale for the quantification of stiffness. RESULTS: The overall stiffness of triceps significantly reduced after cupping therapy with the 45-mm (106.2 ± 7.7, P < 0.05) and 40-mm (109.6 ± 7.1, P < 0.05) cups, but not the 35-mm cup (115.5 ± 10.3, nonsignificant) compared with before cupping (115.8 ± 13.5). The stiffness of superficial layer did not show significantly difference in all three sizes of cup. The stiffness of deep layer significantly reduced after the cupping therapy with the 45- and 40-mm cups. CONCLUSIONS: This is the first study demonstrating that cupping therapy significantly reduced muscle stiffness, especially at the deep layer.