High-throughput screening of mutations affecting SARS-CoV-2 spike functions.

The spike (S) protein of SARS-CoV-2, which is undergoing rapid evolution, plays crucial roles in viral immune escape, infectivity, and transmissibility. To gain clinical insight, Dadonaite et al. developed a novel deep mutational scanning (DMS) platform for mapping the effects of S protein mutations on immune evasion and viral infectivity.

Cytokinins regulate rice lamina joint development and leaf angle.

Leaf angle is determined by lamina joint inclination and is an important agronomic trait that determines plant architecture, photosynthetic efficiency, and crop yield. Cytokinins (CKs) are phytohormones involved in shaping rice (Oryza sativa L.) architecture, but their role in leaf angle remains unknown. Here, we report that CK accumulation mediated by rice CK OXIDASE/DEHYDROGENASE3 (OsCKX3) controls lamina joint development and negatively regulates leaf angle. Phenotypic analysis showed that rice osckx3 mutants had smaller leaf angles, while the overexpression lines (OsCKX3-OE) had larger leaf angles. Histological sections indicated that the leaf inclination changes in the osckx3 and OsCKX3-OE lines resulted from asymmetric proliferation of the cells and vascular bundles in the lamina joint. Reverse transcription quantitative PCR, promoter-fused ß-glucuronidase expression, and subcellular localization assays indicated that OsCKX3 was highly expressed in the lamina joint, and OsCKX3-GFP fusion protein localized to the endoplasmic reticulum. The enzyme assays using recombinant protein OsCKX3 revealed that OsCKX3 prefers trans-zeatin (tZ) and isopentenyladenine (iP). Consistently, tZ and iP levels increased in the osckx3 mutants but decreased in the OsCKX3 overexpression lines. Interestingly, agronomic trait analysis of the rice grown in the paddy field indicated that osckx3 displayed a smaller leaf angle and enhanced primary branch number, grain size, 1,000-grain weight, and flag leaf size. Collectively, our results revealed that enhancing CK levels in the lamina joint by disrupting OsCKX3 negatively regulates leaf angle, highlighting that the CK pathway can be engineered to reduce leaf angle in rice and possibly in other cereals.

Design of MERS-CoV entry inhibitory short peptides based on helix-stabilizing strategies.

Interaction between Middle East respiratory syndrome coronavirus (MERS-CoV) spike (S) protein heptad repeat-1 domain (HR1) and heptad repeat-2 domain (HR2) is critical for the MERS-CoV fusion process. This interaction is mediated by the α-helical region from HR2 and the hydrophobic groove in a central HR1 trimeric coiled coil. We sought to develop a short peptidomimetic to act as a MERS-CoV fusion inhibitor by reproducing the key recognition features of HR2 helix. This was achieved by the use of helix-stabilizing strategies, including substitution with unnatural helix-favoring amino acids, introduction of ion pair interactions, and conjugation of palmitic acid. The resulting 23-mer lipopeptide, termed AEEA-C16, inhibits MERS-CoV S protein-mediated cell-cell fusion at a low micromolar level comparable to that of the 36-mer HR2 peptide HR2P-M2. Collectively, our studies provide new insights into developing short peptide-based antiviral agents to treat MERS-CoV infection.

Expanding the DNA-encoded library toolbox: identifying small molecules targeting RNA.

DNA-encoded library (DEL) technology is a powerful tool for small molecule identification in drug discovery, yet the reported DEL selection strategies were applied primarily on protein targets in either purified form or in cellular context. To expand the application of this technology, we employed DEL selection on an RNA target HIV-1 TAR (trans-acting responsive region), but found that the majority of signals were resulted from false positive DNA-RNA binding. We thus developed an optimized selection strategy utilizing RNA patches and competitive elution to minimize unwanted DNA binding, followed by k-mer analysis and motif search to differentiate false positive signal. This optimized strategy resulted in a very clean background in a DEL selection against Escherichia coli FMN Riboswitch, and the enriched compounds were determined with double digit nanomolar binding affinity, as well as similar potency in functional FMN competition assay. These results demonstrated the feasibility of small molecule identification against RNA targets using DEL selection. The developed experimental and computational strategy provided a promising opportunity for RNA ligand screening and expanded the application of DEL selection to a much wider context in drug discovery.

