We hypothesized that ECCO2R could be enhanced by ventilating the membrane lung with a sodium hydroxide (NaOH) solution with high CO2 absorbing capacity. A computed mathematical model was implemented to assess NaOH–CO2 interactions. Subsequently, we compared NaOH infusion, named “alkaline liquid ventilation”, to conventional oxygen sweeping flows. We built an extracorporeal circuit with two polypropylene membrane lungs, one to remove CO2 and the other to maintain a constant PCO2 (60 ± 2 mmHg). The circuit was primed with swine blood. Blood flow was 500 mL × min−1. After testing the safety and feasibility of increasing concentrations of aqueous NaOH (up to 100 mmol × L−1), the CO2 removal capacity of sweeping oxygen was compared to that of 100 mmol × L−1 NaOH. We performed six experiments to randomly test four sweep flows  for each fluid plus 10 L × min−1 oxygen. Alkaline liquid ventilation proved to be feasible and safe. No damages or hemolysis were detected. NaOH showed higher CO2 removal capacity compared to oxygen for flows up to 1 L × min−1. However, the highest CO2 extraction power exerted by NaOH was comparable to that of 10 L × min−1 oxygen. Further studies with dedicated devices are required to exploit potential clinical applications of alkaline liquid ventilation.Extracorporeal carbon dioxide removal (ECCO This allows independent modulation of minute ventilation and arterial partial pressure of CO2 (PaCO2), which are otherwise physiologically linked [2R has been proposed to facilitate ultra-protective ventilation [2 removed by the extracorporeal support is a crucial determinant of clinical efficacy [2R are still under evaluation due to safety concerns, mainly related to hemorrhagic and thrombotic adverse events [Extracorporeal carbon dioxide removal  to achieve minimally invasive approaches [2 comparable to the amount of CO2 produced by the body in one minute (2), the relatively low CO2 transfer efficiency of conventional MLs significantly reduces the efficacy of these strategies [Several ECCOproaches . Indeed,rategies .2 is the driving force that moves CO2 from blood to the sweeping gases. However, the use of high sweep gas flows, while maximizing the transmembrane gradient, does not increase CO2 clearance significantly. Indeed, during ECCO2R, most of the extracorporeal CO2 removal capacity is achieved for sweep gas flows below 2 L × min−1 since, at higher flows, the system rapidly loses efficiency [The transmembrane gradient of PCOficiency ,14,15,162R techniques are currently undergoing preclinical evaluations. The main aim is to overcome the present limitations to enhance CO2 removal [2 removal through acidification of the blood entering the ML [2 (HCO3−) in favor of dissolved CO2 (PCO2), thus increasing the efficiency of ECCO2R. Nevertheless, these approaches are still experimental, mainly due to safety and technical issues [Several ECCO removal ,19,20,21g the ML ,25,26,27l issues ,29.2 removal could be enhanced through the ventilation of the ML with a sweep fluid with an extremely high CO2 absorbing capacity (sodium hydroxide -NaOH- solutions), thereby preserving the transmembrane CO2 gradient.In the present study, we hypothesized that extracorporeal CO2 is added to dilute NaOH solutions, carbon dioxide first hydrates to carbonic acid (H2CO3), Equation (1), which will subsequently react with NaOH to form sodium bicarbonate (NaHCO3), Equation (2).Indeed, when a high amount of CO2 is added to highly concentrated NaOH solutions, sodium bicarbonate is formed directly, Equation (3), which subsequently forms sodium carbonate, Equation (4).Instead, when CO2 while keeping PCO2 almost down to zero although the elevated pH of the solution causes safety concerns.Consequently, highly concentrated NaOH solutions can absorb a conspicuous amount of CO2 transfer efficacy of membrane lung ventilation with a NaOH solution. This type of ventilation was named “alkaline liquid ventilation”.The aim of the present proof-of-principle study was to evaluate in-vitro the feasibility and the CO2 removal of alkaline liquid ventilation were compared to conventional sweep gas flow.Different concentrations of NaOH were tested and the efficacy and efficiency in CO2 removal.An in vitro setting  was builA closed-loop circuit was assembled with 3/8 and 1/4 inch polyvinylchloride class IV medical tubes , one 4 L reservoir , two polypropylene oxygenators membrane gas exchangers  and one peristaltic pump .® solution  was added to achieve a total volume of about 4 L. Sodium Heparin 25000 I.U. , anticoagulant-citrate-dextrose ACD 300 mL  and cefazolin 1 g  were added to the blood.The circuit was primed with about 3 L of swine blood collected at a local abattoir during usual slaughtering processes in compliance with CE regulations (1069/2009), authorization number 0141051/19 provided by ATS Milano, Regione Lombardia. MultiBic2 to maintain a constant PCO2 of 60 ± 2 mmHg at the inlet of the second oxygenator throughout all experiments. The second oxygenator was employed to remove CO2 through either ventilation with oxygen or a continuous infusion of sodium hydroxide (NaOH) solution, “alkaline liquid ventilation”, into the gas side of the membrane lung. Circuit accesses for blood sampling were positioned upstream (PRE) and downstream (POST) of the second oxygenator.The first gas exchanger downstream the reservoir was ventilated with a gas mixture of air and CONaOH pellets  were diluted in distilled water to achieve the required concentrations. NaOH solutions were stored in disposable parenteral bags  and infused into the gas inlet port in the gas exchanger using a peristaltic pump . NaOH exiting the oxygenator was discarded.The blood temperature was kept stable at 37 °C through heat exchangers connected to the membrane lungs.2 interactions to evaluate the theoretical basis of the study; (2) a safety and feasibility test to evaluate the effects of increasing NaOH concentrations on the membrane lung integrity and CO2 removal; (3) an efficiency test to compare the CO2 removal of similar sweep flows (up to 1 L × min−1) of oxygen vs. NaOH at the concentration selected following the feasibility test; (4) an efficacy test to compare the CO2 removal of the best liquid ventilation flow, selected from the efficiency test, vs. 10 L × min−1 of oxygen.The study was divided into four steps: (1) a mathematical modeling of NaOH and CO−1 of blood flow.All the in-vitro tests were performed with 500 mL × min2 absorption by aqueous NaOH were computed solving a system of equations , including standard mass-action, mass-conservation and electroneutrality laws of the involved species: water, NaOH, CO2  in which we introduced CO2 at different concentrations (from 0 to 100 by 5 mmol × L−1).We simulated a closed system with aqueous NaOH at varying concentrations  was calculated from pH and PCO2 modifying the Henderson-Hasselbalch equation−1 × mmHg−1 (solubility of CO2 in plasma) [Bicarbonate ion concentration ([HCO plasma)  and pK = plasma) ,32,33.−1) was calculated according to the method published by Douglas et al. [Plasma carbon dioxide content PRE and POST membrane lung [TCO22 (in mL × min−1), was calculated from the transmembrane lung TCO2 difference [2PRE represents CO2 content before the membrane lung while TCO2POST is the CO2 content after the membrane lung, blood flow is measured in L × min−1, and the conversion factor is in mL × mmol−1.Carbon dioxide transfer across the membrane lung, fference :(7)V˙CO22 removal of alkaline liquid ventilation at increasing concentrations of NaOH  and increasing ventilating flows  was likewise evaluated. Each combination of NaOH concentration and sweep fluid flow was tested once and for 15 min. At the end of each step, PRE and POST blood samples were collected for blood gas analysis (BGA) .Possible macroscopic detrimental effects on the membrane lung were evaluated. The effect on COIn addition, the integrity of the oxygenator was evaluated through visual inspection of the membrane lung, evaluation of the presence of blood in the NaOH solution exiting the oxygenator, and through analysis of blood sodium, potassium, and methemoglobin as indirect markers of hemolysis. The time-course of methemoglobin was evaluated at 4 time points  while testing aqueous NaOH at different sweep flows during the efficiency and efficacy tests.2 removal efficiency was estimated by computing PCO2 differences across the membrane lung and 2.The COAt the end of the feasibility test, we selected the highest NaOH concentration endured by the membrane lung to perform the subsequent efficiency and efficacy tests.2 equal to 1) and aqueous NaOH at 100 mmol × L−1 (the concentration selected from the feasibility test). Four sweep flows  for each fluid were randomly tested. We also randomized and tested 10 L/min of oxygen flow. Each combination of sweep fluid and flow was applied once during the single experiment.We performed six experiments with blood from 4 pigs. For each experiment, we tested, in random order, two different sweeping fluids, pure oxygen . Two-way repeated measures ANOVA or two-way repeated measures ANOVA on ranks was used, as appropriate, to test safety, feasibility , and efficiency.One-way repeated measures or Friedman repeated measures was used, as appropriate, to test safety and