Home blood pressure telemonitoring (HBPT) is witnessing rapid diffusion worldwide. Contemporary studies documented mainly short-term (6-12 months) effects of HBPT, and there are limited data about its uptake.The aim of this study was to explore the 3-year use and determinants of HBPT, and the interactions with systolic and diastolic blood pressure (SBP/DBP) and overall blood pressure (BP) control rate.HBPT records were obtained from a 3-year cohort of 5658 patients with hypertension in Jieshou, Anhui, China, and data from a structured household survey of a random sample (n=3005) of the cohort. The data analysis comprised (1) timeline trajectories of the rates of monthly active HBPT and mean SBP/DBP for overall and subgroups of patients with varied start-month SBP/DBP; and (2) multivariable linear, logistic, and percentile regression analyses using SBP/DBP, BP control rate, and yearly times of HBPT as the dependent variable, respectively.HBPT was followed by mixed changes in mean monthly SBP/DBP for varied patient groups. The magnitude of changes ranged from –43 to +39 mmHg for SBP and from –27 to +15 mmHg for DBP. The monthly rates of active HBPT all exhibited a rapid and then gradually slower decline. When controlled for commonly reported confounders, times of HBPT in the last year were found to have decreasing correlation coefficients for SBP/DBP  and for BP control rate (from 0.53 to –0.62).HBPT had major and “target-converging” effects on SBP/DBP. The magnitude of changes was much greater than commonly reported. BP, variation in BP, and time were the most important determinants of HBPT uptake. Age, education, duration of hypertension, family history, and diagnosis of hypertension complications were also linked to the uptake but at weaker strength. There is a clear need for differentiated thinking over the application and assessment of HBPT, and for identifying and correcting/leveraging potential outdated/new opportunities or beliefs. Home blood pressure telemonitoring (HBPT) is recommended in current hypertension management guidelines, and is witnessing rapid diffusion worldwide -3. VarioGiven fluctuating BP readings; changing stages   and typeChina has witnessed a rapid increase in the use of HBPT over the past decade. More and more residents are buying and using various types of HBPT devices. However, there is a general paucity of data about the effects and determinants of HBPT. Similar to studies in other countries, the limited publications on HBPT in China have focused primarily on comparing BP differences between the intervention and control groups, with little attention being paid to the determinants and differentiated effects of HBPT.To fill this gap, the aim of this study was to use data from a relatively large-scale (5658 patients with hypertension) and long-term (up to 40 months) cohort in Jieshou, Anhui, China, for performing a relatively in-depth analysis of HBPT, with particular attention placed on comparing its effects and determinants across patient groups with varied levels of BP. As an inland county located in the middle and east of China, Jieshou is representative of the majority of counties in the nation.The study was built upon two related and ongoing projects. The first was initiated by Jieshou Hospital, Anhui province, China, which aimed to improve hypertension management via HBPT. The project covered all patients diagnosed with hypertension (N=5658) in all villages (N=48) served by the Jieshou Hospital Consortium. The HBPT involved an electronic oscillometric upper-arm BP monitor installed with a voice speaker capable of automatically stating the resultant measurements and educational messages to the patient. The monitors were provided by IFLYTEK Co Ltd, and were confirmed to be easily useable by ordinary residents. The readings of the HBPT were synchronously sent to a remote central data center.The second project is an RCT registered in ISRCTN (10999269). This project used a cluster randomized sample (n=3005) of the participants in the above HBPT project to test the efficacy of a novel personalized hypertension management package .By the time this study was carried out, the HBPT project had gathered BP readings from the participants for over 40 months and the RCT had completed the baseline assessment, including a structured baseline household survey.This study used the records from the HBPT project described above and part of the data from the corresponding baseline household survey. Each HBPT record consisted of four items: SBP, DBP, pulse per minute, and measurement date and time. The household survey took place from April to July 2021 via a structured questionnaire administered face to face. This study used 24 items from the questionnaire, soliciting information about: (1) sociodemographic characteristics, including age, sex, and education; (2) body height and weight; (3) age when hypertension was first diagnosed; and (4) hypertension-related symptoms and diagnoses .Data analysis comprised three components: (1) descriptive statistics (numbers and percentages) of study subjects by sociodemographic categories, (2) calculation and presentation (in trajectory lines) of the rates of monthly active HBPT for overall subjects and for subgroups with varied mean SBP/DBP in the first month, (3) multivariable linear and percentile regression modeling of times of HBPT and SBP/DBP in the last year, and (4) multivariable logistic regression modeling of BP control rate.The rate of monthly active HBPT was defined as the proportion of patients who had performed HBPT at least one time in the month under concern. The multivariable linear, logistic, and percentile regression models used similar independent, exposure, and confounder variables. The dependent variables included times of HBPT in the past year for overall participants and subgroups with varied mean SBP/DPB from HBPT in the last year and the BP control rate in the last year. The exposure variables consisted of mean SBP/DBP and variations in the coefficients of SBP/DBP in the last year. The confounder variables comprised sociodemographics and health conditions. The monthly mean SBP/DBP of