Dynamic Reconstruction of Two-Dimensional Defective Bi Nanosheets for Efficient Electrocatalytic Urea Synthesis.

Catalyst surface dynamics drive the generation of active species for electrocatalytic reactions. Yet, the understanding of dominant site formation and reaction mechanisms is limited. In this study, we thoroughly investigate the dynamic reconstruction of two-dimensional defective Bi nanosheets from exfoliated Bi2Se3 nanosheets under electrochemical CO2 and nitrate (NO3 -) reduction conditions. The ultrathin Bi2Se3 nanosheets obtained by NaBH4-assisted cryo-mediated liquid-phase exfoliation are more easily reduced and reconstructed to Bi nanosheets with high-density grain boundaries (GBs; GB-rich Bi). The reconstructed GB-rich Bi catalyst affords a remarkable yield rate of 4.6 mmol h-1 mgcat. -1 and Faradaic efficiency of 32 % for urea production at -0.40 V vs. RHE. Notably, this yield rate is 2 and 8.2 times higher than those of the low-GB Bi and bulk Bi catalysts, respectively. Theoretical analysis demonstrates that the GB sites significantly reduce the *CO and *NH2 intermediate formation energy and C-N coupling energy barrier, enabling selective urea electrosynthesis on the GB-rich Bi catalyst. This work will trigger further research into the structure-activity interplay in dynamic processes using in situ techniques.

Accelerating Industrial-Level NO3 - Electroreduction to Ammonia on Cu Grain Boundary Sites via Heteroatom Doping Strategy.

Although the electrocatalytic nitrate reduction reaction (NO3 - RR) is an attractive NH3 synthesis route, it suffers from low yield due to the lack of efficient catalysts. Here, this work reports a novel grain boundary (GB)-rich Sn-Cu catalyst, derived from in situ electroreduction of Sn-doped CuO nanoflower, for effectively electrochemical converting NO3 - to NH3 . The optimized Sn1% -Cu electrode achieves a high NH3 yield rate of 1.98 mmol h-1 cm-2 with an industrial-level current density of -425 mA cm-2 at -0.55 V versus a reversible hydrogen electrode (RHE) and a maximum Faradaic efficiency of 98.2% at -0.51 V versus RHE, outperforming the pure Cu electrode. In situ Raman and attenuated total reflection Fourier transform infrared spectroscopies reveal the reaction pathway of NO3 - RR to NH3 by monitoring the adsorption property of reaction intermediates. Density functional theory calculations clarify that the high-density GB active sites and the competitive hydrogen evolution reaction (HER) suppression induced by Sn doping synergistically promote highly active and selective NH3 synthesis from NO3 - RR. This work paves an avenue for efficient NH3 synthesis over Cu catalyst by in situ reconstruction of GB sites with heteroatom doping.

Pan-coronavirus fusion inhibitors to combat COVID-19 and other emerging coronavirus infectious diseases.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the currently ongoing coronavirus disease 2019 (COVID-19) pandemic, has posed a serious threat to global public health. Recently, several SARS-CoV-2 variants of concern (VOCs) have emerged and caused numerous cases of reinfection in convalescent COVID-19 patients, as well as breakthrough infections in vaccinated individuals. This calls for the development of broad-spectrum antiviral drugs to combat SARS-CoV-2 and its VOCs. Pan-coronavirus fusion inhibitors, targeting the conserved heptad repeat 1 (HR1) in spike protein S2 subunit, can broadly and potently inhibit infection of SARS-CoV-2 and its variants, as well as other human coronaviruses. In this review, we summarized the most recent development of pan-coronavirus fusion inhibitors, such as EK1, EK1C4, and EKL1C, and highlighted their potential application in combating current COVID-19 infection and reinfection, as well as future emerging coronavirus infectious diseases.

SARS-CoV-2 Omicron XBB subvariants exhibit enhanced fusogenicity and substantial immune evasion in elderly population, but high sensitivity to pan-coronavirus fusion inhibitors.