feasibility (POST–PRE differences) and to compare methemoglobin values at different time points.t-test or Wilcoxon signed rank test was used, as appropriate, to test efficacy. Post-hoc analyses were performed with Bonferroni or Tukey corrections. Statistical significance was defined as p < 0.05. Analysis was performed with SAS software 9.4  and SigmaPlot v.11.0 .Paired 2 of gas/oxygen or distilled water, in a closed system, at increasing concentrations of CO2 raises linearly, see 2 remains close to zero as long as the added CO2 is lower than the amount of added NaOH. When similar amounts of CO2 and NaOH are added, almost all CO2 reacts forming HCO3− and the solution pH is around 8.220–8.230.The PCO2 is lower than NaOH, carbonic acid dissociates to HCO3− which, due to the alkaline milieu, further dissociates to CO32−, thus reducing the concentration of HCO3−. When CO2 is near half or lower than NaOH, almost all CO2 forms CO32− and the solution pH is above 11. Instead, if the TCO2 is higher than NaOH, all hydroxide reacts with CO2 forming HCO3− and the pH decreases below 8.Otherwise, if the added CO2 is higher than twice the NaOH, the PCO2 in the NaOH solution will be higher than the one in a similar gas volume containing the same amount of CO2.Interestingly when the added CO2, the theoretical 2 of an adult, would have a PCO2 of 143 mmHg (713 mmHg × 0.2), while 1 L of water would have a higher PCO2 of 255 mmHg. On the contrary, the same amount of CO2 could be stored in 1 L of NaOH 10 mmol × min−1 solution with a PCO2 close to zero.For example, one liter of gas containing 200 mL (7.86 mmol) of CONo detectable damages to the membrane lung were observed. Moreover, no blood was found in the sweep fluid exiting the oxygenator.2PRE was stable throughout the entire test, 59.0 (58.0–60.0) mmHg. Delta PCO2 across the membrane lung was significantly lower at 10 mmol × L−1 (−32.2 (−38.6–−23.1) mmHg). Otherwise, it showed small increases as NaOH concentration increases (−41.4 (−43.1–−36.8), −47.7 (−49.5–−44), −47.8 (−48.6–−47) and −48.2 (−48.4–−46.6) mmHg at 30, 60, 90, and 100 mmol × L−1 respectively). PCO2POST was reduced to about 12 mmHg with NaOH concentration ≥ 60 mmol × L−1 (12.0 (11.0–15.0), 11.4 (10.2–12.4) and 12.4 (11.3–13.1) mmHg at 60, 90, and 100 mmol × L−1 respectively), subsequently pHPOST increased up to 7.913 (7.885–7.943) at NaOH concentration equal to 100 mmol × L−1. The lowest 2 was also recorded at the lowest NaOH concentration 73.9 (54.3–91.8) mL × min−1. PRE blood sodium and potassium concentration were stable (see p = 0.606).4 −43.1–−.8, −47.72 was observed when 100 mmol × L−1 NaOH was used, this concentration was employed for the efficiency and efficacy tests.As the highest delta PCO2PRE was stable throughout the entire test, 59.6 (58.9–60.4) mmHg. Increasing oxygen flows showed increasing CO2 removal, both as delta PCO2 across the membrane lung and 2, see 2 removal, except for a lower 2 at 1000 mL × min−1 compared to 100 and 250 mL × min−1  mmHg both during NaOH and oxygen steps while PO2POST increased up to 470.5 (452.3–507.0) mmHg only during oxygenation use, while it remained unchanged during liquid ventilation.POBlood electrolytes and lactate concentrations were stable throughout the experiment.2 and delta PCO2, NaOH 250 mL × min−1 was selected as the most performant NaOH flow and compared with 10 L × min−1 of oxygen in the efficacy test. 2 and 2 were similar, suggesting similar extracorporeal CO2 removal (In agreement with the highest −1) of highly concentrated sodium hydroxide solutions into the gas side of conventional polypropylene oxygenators is feasible, despite pH values of the sweeping solution above 12. At low sweep flows, alkaline liquid ventilation showed significantly higher CO2 removal capacity than conventional gaseous ventilation. However, the maximum CO2 removal efficiency achieved through liquid ventilation was not superior to the one achieved with 10 L × min−1 of sweep gas flow.This in-vitro study shows that continuous infusion (from 100 to 1000 mL × min2 absorbing capacity of NaOH solutions. Indeed, in our experimental context, the concentration of NaOH was always significantly higher than the amount of CO2 extracted from the ML. The PCO2 of the alkaline sweep fluid was persistently very close to 0 mmHg, as the added CO2 was instantly hydrated and dissociated to bicarbonate and carbonate. This allowed to keep the PCO2 close to zero and thus optimize the transmembrane PCO2 gradient, favoring the efficiency of extracorporeal CO2 removal.The working hypothesis underlying this study was to exploit the high CO2 while maintaining PCO2 close to zero. On the contrary, the same amount of CO2 added to 10 L of gas would result in a PCO2 around 15 mmHg, therefore reducing the blood-gas CO2 gradient.Indeed, a solution containing 10 mEq of NaOH could absorb 200 mL of CO2 removal. Instead, for NaOH flows greater than 250 mL × min− there was an unexpected reduction in CO2 removal. This reduced efficiency could depend on the density of the sodium hydroxide solution and the mechanics of the membrane lung. Therefore, a clinical application of alkaline liquid ventilation does not seem exploitable using the current technology. The technical complexity and safety profile require further evaluations, although the present tests have recorded no damage to the membrane lung.However, the data showed that increasing NaOH flow did not lead to a linear increase in COAnother important difference between gaseous and liquid ventilation needs to be discussed. Although the oxygenation capacity of low-flow devices using conventional gaseous ventilation is limited by the amount of blood reaching the ML, a certain amount of oxygen is added to the blood. On the contrary, the NaOH infusion does not oxygenate the extracorporeal blood, limiting its potential clinical application to patients with isolated hypercapnic respiratory failure, i.e., able to oxygenate properly through their native lungs.2 extraction capacity resulted more effective [2R to achieve physiological targets. Nevertheless, the clinical application of ECCO2R is still limited and no conclusive indications have been identified mainly because of safety concerns [2R technique to ensure a clinical efficacy with limited extracorporeal blood flows, potentially enabling regional anticoagulation [Although devices with higher COffective ,9,10, nuconcerns ,38. Indegulation ,40. The 2 extraction capacity was estimated both as differences in PCO2 and TCO2 between the blood samples upstream and downstream of the artificial lung [2 showed higher variability than PCO2, as shown in 2 [This study presents several limitations. First, we could not perform any gas analysis of the sodium hydroxide solution. The COial lung . V˙CO2 sin V˙CO2 . Secondl2R through alkaline liquid ventilation of the ML is feasible and safe. The CO2 removal efficiency of alkaline liquid ventilation was higher than conventional gaseous ML ventilation only for low sweep flows. Indeed, at high sweep gas flow, the CO2 removal efficiency was comparable between the two techniques.This in-vitro study showed that ECCOThe development of a dedicated device may be necessary to exploit the potential of this technology. Further studies will be required before any possible clinical application.


HER2+ breast cancer (BC) is an aggressive subtype representing a genetically and biologically heterogeneous group of tumors resulting in variable prognosis and treatment response to HER2-targeted therapies according to estrogen (ER) and progesterone receptor (PR) expression. The relationship with androgen receptors (AR), a member of the steroid hormone’s family, is unwell known in BC. The present study aims to evaluate the prognostic impact of AR expression in HER2+ BC subtypes. A total of 695 BCs were selected and reviewed, AR, ER, PR and HER2 expression in tumor cells were examined by immunohistochemical method, and the SISH method was used in case of HER2 with equivocal immunohistochemical score (2+). A high prevalence of AR expression (91.5%) in BC HER+ was observed, with minimal differences between luminal and non-luminal tumor. According to steroid receptor expression, tumors were classified in four subgroups, including BC luminal and non-luminal HER2+ expressing or not AR. The luminal BC HER2 + AR+ was associated with lower histological grade, lower tumor size, higher PR expression and lower HER2 intensity of expression (2+). Also, the non-luminal tumors AR+ showed lower tumor size and lower prognostic stage but frequently higher grade and higher HER2 intensity of expression (3+). These findings should suggest a different progression of luminal and non-luminal tumors, both expressing AR, and allow us to speculate that the molecular mechanisms of AR, involved in the biology of BC HER2 + AR+, differ in relation to ER and PR expression. Moreover, AR expression may be a useful predictor of prognosis for overall survival (OS) in HER2+ BC subtypes. Our findings suggest that AR expression evaluation in clinical practice could be utilized in clinical oncology to establish different aggressiveness in BC HER2+ subtypes. Breast cancer (BC) is a heterogeneous disease enclosing several entities with different morphologic, prognostic and therapeutic features . InvasivHER2+ BCs include different histological subtypes: invasive BC no special type (IBC-NST), lobular, micropapillary, apocrine, rarely mucinous . ImmunopHER2 protein overexpression or gene amplification, which accounts for ~15–18% of all BC, is frequently associated with high invasiveness and worse prognosis without appropriate therapy . HER2 siThe androgen receptor is a member of the steroid nuclear receptor superfamily, and it is widely expressed in different subtypes of breast cancer, including HER2+ tumors. It could be a promising prognostic and predictive factor and therapeutic target in BC ,17,18,19The aim of the present study was to evaluate AR expression in a large series of HER2+ BC, to establish its prognostic role and clinical-pathological relationship.A retrospective cohort of 695 BC patients, diagnosed between 2012 and 2021, was recruited through a complete review of surgical samples and medical records from the archives of the Department of Histopathology of Oncologic Hospital in Cagliari, Italy. The inclusion criteria were histologically invasive BC, HER2 positive expression determined by immunohistochemistry (IHC) and/or and in situ hybridization (SISH) assay, availability of formalin-fixed, paraffin embedded (FFPE) tumor blocks and clinico-pathological data. The exclusion criteria were in situ BC and HER2 negative status. Three µm-thick tissue sections of FFPE specimens were cut for hematoxylin and eosin staining (H&E), immunohistochemistry, and SISH analysis.The study protocol was approved by the local research ethics committee (File number PG/2021/14264); and followed the Italian law on guidelines for the implementation of retrospective observational studies . Only coded data were collected in order to protect patient confidentiality.The immunohistochemistry analysis was performed using specific antibodies against ER, Clone SP1 ; PR Clone 1E2 ; Ki67, Clone 30-9 ; AR Clone SP107 ; HER2 PATHWAY Clone 4B5 . Immunostaining was performed using the Ventana Benchmark XT staining system with Optiview DAB detection kit. In cases of HER2 with equivocal immunohistochemical score (2+), we performed HER2 gene amplification by ultra-View SISH Detection Kit . Evaluation of immunostaining and SISH for HER2 was based on American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) recommendations . Ki67 waDemographic and clinico-pathological information were extracted from medical records, as well as age at diagnosis, histologic type, histologic grade, tumor site, and TNM classification. Histologic tumor type was established according to the UICC-WHO criteria . Histolop < 0.05. Statistical analysis was carried out using STATA®16 .Quantitative variables were described by median and interquartile range (IQR) according to non–normal distribution of variables, whereas absolute and relative (percentages) frequencies were used for qualitative variables. Statistical differences for qualitative variables were evaluated using Chi2 or Fisher’s exact tests, when appropriate. Logistic regression analysis was performed to evaluate the association between mortality, IHC BC subtypes, clinico-pathological features, and molecular variables. A Kaplan–Meier curve and Log-Rank test were performed to describe OS according to IHC BC subtypes, clinico-pathological and molecular variables. The statistical significance was set up at A total of 695 HER2+ BC patients were selected. Tumors were grouped into four subtypes based on hormone receptors : Luminal B (LB) HER2 + AR+ ; Luminal B HER2 + AR− ; HER2 + AR+ ; HER2 + AR− . The median (range) age at diagnosis was 58 (27–91) years, with 363 (52.2%) equal to or older than 58 years. 590 (85.0%) tumors were ductal, 30 (4.3%) lobular, and 39 (5.6%) apocrine; less common were other histologic types, such as mixed , micropapillary/papillary (2.2%), and mucinous (1.0%). Pathological tumor staging was pT1 in 203 (32.6%) cases, pT2 in 288 cases (46.2%); pT3 in 27 cases (4.3%); and pT4 in 52 cases (8.4%). Pathological lymph node status was distributed as follows: pN0 in 321 (55.9%); pN1 in 134 (23.3%); pN2 in 73 (12.7%); pN3 in 46 (8.0%). A total of 54 (7.8%) were metastatic; 83.7% of HER2-positive BC were G3. Ninety nine out of 695 patients (14.2%) died .AR positivity was detected in 91.5% of all tumors, specifically in 95.2% of luminal and in 84.2% of non-luminal subtypes.p: 0.045) and showed the following features: histological grade 2 was found in 17.3% of AR+ tumors vs. 5.2% of AR− tumors (p: 0.020), pT1 in 33.7% of AR+ tumors vs. 20.8% of AR− ones (p: 0.020), prognostic stage I in 42.8% AR+ BC vs. 16.3% of those with AR negativity (p: 0.001). Moreover, HER2+ AR+ BCs were more frequently associated with ER and PR expression ≥ 10% . There were no significant differences between site, histological type, lymph node status and ratio, metastasis, ki67 and HER2 expression . HER2+ subtypes without AR expression showed the highest pT3 and pT4 rates, accounting for 13.5% and 10.8% (HER2+ AR−), and 12.5% and 12.5% (LB HER2+ AR−), respectively.Prognostic stage I was found more frequently in LB HER2+ AR+ (55.5%), and less frequently in HER2+ AR− (6.3%). On the other hand, stage III was mainly found in HER2+, both AR− and AR+ (28.1% vs. 28.8). HER2+ AR− and HER2+ AR+ subtypes showed a significantly higher frequency of HER2 expression with score 3+ .p: 0.010); age at diagnosis (p < 0.0001); histological type (p: 0.006); histological grade (p: 0.003); tumor size (p < 0.0001); lymph node status and ratio (p < 0.0001 for both); prognostic stage (p < 0.0001); metastasis (p < 0.0001); Ki-67 expression (p: 0.002); ER, PR, and AR expression    . Multivactively) .p: 0.004; p < 0.0001; p < 0.0001; p: 0.040; The Kaplan-Meier curve for OS showed no differences among BC HER2+ subtypes A. HoweveBC HER2+ is an aggressive subtype including heterogeneous tumors with variable prognosis and treatment response to HER2-targeted therapies. On this matter, Staaf et al. identified three genetic BC HER2+ subtypes showing distinct clinical outcomes according to molecular profiling analysis . The samAR is a steroid hormone receptor frequently expressed in BC, including the ER-negative subtypes for which it could represent a complementary target for therapy, though the clinical significance and functional role of AR has not been outlined in BC yet ,41. The AR expression is higher than ER and PR in BC ,43. We fFurthermore, our results showed that AR+ tumors are associated with older age at diagnosis and favorable clinical and pathological features, as well as lower histologic grade (G2), pT1 and prognostic stage (I), suggesting a prognostic value of AR in BC HER2+ subtypes. These findings are supported by previous studies showing the association of AR expression with lower grade, smaller size, more frequently tubule formation, and less pleomorphism and mitotic counts, although they evaluated ER-negative and ER-positive subgroups ,47. BesiMoreover, the luminal BC HER2+ AR+ was associated with lower histological grade (G2) and lower tumor size, higher PR expression, and a lower HER2 intensity of expression (2+). Also, non-luminal tumors AR+ showed smaller size and prognostic stage (I) but higher grade (G3) and higher HER2 intensity of expression (3+). These results agree with the observations of other studies that have detected HER2 overexpression in G3-AR+ carcinomas ,29,49. TTraditionally, the BC HER2+ subtype is distinguished in luminal HER2+ and non-luminal HER2+. In our study, AR positivity was detected in 91.5% of all tumors analyzed. Moreover, AR is expressed in 84.2% of BC HER2+, in the absence of other hormonal receptors, namely ER and PR. From a clinical point of view, tumors with AR expression show a slightly better clinical outcome. These findings suggest that the immunophenotype classification is not completely exhaustive; like TNBC , the preThe role of androgens and AR might vary depending on cancer cell types and/or on the level of expression of other steroid hormone receptors. In ER-positive tumors, AR has an anti-proliferative effect by antagonizing ER, by binding to a subset of estrogen response elements (EREs); it can prevent the activation of target genes which mediate the stimulatory effects of 17-beta-estradiol on breast cancer cells ,52. ReceHowever, androgens may have a proliferative effect through AR in ER- AR+ tumor cells. In HER2+ tumors, AR triggers the WNT/β-catenin pathway causing HER3 upregulation; through HER2/HER3 heterodimers, it can cause the activation of the PI3K/AKT pathway and may lead to cell proliferation through MYC ,54. FurtThe prognostic role of AR in BC ER-negative and PR-negative has been poorly evaluated, in comparison with BC ER-positive PR-positive variants . Our stuAR is frequently expressed in BC HER2+ subtypes. Our results showed that AR expression has a prognostic value in BC HER2+ subtypes, with better clinical outcomes. A better prognosis was highlighted in luminal HER2+ AR+ tumors subtype compared to the non-luminal HER2+ AR+ subtype, based on clinico-pathological data. AR expression should be assessed to evaluate the prognosis of BC HER2+ subtypes. Moreover, the understanding of the complex interactions between AR and the HER2 signaling pathway could pave the way to the use of AR as a therapeutic target in BC HER2+ subtypes.