any patient was defined as their hourly mean SBP/DBP, calculated as the sum of all SBP/DBP readings recorded within a given hour , multiplied by the number of records within the same hour. The BP control rate was computed as the times of BP readings meeting SBP<140 mmHg and DBP<90 mmHg in the past year multiplied by the total BP readings during the same period.The analysis regarding the monthly active HBPT used all participants enrolled in the HBPT project, whereas the regression modeling used all of the participants involved in the baseline survey. The logarithm of times using HBPT in the last year was used to transform the variable into a normal distribution. Detailed value assignment is shown in This study has been approved by Anhui Medical University Biomedical Ethics Committee (number 20200936) and all the participants have signed (for those who are literate) or ticked (for those who are illiterate) the consent form.Of the 3005 participants recruited in the baseline survey, 57% were women. The average age of the participants was 65.50 years. Their duration of hypertension was 9.50 years on average. Over half of the respondents had a family history of hypertension . AlthougP<.001) and DBP . In the percentile regression models, times of HBPT were found to have decreasing correlation coefficients for the two BP variables, from 0.16 to –0.35 and from 0.11 to –0.35 for SBP and DBP, respectively. In the percentile modeling, age also showed significant associations with SBP/DBP for all percentiles (positive for SBP and negative for DBP), whereas almost no significant relations were found for education, family history, and number of hypertension-related symptoms and diagnoses to both SBP and DBP . Sex was associated with DBP but not to SBP, whereas the duration of hypertension and BMI exhibited statistically significant links on apparently more percentiles for SBP than for DBP.P values of the patients under concern. The magnitude of changes ranged from –43 to +39 mmHg for SBP and from –27 to +15 mmHg for DBP. When controlled for commonly reported confounder variables such as sex, age, education, duration of hypertension, BMI, family history of hypertension, and numbers of hypertension-related symptoms and diagnoses, the differentiated effects of HBPT on SBP/DBP were still observable. In the multivariable percentile regression model  and long-term cohort. Relatively in-depth analysis of the determinants of HBPT was performed, with particular attention paid to subtle and differential interactions with the resultant BP outcome. This study thus produced useful trajectories of monthly mean SBP/DBP and monthly active rates of HBPT for up to 40 months. Multivariable linear, percentile, and logistic regression modeling of times of HBPT, mean SBP/DBP, and BP control rate as the dependent variables, respectively, enabled cross-checks and comparisons of the results.P values from HBPT are prone to various influences. Second, the study population was relatively old (65.50 years on average) and the findings should be generalized with caution. Third, the study considered only SBP/DBP as the outcome variables without considering others  and lacked comparison with patients who had not used HBPT. Fourth, BP readings are susceptible to diurnal and intraobserver variations, which can lead to measurement biases, although our use of monthly and hourly mean SBP/DBP may have helped to reduce these biases to some extent. Our further research activities in response to these shortcomings include performing household surveys/observations to help identify the factors influencing HBPT readings, extend the HBPT to younger populations, and perform further analyses linking HBPT with major adverse cardiovascular events  and quality of life.This study also suffers from drawbacks. First, being performed at home by ordinary residents, the BHBPT had major and “target-converging” effects on SBP/DBP. The “target” was the widely validated and accepted defining values of hypertension control . HBPT was followed by SBP/DBP reductions or increases for cohorts with a mean BP higher or lower than the “target,” respectively. The magnitude of changes was a few times greater than commonly documented. These differentiated effects remained observable into the third year after initiation of HBPT. BP, variation in BP, and time were the most important determinants of HBPT uptake, whereas age, education, duration of hypertension, family history, and diagnosis of hypertension complications were also linked to the uptake but at apparently weaker strength. HBPT displayed stronger associations with the variation in SBP than in DBP.There is a clear need for differentiated thinking over the application and assessment of HBPT. First, the traditional approach of simply comparing the effects in the intervention group as a whole with that in the control group is prone to underestimation of the actual influences of HBPT, since decreases in a portion of the patients were offset by increases in others. HBPT leads to BP decrease, stability, or increase depending on the complex and dynamic context of the patient under concern. These varied effects may not necessarily all be beneficial and merit careful scrutiny in the future. This study thus highlights the need for correcting outdated beliefs or practices and leveraging new opportunities with the application of HBPT. Second, the difference in the “white coat” effect suggests lower than traditional cut-off values of hypertension control when readings from HBPT were used. In other words, patients should be better educated about the “white coat” effect and that they need to exert further efforts to maintain their HBPT readings slightly below 140/90 mmHg. Third, the varied responses toward different levels of HBPT readings indicate selective telemonitoring, group-specific “targets,” or even personalized interventions. Fourth, relatively less attention paid to DBP than to SBP implies that additional efforts are needed to promote balanced awareness among patients. In particular, patients should be informed that DBP is as important as SBP and thus merits equal attention in self-monitoring.