Numerous emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariants have shown significant immune evasion capacity and caused a large number of infections, as well as vaccine-breakthrough infections, especially in elderly populations. Recently emerged Omicron XBB was derived from the BA.2 lineage, but bears a distinct mutant profile in its spike (S) protein. In this study, we found that Omicron XBB S protein drove more efficient membrane-fusion kinetics on human lung-derived cells (Calu-3). Considering the high susceptibility of the elderly to the current Omicron pandemic, we performed a comprehensive neutralization assessment of elderly convalescent or vaccine sera against XBB infection. We found that the sera from elderly convalescent patients who experienced with BA.2 infection or breakthrough infection potently inhibited BA.2 infection, but showed significantly reduced efficacy against XBB. Moreover, recently emerged XBB.1.5 subvariant also showed more significant resistance to the convalescent sera of BA.2- or BA.5-infected elderly. On the other hand, we found that the pan-CoV fusion inhibitors EK1 and EK1C4 can potently block either XBB-S- or XBB.1.5-S-mediated fusion process and viral entry. Moreover, EK1 fusion inhibitor showed potent synergism when combined with convalescent sera of BA.2- or BA.5-infected patients against XBB and XBB.1.5 infection, further indicating that EK1-based pan-CoV fusion inhibitors are promising candidates for development as clinical antiviral agents to combat the Omicron XBB subvariants.

Repurposing Navitoclax to block SARS-CoV-2 fusion and entry by targeting heptapeptide repeat sequence 1 in S2 protein.

Along with the long pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has come the dilemma of emerging viral variants of concern (VOC), particularly Omicron and its subvariants, able to deftly escape immune surveillance and the otherwise protective effect of current vaccines and antibody drugs. We previously identified a peptide-based pan-CoV fusion inhibitor, termed as EK1, able to bind the HR1 region in viral spike (S) protein S2 subunit. This effectively blocked formation of the six-helix bundle (6-HB) fusion core and, thus, showed efficacy against all human coronaviruses (HCoVs). EK1 is now in phase 3 clinical trials. However, the peptide drug generally lacks oral availability. Therefore, we herein performed a structure-based virtual screening of the libraries of biologically active molecules and identified nine candidate compounds. One is Navitoclax, an orally active anticancer drug by inhibition of Bcl-2. Like EK1 peptide, it could bind HR1 and block 6-HB formation, efficiently inhibiting fusion and infection of all SARS-CoV-2 variants tested, as well as SARS-CoV and MERS-CoV, with IC50 values ranging from 0.5 to 3.7 µM. These findings suggest that Navitoclax is a promising repurposed drug candidate for development as a safe and orally available broad-spectrum antiviral drug to combat the current SARS-CoV-2 and its variants, as well as other HCoVs.

HPV-infection status and urinary incontinence: a population-based analysis of the NHANES 2005-2016.

PURPOSE: Urinary incontinence is a common condition and reduces the quality of life. The purpose of this study was to assess the association between HPV infection and urinary incontinence among adult women in the USA. METHODS: We examined a cross-sectional study using the National Health and Nutrition Examination Survey database. Women who had valid HPV DNA vaginal swab test results and answered the questionnaire about urinary incontinence were selected from six consecutive survey cycles (2005-2006 to 2015-2016). The association between HPV status and urinary incontinence was analyzed using weighted logistic regression. Models adjusted for potential variables were established. RESULTS: In total, 8348 females aged between 20 and 59 years old were enrolled in this study. 47.8% of participants had a history of urinary incontinence and 43.9% of women were HPV DNA positive. After adjusting for all confounders, women with HPV infection were less likely to have urinary incontinence (OR = 0.88, 95%CI 0.78-0.98). Low-risk HPV infection correlated with a lower incidence of incontinence (OR = 0.88, 95%CI 0.77-1.00). For women aged below 40 years, low-risk HPV infection negatively correlated with stress incontinence (20-29ys: OR = 0.67, 95%CI 0.49-0.94; 30-39ys: OR = 0.71, 95%CI 0.54-0.93). However, low-risk HPV infection positively correlated with stress incontinence (OR = 1.40, 95%CI 1.01-1.95) for women 50-59 years old. CONCLUSION: This study revealed a negative association between HPV infection and urinary incontinence in females. Low-risk HPV correlated with stress urinary incontinence, with the reverse trend for participants of different ages.