Prostate cancer is the second most common cancer in males. In prostate cancer cells, androgens bind and activate the intracellular mediator called Androgen Receptor that control cell proliferation and survival. Hormone deprivation therapy is administrated to reduce androgen levels and consequently tumour growth. Unfortunately, most patients develop resistance to hormone treatment over the years and novel hormonal agents, such as Abiraterone or Enzalutamide, are administered. However, many patients do not initially respond or become resistant to these drugs quickly. Firstly, we demonstrated that in hormonal sensitive human prostate cancer cells the combination therapy of Abiraterone plus Enzalutamide reduced cell growth and survival. Moreover, starting from these prostate cancer cell lines, we generated cellular models of resistance to hormonal deprivation alone or in combination with the novel hormonal agents. In all the cases, resistant cell lines restore Androgen Receptor expression, Androgen Receptor functionality, cell proliferation and migration in the absence of androgens. Importantly, these novel cellular models acquire cross-resistance to each other. These results are consistent with clinical trials in castration resistant prostate cancer patients and suggest the biological rationale to test the combination therapy of Abiraterone plus Enzalutamide as first-line treatment in hormone-sensitive prostate cancer patients before becoming hormonal resistant.AR-V7 and AR-V9) has been associated to CRPC. However, resistance mechanisms to novel NHAs are not yet well understood. Androgen-dependent PCa cell lines were used to generate resistant models to ADT only or in combination with Abiraterone and/or Enzalutamide (concomitant models). Functional and genetic analyses were performed for each resistance model by real-time cell monitoring assays, flow cytometry and RT-qPCR. In androgen-dependent PCa cells, the administration of Abiraterone and/or Enzalutamide as first-line treatment involved a critical inhibition of AR activity associated with a significant cell growth inhibition. Genetic analyses on ADT-resistant PCa cell lines showed that the CRPC phenotype was accompanied by overexpression of AR full-length and AR target genes, but not necessarily AR-V7 and/or AR-V9 isoforms. These ADT resistant cell lines showed higher proliferation rates, migration and invasion abilities. Importantly, ADT resistance induced cross-resistance to Abiraterone and/or Enzalutamide. Similarly, concomitant models possessed an elevated expression of AR full-length and proliferation rates and acquired cross-resistance to its alternative NHA as second-line treatment.Androgen deprivation therapy (ADT) and novel hormonal agents (NHAs)  are the goal standard for metastatic prostate cancer (PCa) treatment. Although ADT is initially effective, a subsequent castration resistance status (CRPC) is commonly developed. The expression of androgen receptor (AR) alternative splicing isoforms ( Androgen deprivation therapy (ADT) is still the main treatment option for advanced PCa although most patients will eventually develop castration-resistant prostate cancer (CRPC) [Prostate cancer (PCa) is the most common cancer in non-smoking men worldwide and the third cause of cancer-related death after lung and colorectal cancers (r (CRPC) ,2.CRPC patients are frequently treated with novel hormonal agents (NHAs), such as Abiraterone Acetate (AA) and Enzalutamide (Enz) ,4. AA blAR overexpression, AR amplification, AR mutations, loss of AR expression by hypermethylation of the AR promoter or expression of AR splice variants (AR-Vs) [AR-Vs are originated by alternative splicing of cryptic exons located on intron 3 in the AR locus, and the resulting protein isoforms conserve the N-terminal activation domain but lose the C-terminal LBD acting as an androgen-independent transcription factor. AR variant 7 (AR-V7) is the most commonly studied variant in PCa, and its detection in circulating tumour cells (CTCs) has been described as a prognostic marker for AA and Enz resistance [AR-V9 was shown to share a common 3′ terminal cryptic exon with AR-V7 and was recently described to be co-expressed in AA-resistant PCa metastatic patients [Several molecular mechanisms associated to CRPC and AR have been described: increased testosterone synthesis in the adrenal glands or prostatic tissue,  (AR-Vs) ,14,15,16sistance . Recentlsistance . In addipatients .AR expression levels, AR transcriptional activity, functionality (cell migration and invasion) and the cross-resistance among the different NHA therapies in all new CRPC models.The main aims of this work were to generate and to characterize novel CRPC cellular models from androgen sensitive PCa cell lines: (a) ADT-resistant PCa cell lines (R-ADT) selected in the absence of androgens; (b) Concomitant ADT-NHA-resistant PCa cell lines  obtained through the continuous growth in the presence of NHAs and the absence of androgens. We evaluated the proliferation rates and cell cycle, MEM Non-Essential Amino Acids Solution (Gibco), 1% glutaMAX (Gibco) and 1% Penicillin-Streptomycin Solution 100X (BioWest) in a humid atmosphere incubator with 5% CO2. The cell lines were mycoplasma-free and periodically checked for Mycoplasma by the Cell Culture Unit at GENyO.Three different human PCa cell lines were used: LNCaP (androgen-sensitive adenocarcinoma cells derived from supraclavicular lymph node metastasis) and 22RV1 , both purchased from the American Type Culture Collection , and PC-3 (androgen-independent cell line originated from a bone metastasis of prostatic adenocarcinoma), that was kindly provided by Dr Ignacio Gil Bazo  as CRPC model. All cell lines were authenticated using STR at the Laboratory of Genetic Identification  at the University of Granada. The three cell lines were maintained with an RPMI 1640 medium (BioWest) containing 10% Fetal Bovine Serum (FBS), 1% LNCaP and 22RV1 ADT-resistant cell lines (R-ADT) were generated after exposing the parental sensitive cells to a hormone-reduced medium ) for 6 months . Once thThe tumour cells lines resistant to ADT/NHAs were obtained by the continuous exposure of R-ADT cells to increasing AA and/or Enz concentrations. Growth mediums containing fresh NHAs were changed every 5 days in order to maintain a consistent drug concentration during the selection process. To avoid the initial lethality of both treatments, cells were grown in a hormone-reduced medium with increasing treatment concentrations at different time points. Treatment resistance was acquired after 6 months . The finThe use of AA or Enz as second-line therapy was done after concomitant treatment . Regarding R-ADT/AA, cells were treated with 40 μM Enz, while for R-ADT/E cells were exposed to 20 μM AA  . Cells were monitored on the RTCA system for 5 days, and impedance was recorded as a measurement of Cell Index (CI). At least four experimental replicates for each experimental condition were performed as recommended by the manufacturer.0/G1 and G2/M phases, respectively, using the BDFACSuite™, ModFit LT™ and GraphPad Prism™ software.To study the effect of each treatment in the cell cycle, sensitive and resistant cell lines were dissociated after 5-day cultures, washed with PBS and fixed on ice-cold 70% ethanol. The cells were incubated overnight at –20° C and then incubated with propidium iodide buffer (propidium iodide 50 µg/mL and RNase 100 µg/mL in PBS). The cell cycle distribution was analysed on a BD FACSVerse™. Fluorescent intensity, indicating that N and 2N ploidy were represented as indicators of G5 cells/mL. 200 μL of the cell suspension were seeded on top of a 24-well Transwell with a pore size of 8 μm . The lower chamber was filled with 700 μL of RPMI medium supplemented with 10% FBS. The cells were kept under these conditions for 48 h. Then, non-traversed cells from the upper compartment of the transwell were removed using wet swabs. Traversed cells from the lower side of the transwell were fixed in methanol and stained with 0.5% crystal violet solution. For cell invasion assays, the same method was performed, with the exception that a layer of MatrigelTM Matrix , simulating an extracellular matrix, was added in the upper chamber. To calculate migration/invasion rates, the total number of cells per insert was determined calculating the number of cells by the area of the microscope-viewing field. An average from 5 random fields at 10× magnification using a microscope  was used to estimate the cell number per field. Then, the total number of traversed cells was recalculated for the entire area of the transwell insert. The results were expressed as the number of traversed cells for every 1 × 105 cells seeded from three independent experimental replicas.The cells were resuspended in RPMI + 10% CSS medium at a density of 2 × 10AR full-length and AR-V7. qPCR conditions were 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Standard curves were used to assess the primer efficiency and average change in threshold cycle (ΔCT) values determined for each sample relative to endogenous GAPDH levels and compared to control cultures for fold change calculations 2(−ΔΔCt). The experiments were performed in triplicate to determine the mean standard error, and the student’s t-tests were performed with normalization to control for p-values.The total RNA was isolated using TRI Reagent (Life Technologies), and the quality tested in an Agilent Bioanalyzer 2010 (Agilent Technologies). Reverse transcription was performed with 0.5 μg of the total RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche Life Science). The resulting cDNA was used for qPCR using iTaq Universal SYBR Green Supermix in a HT7900 Fast Real-Time PCR System (Applied Biosystems) using custom primers . PrimersAR splice variants, conventional PCR under the same qPCR conditions was performed, and the PCR products were examined by standard TA subcloning in a pCR2.1 vector (Thermo Fisher Scientific) and Sanger sequenced using the M13 FW primer in an ABI Prism 3130 genetic analyser (Applied Biosystems) (To confirm the amplification of the desired systems) .AR was indirectly measured by the qPCR of a selected panel of AR-regulated genes . The qPCR conditions and procedures were as described above, and the designed primers are shown in The transcriptional activity of t-test. Values were considered statistically significant at p < 0.05.All data are expressed as mean ± SD. Statistical comparisons were performed with a paired student’s 0/G1 for both LNCaP and 22RV1 was observed  during 5 days on the three PCa cellular models was analysed. Hormonal-deprivation-produced growth rates decreased in both androgen sensitive cell lines , while the PC-3 cell line showed no differences over control cells at the proliferative level A. Regardobserved . MoreoveaP cells B, indicaaP cells .AR total and AR full-length was observed, while the expression of most AR target genes was diminished   were not detected.The genetic analyses showed that, in the case of LNCaP, an increase in the mRNA levels of minished C. For thelevated C. In addA levels C. Finall in FBS) C. Furthep < 0.05) and 22RV1 cells . In addition, the combination of Enz with AA (E + AA) had a synergistic effect inhibiting cell growth at a higher rate compared to AA or Enz alone in LNCaP  and in 22RV1 . Again, LNCaP cells are more sensitive to NHA therapy than 22RV1 cells. In PC-3 cells, no differences were observed at the proliferative level, demonstrating that this cell line is also resistant to NHA therapies , Enz (40 μM) or the combination of both for 5 days. When cell proliferation was analysed, we observed that AA was significantly more efficient reducing proliferation rates than Enz in both LNCaP , while the combination produced a dramatic cell death increase  significantly increased the expression of AR-V7 and AR-V9 isoforms and, although to a lesser extent, of AR full-length and AR total , while 22RV1 R-ADT reached a proliferative rate identical to that of their respective wild-type counterparts   for 6 months. The generated cell lines, denominated LNCaP R-ADT and 22RV1 R-ADT, were able to grow efficiently in the absence of androgens. LNCaP R-ADT showed a significantly higher proliferation rate than wild-type LNCaP  A. Regard%, n.s.) B. ImportAR total and AR full length at the mRNA level, while the AR-V7 and AR-V9 isoforms were only slightly increased   increased considerably in 22RV1 R-ADT cells  C. In conDT cells C. Again,DT cells C.Next, we wondered whether the acquisition of resistance to ADT conditioned the response to second-generation NHA therapies in PCa cells. For this purpose, AA and Enz were used as second-line treatment after ADT resistance acquisition A. In LNCConcerning 22RV1 R-ADT, the AA treatment involved a decrease of growth rate to 44.2%, while for the Enz treatment the growth rate remained at 88.5% with respect to control 22RV1 R-ADT B. When tp < 0.05) , while most of the remaining AR target genes maintained levels similar to those of wild-type cells   A. Regard PMEPA1) B.p < 0.05)  ( < 0.05) A. qPCR aR-ADT/AA B. Interep < 0.05), while 22RV1 R-ADT/AA cells proliferated significantly less than their control 22RV1 wild-type counterpart (73.1% vs. 100%) (p < 0.05). Additionally, the effect of the concomitant treatment with double treatment (R-ADT/E + A) showed a significant reduction of the proliferation rate compared with the wild-type cell line   A (p < 0. ≤ 0.05) A, suggesAR full-length and AR-V7 expressions. However, an additional increase of AR-V9 was observed in 22RV1 R-ADT/AA and 22RV1 R-ADT/E + A, while AR-V9 expression was not detected in 22RV1 R-ADT/Enz cells , while it was also observed, to a lesser extent, in both concomitant cellular models: LNCaP R-ADT/E and LNCaP R-ADT/AA cells (p < 0.01). In the case of invasion assays, a large increase in invasive capacity was only observed in the LNCaP R-ADT cell line (p < 0.01)  and the concomitant exposure to ADT and NHAs affected PCa aggressiveness. Importantly, all three LNCaP cell lines showed a significant increased migration capacity in comparison with the control cell line (LNCaP wild-type) A. This i < 0.01) B. In con < 0.01) B.p < 0.01) (p < 0.01) or in combination with AA (22RV1 R-ADT/E + A) (p < 0.05). As previously shown in LNCaP cellular models, only ADT-resistant cells (22RV1 R-ADT) possessed potentiated invasive capabilities (p < 0.01)  A. All th < 0.01) B, while Once resistance to concomitant treatment schedules has been achieved, we evaluated the sensitivity of each cell line to the alternative NHA. The proliferation rate in LNCaP R-ADT/E cells treated with AA under ADT conditions was even slightly higher than in the presence of ADT and Enz (117.4% vs. 100%) , we observed an increase in AR total but not in AR full length or the AR splicing variants AR-V7 or AR-V9, suggesting that other non-studied alternative AR isoforms could be up-regulated. Importantly, those alternative isoforms are not able to increase the gene expression of the evaluated AR target genes  did not modify the p < 0.05) , the sequential use of NHAs, AA or Enz, respectively, as a second-line treatment promoted a severe down-regulation of all et genes D.In summary, we developed functional and genetic analyses on hormone-sensitive and resistant tumour cell lines, demonstrating that the previous treatment with ADT, and the subsequent resistance acquisition, decreases AA and Enz efficiency. In addition, we also showed that an increased AR transcriptional activity is associated to AA and Enz resistance in the novel PCa cellular models generated in this study .The idea that androgenic signalling is essential for the growth and maintenance of prostate homeostasis is widely accepted. For decades, ADT has been the most important treatment for men with prostate cancer, especially for those with metastatic disease ,2. This AR mRNA and AR target genes in CRPC cell lines and primary samples [0/G1 phase arrest and the accumulation of death cells (Sub-G0 peak). Moreover, 22RV1 cell lines had a 35kb tandem duplication containing exon 3 from an AR gene [There are many evidences that ADT increased  samples ,22,23,24 samples . Similar samples ,27. Howe AR gene . Althoug AR gene , we obseHowever, the existence of androgen-independent PCa cells such as PC-3 is an example of the fact that the insensitivity to hormonal therapies is a reflection of the variability and complexity of the therapeutic response of prostate cancer patients, depending on the initial characteristics of the tumour cells . While tImportantly, NHA-mediated cell growth inhibition in hormone-sensitive PCa cell lines was reversed with the acquisition of ADT resistance after 6 months of selective pressure. In fact, we observed that growth rates in the initial androgen-dependent tumour cell lines LNCaP and 22RV1 not only increased but also promoted a more aggressive behaviour, amplifying growth rates even more than the androgen-resistant cell line PC-3.0/G1 that was not accompanied by cell death.The implication of AR signalling in the development of ADT resistance in PCa is complex, involving different types of genomic alterations and leading to a host of selective/adaptive responses. For example, ADT induces the reduction of DHT levels, which is the main mediator of AR signalling in prostatic tissue. Consequently, most of the prostate cells undergo apoptosis, but some of them can escape by entering a dormant status . These tAR have been strongly associated with ADT resistance as well as with AA and Enz resistance [AR-V9 mRNA. Our experimental design allowed us to detect and differentiate with certainty AR-V7 and AR-V9 isoforms. Cloning and sequencing of the two independent amplicons confirmed the effectiveness of our approach and guaranteed the qPCR expression results. We propose that other independent laboratories validate this new strategy in order to standardize AR-Vs detection methodologies and to clarify the current controversy.Progression in the CRPC patients involves modifications of genetic pathways directly or indirectly associated to the AR signalling. These modifications include amplification, overexpression, epigenetic changes or mutations of the AR gene. Splicing variants of sistance . There isistance ,3,4,5,17sistance ,12,13,14sistance , the cryAR full-length but not necessarily by the overexpression of the splice variants, AR-V7 and AR-V9, suggesting that these splice variants might not be essential for the acquisition of ADT resistance. In this context, it was suggested that the growth of tumour cells with high AR-Vs expression did not require the presence of AR full-length to induce proliferation of genes associated to AR-Vs [AR full-length was associated to an increase of AR-Vs. Moreover, AR-V7 and AR-V9 isoforms do not always maintain the same pattern of transcriptional regulation with each other. For example, in PC-3 wild-type cells treated for 5 days with Enz, AR-V7 was totally repressed, while AR-V9 was slightly induced; on the contrary in 22RV1 R-ADT/E cells both AR-Vs followed the opposite regulation pattern. However, all our CRPC cellular models showed AR activation, independently of the AR-V status, in contrast to Cato L et al.’s results in preclinical models, that conclude that AR-V7 heterodimerises with AR full-length and is necessary for CRPC [In our results, we observed how the tumour cells lines, LNCaP and 22RV1, initially hormone-sensitive, became ADT-resistant after a 6-month treatment. This resistance was accompanied by the overexpression of to AR-Vs . In factfor CRPC ,43. ThusAR full-length and the acquisition of castration resistance was previously evaluated by Shiota M et al. [AR full-length and the Epithelial to Mesenchymal Transition (EMT) process as a new mechanism of castration resistance. Our results demonstrated that the acquisition of ADT resistance increases the ability to migrate, a property acquired during EMT. This characteristic was more evident in LNCaP than in 22RV1 CRPC models. These results coincide with recent results published by Miao L et al. in 2017, who demonstrated that the induction of EMT was an adaptive response to Enz with implications for therapy resistance [The relationship between M et al. . They idsistance .Therefore, the question we need to answer is: What is the best treatment combination according to the resistance mechanisms induced by previous treatments ? This quRegarding the potential concomitant use of ADT plus AA or Enz as first-line treatment, the findings from CHAARTED, LATITUDE, and ARCHES trials have demonstrated the improvement of OS and PFS in mCRPC . Our expAR full-length and TMPRS2 were the common denominator in the emergence of resistances with any treatment, occurring in both LNCaP and 22RV1 CRPR cellular models. The emergence of resistance was accompanied by constitutive AR activation, detected as an up-regulation of several AR target genes. However, the induction of AR-V7 and AR-V9 variants was not necessarily accompanied by greater AR activation. In addition, AR-V9 up-regulation is always associated to AR-V7 increase. On the contrary, AR-V7 induction was not always correlated with higher AR-V9 expression levels, as Kohli’s study suggested [AR-V9 was associated with resistance to AA in 22RV1 CRPR cellular models, but this correlation was not observed in LNCaP ones. In fact, we observed that resistance to ADT in combination with AA (R-ADT/A) did not involve the up-regulation of AR-V9. Importantly, AA was more effective in LNCaP than in 22RV1 cellular models, while Enz was more efficient in 22RV1 cells. In this case, AR-V9 up-regulation was observed. These results suggest the need to detect the expression levels of both AR-V7 and AR-V9 in PCa patients in order to choose the best therapeutic option (AA or Enz).In order to characterize the molecular pathways involved in concomitant cellular models, we evaluated AR expression and AR target genes. We observed that only uggested . InteresAR-V7 but also AR-V9 expression to correctly select the most effective anti-androgen to be administrated.In conclusion, our work identified that the treatment with AA or Enz could be more effective if used as first-line therapy in androgen-sensitive PCa, since ADT induces additional resistant mechanisms that reduce the efficiency of these drugs as a second-line treatment. Our CRPC cellular models recapitulate the acquisition of cross-resistance between NHAs observed in mCRPC patients. In addition, we suggest the need to identify not only