Decoy nanoparticles protect against COVID-19 by concurrently adsorbing viruses and inflammatory cytokines.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has highlighted the urgent need to rapidly develop therapeutic strategies for such emerging viruses without effective vaccines or drugs. Here, we report a decoy nanoparticle against COVID-19 through a powerful two-step neutralization approach: virus neutralization in the first step followed by cytokine neutralization in the second step. The nanodecoy, made by fusing cellular membrane nanovesicles derived from human monocytes and genetically engineered cells stably expressing angiotensin converting enzyme II (ACE2) receptors, possesses an antigenic exterior the same as source cells. By competing with host cells for virus binding, these nanodecoys effectively protect host cells from the infection of pseudoviruses and authentic SARS-CoV-2. Moreover, relying on abundant cytokine receptors on the surface, the nanodecoys efficiently bind and neutralize inflammatory cytokines including interleukin 6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM-CSF), and significantly suppress immune disorder and lung injury in an acute pneumonia mouse model. Our work presents a simple, safe, and robust antiviral nanotechnology for ongoing COVID-19 and future potential epidemics.

Exosomal miR-625-3p secreted by cancer-associated fibroblasts in colorectal cancer promotes EMT and chemotherapeutic resistance by blocking the CELF2/WWOX pathway.

Migration, invasion, epithelial-mesenchymal transformation (EMT), and chemotherapeutic resistance are the leading causes of therapeutic failure in people with colorectal cancer (CRC). The migration of exosomal miRNA between cancer cells and the tumor microenvironment is directly associated with malignant behavior in cancer-associated fibroblasts (CAFs). In the context of earlier research, the purpose of the current study was to assess the role and potential mechanism of miR-625-3p released by CAFs in CRC cells. Exosomes were extracted and purified from CAFs conditioned medium by ultracentrifugation. Western blot, immunohistochemistry, CCK-8, transwell assay, H&E staining, Tunnel, real-time PCR, double luciferase assay, RNA-binding protein immunoprecipitation (RIP), and immunofluorescence double staining experiments were used to investigate the effects of CAFs-Exo and miR-625-3p on CRC cell invasion, migration, proliferation, EMT, chemotherapeutic resistance, and molecular mechanisms. The current results indicated that CAFs-Exo was directly internalized by CRC cells, and exosomal miR-625-3p derived from CAFs might promote migration, invasion, EMT and chemotherapeutic resistance in CRC cells by inhibiting the CELF2/WWOX pathway, providing a potential candidate for CRC prediction and treatment.

Coronavirus Entry Inhibitors.

Coronaviruses (CoVs) are enveloped RNA viruses that widely exist in the environment. Several CoVs possess a strong ability to infect humans, termed as human coronavirus (HCoVs). Among seven known HCoVs, SARS-CoV-2, SARS-CoV, and MERS-CoV belong to highly pathogenic HCoVs, which can cause severe clinical symptoms and even death. Especially, the current COVID-19 pandemic severely threatens human survival and health, which emphasizes the importance of developing effective CoV vaccines and anti-CoV agents to protect humans from HCoV infections. Coronavirus entry inhibitors can block various processes in viral entry, such as receptor binding, proteolytic activation of spike protein, or virus-cell membrane fusion. Coronavirus entry inhibitors, alone or in combination with other drugs, play important roles in the treatment of coronavirus diseases. Thus, we summarize and discuss the development of coronavirus entry inhibitors in this chapter.

Ratiometric Detection of Glutathione Based on Disulfide Linkage Rupture between a FRET Coumarin Donor and a Rhodamine Acceptor.

Abnormal levels of glutathione, a cellular antioxidant, can lead to a variety of diseases. We have constructed a near-infrared ratiometric fluorescent probe to detect glutathione concentrations in biological samples. The probe consists of a coumarin donor, which is connected through a disulfide-tethered linker to a rhodamine acceptor. Under the excitation of the coumarin donor at 405 nm, the probe shows weak visible fluorescence of the coumarin donor at 470 nm and strong near-infrared fluorescence of the rhodamine acceptor at 652 nm due to efficient Forster resonance energy transfer (FRET) from the donor to the acceptor. Glutathione breaks the disulfide bond through reduction, which results in a dramatic increase in coumarin fluorescence and a corresponding decrease in rhodamine fluorescence. The probe possesses excellent cell permeability, biocompatibility, and good ratiometric fluorescence responses to glutathione and cysteine with a self-calibration capability. The probe was utilized to ratiometrically visualize glutathione concentration alterations in HeLa cells and Drosophila melanogaster larvae.

IgG Fc-binding motif-conjugated HIV-1 fusion inhibitor exhibits improved potency and in vivo half-life: Potential application in combination with broad neutralizing antibodies.

The clinical application of conventional peptide drugs, such as the HIV-1 fusion inhibitor enfuvirtide, is limited by their short half-life in vivo. To overcome this limitation, we developed a new strategy to extend the in vivo half-life of a short HIV-1 fusion inhibitory peptide, CP24, by fusing it with the human IgG Fc-binding peptide (IBP). The newly engineered peptide IBP-CP24 exhibited potent and broad anti-HIV-1 activity with IC50 values ranging from 0.2 to 173.7 nM for inhibiting a broad spectrum of HIV-1 strains with different subtypes and tropisms, including those resistant to enfuvirtide. Most importantly, its half-life in the plasma of rhesus monkeys was 46.1 h, about 26- and 14-fold longer than that of CP24 (t1/2 = 1.7 h) and enfuvirtide (t1/2 = 3 h), respectively. IBP-CP24 intravenously administered in rhesus monkeys could not induce significant IBP-CP24-specific antibody response and it showed no obvious in vitro or in vivo toxicity. In the prophylactic study, humanized mice pretreated with IBP-CP24 were protected from HIV-1 infection. As a therapeutic treatment, coadministration of IBP-CP24 and normal human IgG to humanized mice with chronic HIV-1 infection resulted in a significant decrease of plasma viremia. Combining IBP-CP24 with a broad neutralizing antibody (bNAb) targeting CD4-binding site (CD4bs) in gp120 or a membrane proximal external region (MPER) in gp41 exhibited synergistic effect, resulting in significant dose-reduction of the bNAb and IBP-CP24. These results suggest that IBP-CP24 has the potential to be further developed as a new HIV-1 fusion inhibitor-based, long-acting anti-HIV drug that can be used alone or in combination with a bNAb for treatment and prevention of HIV-1 infection.

25-Hydroxycholesterol-Conjugated EK1 Peptide with Potent and Broad-Spectrum Inhibitory Activity against SARS-CoV-2, Its Variants of Concern, and Other Human Coronaviruses.

The COVID-19 pandemic caused by SARS-CoV-2 infection poses a serious threat to global public health and the economy. The enzymatic product of cholesterol 25-hydroxylase (CH25H), 25-Hydroxycholesterol (25-HC), was reported to have potent anti-SARS-CoV-2 activity. Here, we found that the combination of 25-HC with EK1 peptide, a pan-coronavirus (CoV) fusion inhibitor, showed a synergistic antiviral activity. We then used the method of 25-HC modification to design and synthesize a series of 25-HC-modified peptides and found that a 25-HC-modified EK1 peptide (EK1P4HC) was highly effective against infections caused by SARS-CoV-2, its variants of concern (VOCs), and other human CoVs, such as HCoV-OC43 and HCoV-229E. EK1P4HC could protect newborn mice from lethal HCoV-OC43 infection, suggesting that conjugation of 25-HC with a peptide-based viral inhibitor was a feasible and universal strategy to improve its antiviral activity.

Ratiometric Near-Infrared Fluorescent Probes Based on Hemicyanine Dyes Bearing Dithioacetal and Formal Residues for pH Detection in Mitochondria.

Ratiometric near-infrared fluorescent probes (AH+ and BH+) have been prepared for pH determination in mitochondria by attaching dithioacetal and formal residues onto a hemicyanine dye. The reactive formyl group on probe BH+ allows for retention inside mitochondria as it can react with a protein primary amine residue to form an imine under slightly basic pH 8.0. Probes AH+ and BH+ display ratiometric fluorescent responses to pH changes through the protonation and deprotonaton of a hydroxy group in hemicyanine dyes with experimentally determined pKa values of 6.85 and 6.49, respectively. Calculated pKa values from a variety of theoretical methods indicated that the SMDBONDI method of accounting for solvent and van der Waals radii plus including a water molecule located near the site of protonation produced the closest overall agreement with the experimental values at 7.33 and 6.14 for AH+ and BH+ respectively.