We report the possible therapeutic efficacy of immersive virtual reality (VR) rehabilitation for the treatment of ideomotor apraxia in a patient with stroke.A 56-year-old man with sudden weakness of his left side caused by right frontal, parietal, and corpus callosal infarction was transferred to rehabilitation medicine center for intensive rehabilitation. Although his left-sided weakness had almost subsided 10 days after the onset of symptoms, he presented difficulty using his left hand and required assistance in most activities of daily living.Ideomotor apraxia in a patient with right hemispheric infarction.VR content was displayed to the study participants using a head-mounted display that involved catching of moving fish in the sea by grasping. Before and after of rehabilitative intervention including VR, functional measurements incorporating the Test of Upper Limb Apraxia (TULIA) were conducted. To directly compare therapeutic potencies under different conditions, success rates of consecutive grasping gesture performance were observed in VR, conventional occupational therapy setting, and augmented reality intervention.The patient demonstrated remarkable amelioration of apraxic symptoms while performing the task in the VR environment. At 1 and 3 months after the training, he showed significant improvement in most functions, and the TULIA score increased to 176 from 121 at the initiation of therapy. The number of successful grasps during 30 trials of each grasp trial was 28 in VR, 8 in the occupational therapy setting, and 20 in augmented reality.This case report suggests the possible therapeutic efficacy of immersive VR training as a rehabilitative measure for ideomotor apraxia. Although patients understand the actions to be performed, they cannot translate it to actual movement. The primary disability in patients with apraxia is an “automatic-voluntary dissociation,” which is the inability to perform voluntary movements, although the patient may carry out such movements. These patients are unable to initiate automatic motor responses in real-world rehabilitation settings. Virtual reality (VR) is a form of information and communication technology that displays simulated environments and interactions with the user. It has shown promising results in rehabilitation therapy by improving patients’ motivation and willingness to participate in. Among the domains of VR training for patients with stroke, functional recovery of the upper extremity was reported to be most significant. Rehabilitation for the treatment of apraxia should include facilitation of automatic responses, which may be induced by VR. We present a case of dramatic amelioration of apraxic symptoms in a patient with stroke when immersive VR was applied. The immediate responses of automatic-voluntary dissociation were compared in three different settings, and final functional improvements after VR training rehabilitation and changes in the Test of Upper Limb Apraxia (TULIA) score are presented.Ideomotor apraxia refers to the inability to mimic limb gestures, despite the absence of paralysis.22.1 As for gross motor ability, he could stand independently but could not walk. Despite the absence of remarkable paralysis, he experienced difficulty using his left hand and required assistance in most activities of daily living (ADL). The scores of the Mini-Mental State Examination, Montreal Cognitive Assessment, and intelligence quotient of Wechsler Adult Intelligence Scale-IV were 26, 23, and 80, respectively, showing fair cognitive function. He could not mimic some gestures after verbal instructions; however, he could understand and explain the motor sequences required for those movements. For example, he could not grasp the examiner's hand when verbally instructed and required a long time to shake the examiner's hand. After initiation of the grasping hand movement, his hand muscles showed excessive rigidity, and he was unable to control the movement and release the hand. The TULIA score confirmed apraxia (score 124 on 240 points). During the TULIA assessment, the score of the imitation part was higher than that of pantomime. For example, the item “put the index finger on top of the nose” was scored as 4 out of 5, while the scores for items “comb hair” and “drink from a glass” were lower with 2 out of 5. Within imitation and pantomime, a descending trend was observed with the highest score in non-symbolic and intransitive, and lowest in transitive movements. Errors in movement were observed in every task of TULIA, and the intended gestures were not achieved. Written informed consent was obtained from the patient. This study was approved by the institutional review board of the study hospital.A 51-year-old right-handed man presented with sudden weakness of his left side and was diagnosed with right frontal, parietal, and corpus callosal infarction. He was transferred to the Department of Rehabilitation Medicine 10 days after the onset of symptoms, and his left-sided weakness had subsided .2.22, matrix size = 256 × 256, slice thickness = 2.0 mm. DTI data were analyzed using DSI studio software (http://dsi-studio.labsolver.org). Regions of interests were defined automatically based on an anatomical atlas loaded into the DSI studio program. The fractional anisotropy (FA) values, tract volume, and number of fibers were obtained. Analysis results revealed decreased value of FA in the right cingulate gyrus, superior longitudinal fasciculus (SLF) I, and decreased value of tract volume and number of fibers in the right SLF II, SLF III, uncinate fasciculus, and corticospinal tract compared with the left side  at 1 month after onset. Axial T2-weighted, axial T1-weighted fast spoiled gradient recalled echo and diffusion tensor images (DTI) sequences were included. T1, T2-weighted showed subacute to chronic infarction in the right corpus callosum, right frontal lobe , and right parietal lobe. It also showed old infarction in the right centrum semiovale and basal ganglia with ipsilateral Wallerian degeneration , which involved grasping and releasing actions of the hands. Passive cueing was provided with physical guidance or verbal instructions for motor sequences. However, the progress of learning was delayed for sequential activities in most ADL. He received VR training as well as conventional occupational therapy (OT), which included training in reaching, grasping, manipulation, and releasing objects and practices of dressing, eating, drinking, washing, brushing teeth, combing hair, etc. The VR training system adopted a head-mounted display, Vive , which uses the “Ocean Catch” software , ideated by the corresponding author of this report. This immersive VR content simulated an under the sea virtual environment and provided opportunities for grasping movements to catch moving fish Fig. . The cor2.4After 4 weeks of VR training, the TULIA score increased to 161 from 121 at the initiation of the therapy in both the pantomime and imitation domains , and VR environments. For the real-world setting, the block manipulation activity was assessed. RAPAELSmart Glove  was used as the AR instrument. We observed an incomparably best motor response of the left hand during the VR condition. The number of successful maneuvers using OT, AR, and VR was 8 (26.7%), 20 (66.7%), and 28 (93.3%), respectively.3 However, the severity of apraxic symptoms correlates with reduced independence in daily living skills and reduced improvement in independent functioning.,12 This case also showed improvement in ADL with an elevated MBI score along with a decrement in apraxic features that occurred during the VR training.The immersive VR training seemed to have significantly ameliorated ideomotor apraxia in the present case. Compared with other conditions, such as block manipulation in the real world and conducting an AR task using a smart glove, the patient showed immediate improvement in grasp and release performance in the VR condition. This means that the automatic motor response is more easily induced by the VR environment. Given the basic characteristics of apraxia (automatic-voluntary dissociation), its impact on daily life might be overlooked. In the present case, we observed parietal and frontal cortex defects in the right hemisphere and corpus callosum. The reduced FA values and tract volume revealed partial injury in the right cingulate gyrus, SLF, and uncinate fasciculus, which contain projections between frontal and parietal cortex. These brain lesions seemed to cause apraxic symptoms in patients and might have affected the VR rehabilitation outcome in this case. The follow-up study of DTI, which identify for changes in values of FA, would be helpful to confirm the effectiveness of the VR rehabilitation.Apraxic symptom has been associated with damage to the parietal and frontal cortex as well as the white matter connections between them. Furthermore, anterior callosal lesions and basal ganglia were reported as the apraxia-related area. The VR used in this study also provided feedback immediately after each training session. Above all, it seems that the fun and motivation induced by the immersive VR may have strengthened the intensity of response to rehabilitation. According to a recent Cochrane review, VR may be beneficial for improving upper limb function and ADLs in post-stroke patients when used as an adjunct to usual care.,16 This positive effect could have been induced by increased motivation of the patients while using VR. Therefore, we concluded that immersive VR provided a motivating condition that enabled the patient to overcome automatic-voluntary dissociation, which might be corrected only by activating the function at an unconscious level.VR enables repetitive practice, provides feedback on the performance, and motivates the user to endure the training for motor rehabilitation. Feedback on the performances given by the virtual environment would be stronger than that given during real-world practice.Consequently, his dexterity and ADLs improved over a relatively short period (4 weeks). This improvement persisted even 12 weeks after rehabilitation. Although the patient received additional conventional rehabilitation, considering the immediate responses to VR training, it is reasonable to conclude that VR training was beneficial in this case.4,18 A recent review suggested that rehabilitative training alone would be insufficient for a sustained benefit. However, our patient showed significant recovery from apraxia 3 months after completion of our intervention. More studies and large-scale clinical trials are needed to determine the therapeutic efficacy and its mechanism in VR application for apraxia. To the best of our knowledge, no studies have reported on the treatment of limb apraxia using immersive VR. This case report shows the beneficial effects of immersive VR-based rehabilitation in a patient with severe apraxia.Currently, there is no specific treatment for patients with motor apraxia after stroke.Conceptualization: MinYoung Kim.Data curation: Wookyung Park, Jongwook Kim.Supervision: MinYoung Kim.Writing – original draft: Wookyung Park.Writing – review & editing: MinYoung Kim.