Fluorescent probes with high pKa values based on traditional, near-infrared rhodamine, and hemicyanine fluorophores for sensitive detection of lysosomal pH variations.

Sterically hindered fluorescent probes (A-C) have been developed by introducing 2-aminophenylboronic acid pinacol ester to a traditional, A, a near-infrared rhodamine dye, B, and a near-infrared hemicyanine dye, C, forming closed spirolactam ring structures. Probe A was non-fluorescent under basic pH conditions whereas probes B and C were moderately fluorescent with fluorescence quantum yields of 9% and 5% in pH 7.4 PBS buffer containing 1% ethanol, respectively. With all probes increasing acidity leads to significant increases in fluorescence at 580 nm, 644 and 744 nm for probes A, B and C with fluorescence quantum yields of 26%, 21% and 10% in pH 4.5 PBS buffer containing 1% ethanol, respectively. Probes A, B and C were calculated to have pKa values of 5.81, 5.45 and 6.97. The difference in fluorescence under basic conditions is ascribed to easier opening of the closed spirolactam ring configurations due to significant steric hindrance between the 2-aminophenylboronic acid pinacol ester residue and an adjacent H atom in the xanthene derivative moiety in probe B or C. The probes show fast, reversible, selective and sensitive fluorescence responses to pH changes, and are capable of sensing lysosomal pH variations in living cells.

Association between obstructive sleep apnea syndrome and nocturia: a meta-analysis.

BACKGROUND: The relationship between obstructive sleep apnea (OSA) and the risk of nocturia remains unclear. Therefore, we sought to identify whether or not OSA affects the incidence of nocturia. METHODS: A thorough literature search was executed in September 1st 2018 from PubMed, Web of Science database, and Embase. We used DerSimonian and Laird random-effects to calculate the pooled relative ratio (RR). RESULTS: Total of 13 studies met inclusion criteria and in total comprised, 406 patients and 9518 controls. There was a significant association between OSA and the risk of nocturia (RR = 1.41, 95% CI 1.26-1.59). Through subgroup analysis by different severity of OSA, we found patients who had severe OSA were at high risk of nocturia. Through another subgroup analysis, we found a statistically significant association between OSA and risk of nocturia in the men (RR = 1.487, 95% CI 1.087-2.034, P = 0.013). However, there was no significant relationship between OSA and nocturia in the women (RR = 1.537, 95% CI 0.831-2.842, P > 0.05). Subgroup analysis of different diagnostic methods indicated that OSA was significantly associated with the risk of nocturia regardless what method was used to diagnose OSA (P < 0.05). CONCLUSION: The findings suggest that men with OSA have a high incidence of nocturia. A large multicenter study may be useful to explore the relationship between OSA and nocturia, in order to elucidate its causes.

Androgen Deprivation Therapy and the Risk of Stroke in Patients with Prostate Cancer: An Updated Systematic Review and Meta-Analysis.

BACKGROUND: The aim of this article was to review the association between androgen deprivation therapy (ADT) and the risk of stroke in patients with prostate cancer (PC). Thus, we performed this study to understand the impact of ADT on the incidence of stroke in PC patients. METHODS: A comprehensive literature search was performed in June 2019 based on PubMed, EMBASE, and Web of science databases. Pooled rate ratio (RR), hazard ratio (HR), and their 95% confidence intervals (95% CIs) were calculated with a DerSimonian and Laird random effects. RESULT: A total of 239,099 patients from 10 studies were included in this analysis. There was no significant association in pooled RR analysis. Pooled HR analysis showed that ADT treatment increased the risk of stroke (HR = 1.129, 95% CI: 1.019-1.251, p = 0.02). In a subgroup analysis of RR results, we found that different ADT treatments had no significant effect on increasing the risk of stroke. And in the subgroup analysis of HR results, only PC patients treated with gonadotropin-releasing hormone (GnRH) agonists, orchiectomy, or oral antiandrogen had significantly higher risk of stroke. In addition, we observed the result from another comparison that PC patients treated with GnRH agonists combined with oral antiandrogens might have a lower risk of stroke compared with using GnRH agonists alone. CONCLUSION: Our results showed that GnRH agonists, orchiectomy, or oral antiandrogen might play an important role in the incidence of stroke. We still need further studies to clarify the role of ADT in the increased risk of stroke.