Diabetic retinopathy has recently been defined as a highly specific neurovascular complication of diabetes. The chronic progression of the impairment of the interdependence of neurovascular units (NVUs) is associated with the pathogenesis of diabetic retinopathy. The NVUs consist of neurons, glial cells, and vascular cells, and the interdependent relationships between these cells are disturbed under diabetic conditions. Clinicians should understand and update the current knowledge of the neurovascular impairments in diabetic retinopathy. Above all, neuronal cell death is an irreversible change, and it is directly related to vision loss in patients with diabetic retinopathy. Thus, neuroprotective and vasoprotective therapies for diabetic retinopathy must be established. Understanding the physiological and pathological interdependence of the NVUs is helpful in establishing neuroprotective and vasoprotective therapies for diabetic retinopathy. This review focuses on the pathogenesis of the neurovascular impairments and introduces possible neurovascular protective therapies for diabetic retinopathy. According to the ninth edition of the International Diabetes Federation diabetes atlas, the estimated prevalence of diabetes will be 10.2% (578 million) by 2030 and 10.9% (700 million) by 2045 in the world . I. I121]. Suppressor of cytokine signaling (SOCS) 1 is one of the family members of the SOCS, intracellular cytokines inducible protein. SOCS1 is a negative regulator of IL-6 and TNF-α and regulates cytokine receptor signaling by a negative feedback loop . SOCS3 iMicroglia play a pivotal role in the inflammatory responses in diabetic retinopathy . MinocycBDNF is one of the nerve growth factor family members and is associated with the pathogenesis of diabetic retinopathy and one of the therapeutic options. The BDNF level in the serum and the aqueous humor in patients with diabetes is significantly reduced , which cPEDF, an intrinsic antioxidant, is secreted by various retinal cells, including the retinal pigment epithelial cells, and it has two actions, viz., vasoprotection and neuroprotection. In diabetic animal models, intravitreal injection of PEDF reduces retinal neuronal damage and VEGF expression as an antioxidant . FurtherSomatostatin has both antiangiogenic and neuroprotective effects and it acts through somatostatin receptor 2. Topical administration of somatostatin ameliorates neuronal degeneration in diabetic murine models . HoweverGlucagon-like peptide-1 (GLP-1) and GLP-1 receptors are expressed in the retina , and then = 9795 and 10,251, respectively; Although it is not a neurotrophic factor, fenofibrate may be an ideal drug to prevent the development and the progression of diabetic retinopathy. Fenofibrate, a peroxisome proliferator-activated receptor alpha (PPARα), is used as a drug to treat hyperlipidemia worldwide. Two randomized clinical trials found that fenofibrate significantly reduced the progression of diabetic retinopathy ,149 (n =DME is one of the common causes of vision decrease in the early stage of diabetic retinopathy. Although several therapeutic procedures are effective for improving DME, anti-VEGF therapies have become the gold standard for the management of DME ,70,71,72Diabetic retinopathy is a disease accompanied by NVU impairments, and all the pathological changes, including to vascular cells, glial cells, and neuronal cells, should be ameliorated so to prevent the development and progression of diabetic retinopathy. Neuronal cell death is an irreversible event which directly results in vision loss. Thus, neuroprotective therapies for diabetic retinopathy should be established. However, the target of ideal therapies for a neurovascular disease such as diabetic retinopathy should focus on the NVU impairments; thus, both vasoprotective and neuroprotective therapies are required for preventing vision loss in patients with diabetic retinopathy. Understanding the NVU impairments and establishing therapies for neurovascular protection should be helpful for establishing new ways of managing diabetic retinopathy in the future.


Morus alba (MA) and Aronia melanocarpa (Michx.) (AR) against high-fat diet induced obesity. Four-week-old male C57BL/6J mice were randomly divided into five groups that were fed for 14 weeks with a normal diet (ND), high-fat diet (HD), HD with M. alba 400 mg/kg body weight (MA), HD with A. melanocarpa 400 mg/kg body weight (AR), or HD with a mixture  of M. alba and A. melanocarpa (400 mg/kg) (MA + AR). Treatment with MA, AR, and MA + AR for 14 weeks reduced high fat diet-induced weight gain and improved serum lipid levels, and histological analysis revealed that MA and AR treatment markedly decreased lipid accumulation in the liver and adipocyte size in epididymal fat. Furthermore, micro-CT images showed MA + AR significantly reduced abdominal fat volume. Expression levels of genes involved in lipid anabolism, such as SREBP-1c, PPAR-γ, CEBPα, FAS, and CD36 were decreased by MA + AR treatment whereas PPAR-α, ACOX1, and CPT-1a levels were increased by MA + AR treatment. Protein expression of p-AMPK and p-ACC were increased in the MA + AR group, indicating that MA + AR ameliorated obesity by upregulating AMPK signaling. Together, our findings indicate that MA and AR exert a synergistic effect against diet-induced obesity and are promising agents for managing obesity.The present study investigated the synergic effect of extracts of  Obesity in Asian countries is diagnosed as a body mass index , also known as silkworm mulberry, common mulberry or white mulberry is a plant in the family Moraceae. This species, which is native to India and northern China, has many beneficial health effects [ effects . Compoun effects . Extract effects . Further effects  reported effects .Aronia melanocarpa (Michx.) (AR), widely known as black chokeberry, is native to eastern North America and eastern Canada and is a member of the Rosaceae family. It is easy to cultivate, and its fruits have numerous bioactive compounds. AR has been shown to have anti-T2DM effects [ effects , anti-fi effects , anti-in effects , as well effects  properti effects , in addi effects . Bioacti effects . Zhu et  effects  reportedPatrinia scabiosaefolia and Hippophae rhamnoides extracts had a synergistic anti-obesity effect [Although a few studies have investigated the anti-obesogenic effects of MA or AR alone ,10,17,18y effect . Similary effect . Given tPowdered forms of MA leaves and AR fruits were obtained from Jinan-gun, Korea and were extracted twice, three hours from reflux extractor, each with 700 mL of 80% ethanol in 70 g powder. The extract was filtered with whatman N0.41 and concentrated with a rotary evaporator to remove the ethanol, freeze-dry, and manufactured in powder form. The compounds in the MA and AR was quantified by HPLC.The compounds of MA were determined using Agilent 1200 infinity  equipped with a FLD detector. A Capcell pak ADME 3UM column (4.6 × 150 mm) was used. Flow rate was set at 1.0 mL/min. The column temperature was continued at 35 °C. Injection volume was 10 μL. Excitation wavelength and emission wavelength were set to be 254 and 322 nm, respectively, for the analysis. A gradient elution using solvent A (0.1% formic acid in DW) and solvent B (acetonitrile) was used as follows: 0–10 min, 60% A and 40% B. The compounds of AR were determined using Agilent 1200 infinity  equipped with a DAD detector. A Capcell pak ADME 3UM column (4.6 × 150 mm) was used. Flow rate was set at 1.0 mL/min. The column temperature was continued at 25 °C. Injection volume was 10 μL. Wavelength was set to be 310 nm. A gradient elution using solvent A (0.1% formic acid in DW) and solvent B (acetonitrile) was used as follows: 0–4 min, 90% A and 10% B; 4–15 min, 90% A and 10% B; 15–25 min, 70% A and 30% B; 25–30 min, 70% A and 30% B; 30–40 min, 90% A and 10% B. These were performed by the Institute of Jinan Red Ginseng .v/v ratio. Samples were dissolved in distilled water and orally administered 100 μL per mouse for 14 weeks, once a day. The control groups received oral administration of the same volume of distilled water.All experimental protocols related to animals were endorsed by the Animal Ethics Committee of Jeonbuk National University (CBNU 2020–0123). Four-week-old C57BL/6J mice were purchased from Central Lab, Animal, Inc.  and experimental diets obtained from DooYeol Biotech . Upon arrival, mice were housed in cages under a 12 h light, 12 h dark cycle within the temperature (range of 25 ± 2 °C) and humidity of 50 ± 5%. After one week of adaptation, mice were divided into five groups each containing 10 mice: ND , HD (high-fat diet control), MA (high-fat diet supplemented with MA extract), AR (high-fat diet supplemented with AR extract), and MA + AR (high-fat diet supplemented with a mixture of MA + AR). The normal-diet (ND) group was fed a diet containing 10% kcal from fat (D12450B), and the high-fat diet groups were fed a diet containing 60% kcal from fat (D12492) for 14 weeks and the treatment with samples were started at the same time of experimental diet feeding. MA and AR groups received oral administration of individual extracts at 400 mg/kg body weight (bw), while the MA + AR group received extracts (400 mg/kg bw each) mixed at a 1:1 Body weight was measured once a week. Feed intake was calculated thrice a week, by subtracting the remaining amount from the feed given per mice per cage. Mice were sacrificed after the experimental period after 12 h of overnight fasting and blood, liver, epididymal white adipose tissue were collected. Serum was collected by keeping the blood at room temperature for 1 h followed by centrifugation at 3000 rpm for 15 min at 4 °C. Epididymal fat and liver were dissected, weighed, and stored a −72 °C until further assay.Serum triglyceride (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-C) levels were determined by enzymatic methods using commercial kits . Very-low-density lipoprotein cholesterol (VLDL-C) and low-density lipoprotein cholesterol (LDL-C) levels were calculated using Friedewald’s formula . Serum free fatty acids (FFAs) and glycerol concentrations were determined with assay kits  . Serum insulin, adiponectin, and leptin concentrations were determined using ELISA kits according to the manufacturer’s protocols . Absorbance was measured using a microplate reader .Hepatic lipids were extracted as described previously . To measMicro-CT was conducted using a high-resolution in vivo micro-CT system  at the Center for University-wide Research Facilities (CURF) of Jeonbuk National University to analyze visceral white adipose tissue (VAT) volume. Fat volume was calculated using Image J software.Liver and adipocyte tissue were fixed with 10% formalin for 48 h and samples were processed further by the KP&T Company . Hematoxylin and eosin (H&E) staining and Oil Red O (ORO) staining were performed. Stained samples were analyzed by optical microscopy  in the CURF of Jeonbuk National University. Image-J software  was used to determine adipocyte area.Expression levels of genes associated with lipid metabolism in the liver and adipose tissue were analyzed using real time-PCR . The following PCR cycle was performed: Stage 1; 95 °C for 10 min, Stage 2;  × 40 cycles. Briefly, mRNA from liver and adipose tissue was isolated using Trizol reagent . After quantification of RNA concentration, cDNA was synthesized using the PrimeScript RT Master Mix . Real time-PCR was conducted using SYBR green qPCR mix . Primers used in RT-PCR are listed in g, 15 min), and the supernatant was then collected. After measuring protein concentrations, equal concentration of samples was heated to 95 °C after mixing with 5X protein buffer. Then, proteins were electrophoresed on 8–10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes  followed by blotting with antibodies.Protein levels of p-AMPK, AMPK, ACC, p-ACC , and β-actin  in the liver were determined by immunoblotting. Liver tissue (50–100 mg) was homogenized in RIPA lysis buffer  containing 1% protease inhibitor and 1% phosphatase inhibitor cocktail , centrifuged  indicate statistically significant differences among groups.Data are expressed as means ± standard deviation of the mean (S.D). Statistical significance was verified using Duncan’s test with one-way ANOVA in SPSS version 17.0 , and results were considered significant at For HPLC analysis, the most commonly detected substance in the 80% ethanol extract of MA is 1-deoxynojirimycin. The amount of 1-deoxynojirimycin is 21.45 mg/g extract A. As shoAs shown in As shown in p < 0.05). There was a significant difference in TC between the HD group and the groups treated with MA or AR alone. Hepatic TG were significantly reduced in all treatment groups compared to the HD group.Hepatic lipid profiles are shown in M. alba extract (400 mg/kg bw), A. melanocarpa extract (400 mg/kg bw), or mixture of these two extracts was orally administered to experimental mice fed a high-fat diet for 14 weeks. TC, total cholesterol, TG, triglycerides. ND, normal diet, HD, high-fat diet, MA, high-fat diet + M. alba 400 mg/kg bw, AR, high-fat diet + A. melanocarpa 400 mg/kg bw, MA + AR, high-fat diet + mixture of M. alba and A. melanocarpa (400 mg/kg bw). Results are expressed as means ± S.D (n = 10). Means with different superscript letters  are significantly different from each other by Duncan’s test of ANOVA at p < 0.05. “a” denotes the highest value and “b” represents the lowest value.Micro-CT was used to investigate changes in visceral fat volume after treatment. Visceral fat volume of the HD group was higher than that of the ND group . CompareThe mRNA expression of genes involved in lipid metabolism in the liver was analyzed using real time-PCR A. ExpresAs shown in The prevalence of obesity has increased during the past 35 years to the extent that more than one third of the world’s population are overweight or obese . ObesityIn the present study, we investigated the anti-obesity effects of the combination of MA and AR in a HD-induced obese mouse model. MA, AR, or a mixture of these two extracts was administered orally to mice for 14 weeks. Treatment with MA and AR, reversed obesity-induced changes in blood lipids, serum hormones, liver weight, and epididymal fat weight, as much as individual treatment with these extracts. Moreover, the expression of lipolysis-related genes in liver and adipose tissue was significantly increased by MA + AR treatment.Continuous consumption of a high-fat diet leads to a continual increase in body weight and fat mass . The incPersistent dyslipidemia increases the risk of T2DM and vascular diseases, such as stroke, hypertension, and coronary artery disease (CAD) . In the Hormones and adipokines produced by adipose tissue play a central role in obesity. PPAR-γ and C/EBP-α play key roles in pre-adipocyte differentiation and adipogenesis . ExpressActivation of AMPK has been shown to reduce body weight gain HD-induced obese mice . AMPK isThe activation of AMPK was noticeably increased in MA + AR group indicating that action of MA + AR against HD-induced obesity is through down regulation of lipogenesis and upregulation of β-oxidation. Clinical trials of MA extracts have been conducted in reducing cholesterol levels and blood glucose on the hypolipidemic and hypoglycemic effects . AR has In summary, we observed that MA and AR interacted synergistically to ameliorate the negative effects of obesity compared to each of these extracts individually in C57BL/6 mice. MA and AR extracts significantly reduced the HD-induced increase in body weight. Serum and liver biochemical parameters, such as TG, LDL-C, VLDL-C, total-cholesterol, FFAs, glycerol, and adiponectin improved upon MA and AR treatment. These changes were associated with changes in the expression of transcription factors and coactivators related to adipogenesis and lipolysis. Furthermore, both MA and AR stimulated AMPK and ACC phosphorylation in liver tissue in a synergistic manner. Taken together, these results demonstrated that MA and AR had a synergistic anti-obesity effect.


Castration-resistant prostate cancer (CRPC) is a highly aggressive and advanced prostate cancer that is currently incurable with conventional therapies. The recurrence and chemotherapy-resistant properties of CRPC are attributed to prostate cancer stem cells (CSCs). On the other hand, the factors regulating the prostate CSC-like properties have not been studied extensively. Previously, ubiquitin C-terminal hydrolase-L1 (UCH-L1) and ubiquitin C-terminal hydrolase-L3 (UCH-L3) were reported to be involved in prostate cancer cell progression through the epithelial-to-mesenchymal transition (EMT) regulation. Here, the differential regulation on the CSC-like properties by UCH-L1 and UCH-L3 were identified in prostate cancer cells. The CSC-like characteristics, such as the expression of pluripotency markers, chemoresistance, and sphere-forming ability, were promoted by UCH-L1, whereas those were repressed by UCH-L3. Moreover, the modulation of CSC-like properties by UCH-L1 and UCH-L3 was through the PI3 K/Akt signaling pathway. The CSC-like properties induced by UCH-L1 overexpression or UCH-L3 depletion were suppressed by the PI3 K/Akt pathway inhibitor. In conclusion, UCH-L1 and UCH-L3 are novel regulators of the CSC-like properties and shed light on new therapeutic strategies to overcome CSCs in prostate cancers. Prostate cancer is the most prevalent and major life-threatening cancer among men in western countries  and ubiquitin C-terminal hydrolase-L3 (UCH-L3) are deubiquitinating enzymes (DUBs) that remove ubiquitin (Ub) from the target proteins . Recent studies have proposed that EMT induces CSC-like features in cancer cells. During cancer development, activation of the EMT program increases the tumor initiation capacity and imparts cancer cells with resistance to a range of therapeutic agents . RWPE1 cells were maintained in Keratinocyte-serum-free medium  containing EGF (1.25 g/L) and bovine pituitary extract (BPE) (12.5 mg/L). RPMI1640 and K-SFM were supplemented with 10% FBS and 0.1 mg/mL streptomycin plus 100 U/mL penicillin.The antibodies used and their manufacturers are as follows. OCT4 (sc-9081), BMI1 (sc-390443), SOX2 (sc-17320), Nanog (sc-134218), AKT (sc-8312), 4EBP1 (sc-9977), p70 S6 kinase (sc-8418), and UCH-L1 (sc-58593) were purchased from Santa Cruz. KLF4 (ab151733), phospho-AKT (ab81283), and UCH-L3 (ab87154) were obtained from Abcam. Phospho-4EBP1 (2855) and phospho-p70 S6 kinase (9205) were purchased from Cell signaling. Flag M2 (F3165) and β-actin (A1978) were supplied by Sigma-Aldrich. The following chemicals were used: BEZ235 , LY294002 , doxorubicin , etoposide , and mitoxantrone dihydrochloride (MTX) .The procedures for the stable overexpression of UCH-L1, UCH-L1 C90S, UCH-L3, or UCH-L3 C95S and the knockdown of UCH-L1 or UCH-L3 in RWPE1 or DU145 were referenced from previous reports  primers and RevertAid reverse transcriptase. The SYBR green and QuantStudio 1 Real-Time PCR System was used for the semiquantitative real-time RT–PCR experiments. The data were analyzed using the ΔΔCt method and was normalized to the GAPDH gene. The sequences of the primers used in the study were referenced as follows: After isolating the RWPE1 and DU145 cells into single cells, 100 cells were seeded in 96-well Ultra-low Attachment plates. The media for the sphere forming assay was serum-free DMEM/F12 K medium containing B27, 20 ng/mL EGF and bFGF, and 4 μg/mL insulin. After 14 days for RWPE1 or 7 days for DU145, the number of spheres with a diameter exceeding 100 μm was counted under a microscope at 200× magnification.The TCGA-PRAD clinical data and RNA sequencing data were downloaded from the cBioPortal. The samples from 491 patients were examined with the mRNA levels of UCH-L1 and UCH-L3 to classify them into low- (n = 245) and high-expression groups (n = 246) using the median expression level as the cut-off for each mRNA. For statistical analysis, the log-rank test was used. The Kaplan-Meier method was used to obtain the disease-free survival curves using R software.As EMT progresses, cancer cells undergo dedifferentiation and adopt a CSC status , p4EBP1(Thr37/46), and pS6 K(Thr389). On the other hand, UCH-L3 C95S did not have any significant effects  . The TCGA cohort studies suggested that the expression level of UCH-L1 and UCH-L3 affects the survival and recurrence of prostate cancers. Overall, these results showed that UCH-L1 and UCH-L3 could be effective therapeutic targets to eliminate the recurrence caused by CSCs in prostate cancers.As the relapse of CRPC is attributed to CSCs, we examined the relationship between the disease-free survival and the level of UCH-L1 and UCH-L3 in prostate cancer patients. The results of Kaplan-Meier analysis of 491 prostate cancer patients showed that patients expressing high UCH-L3 had higher disease-free survival compared to patients expressing relatively low UCH-L3 levels . The implications of UCH-L1 and UCH-L3 in cancer development have been reported  is a receptor tyrosine kinase that activates multiple downstream signaling cascades, including PI3 K/Akt. EGFR signaling plays a pivotal function in supporting the CSC properties, and aberrant EGFR expression is implicated in the progression of CRPC (Berger et al. In this study, UCH-L1 and UCH-L3 differentially regulated the CSC-like properties of prostate cancer cells via the PI3 K/Akt signaling pathway. The CSC-like properties were enhanced by UCH-L1 but inhibited by UCH-L3. TCGA dataset analysis showed that a high level of UCH-L3 expression is positively correlated with the disease-free survival. By contrast, a high level of UCH-L1 expression had an inverse correlation with the survival rate. This suggests that these two DUBs could be promising diagnostic and therapeutic targets for treating prostate cancers. Although the differential effects of UCH-L1 and UCH-L3 on CSC-like behaviors have been identified, it is essential to identify the specific targets for these two deubiquitinating enzymes to achieve a deeper understanding underlying the molecular mechanism of prostate cancer progression.