Dear Editor,Venetoclax is an oral drug that is BH3-mimetic and a selective B-cell lymphoma 2 (BCL-2) inhibitor. Inhibition of BCL-2 induces apoptosis in hematological malignancies . Venetoc2, day 1, ifosfamide 1.7 g/m2, days 2–3, 1.6 g/m2, day 4, carboplatin 5 × AUC, day 2, and etoposide 100 mg/m2, days 2–4, for 3 cycles), and rituximab . The IgM level decreased from 3270 to 2020 mg/dL after these treatments. We assessed him as a minor response according to the International Workshops for WM (IWWM) criteria [2, days 1–14, and aclarubicin 14 mg/m2, days 1–4, for 1 cycle) was administered for t-AML. Although complete remission with incomplete hematologic recovery (CRi) was achieved, fever, serum IgM elevation, and splenomegaly progressed. WBC, Neu, Hgb, and Plt were 3200/μL, 300/μL, 7.6 g/dL, and 49,000/μL. The IgM level rose to 1791 mg/dL. It was thought that cytarabine and aclarubicin controlled t-MDS/AML but not WM. Rituximab  was administered as treatment of WM, but no response was observed. He developed bilateral cataplexy of brachial and antebrachial arms and was suspected to be Bing-Neel syndrome due to WM progression [A 74-year-old male diagnosed as having WM more than 20 years previously had been administered several cytotoxic agents including MP  at a previous hospital, R-ICE . Hyperuricemia and hyperphosphatemia associated with laboratory tumor lysis syndrome were found, but these abnormalities improved. After initiation of Ven/Aza treatment, blast cells disappeared and anemia was improved. In addition, splenomegaly tended to improve and serum IgM levels decreased. CRi was achieved for t-AML and PR was achieved for WM. WBC, Neu, Hgb, and Plt were 2100/μL, 290/μL, 7.0 g/dL, and 70,000/μL, respectively. Treatment of both t-MDS/AML and WM by Ven/Aza is ongoing.At this time, because Ven/Aza  was apprVenetoclax has been suggested to be effective in the treatment of AML, including t-AML , and B-cIn conclusion, further data is needed; however, our study suggests that Ven/Aza is an effective treatment regimen for patients with t-MDS/AML with concomitant WM.


Pseudomonas aeruginosa contributes to many chronic infections and has been found to be resistant to multiple antibiotics. Pseudomonas use a quorum sensing system (QS) to control biofilm establishment and virulence factors, and, thus, quorum sensing inhibitors (QSIs), such as meta-bromo-thiolactone (mBTL), are promising anti-infective agents. Accordingly, this study intended to investigate the antibacterial and anti-virulence activity of mBTL-loaded calcium alginate nanoparticles (CANPs) against Pseudomonas aeruginosa and different QS mutants. The results show that the mBTL-CANPs had higher antibacterial activity, which was made evident by decreases in all tested strains except the ∆lasR/∆rhlR double mutant, with MIC50 (0.5 mg/mL) of mBTL-CANPs compared with free mBTL at MIC50 (˃1 mg/mL). The biofilm formation of P. aeruginosa and some QS-deficient mutants were reduced in response to 0.5–0.125 mg/mL of mBTL-encapsulating CANPs. The pyocyanin production of the tested strains except ∆lasA and ∆rhlR decreased when challenged with 0.5 mg/mL of mBTL-loaded NPs. The subsequent characterization of the cytotoxic effect of these NPs on human lung epithelial cells (A549) and cystic fibrosis fibroblast cells (LL 29) demonstrated that synthesized NPs were cytocompatible at MIC50 in both cell lines and markedly reduced the cytotoxic effect observed with mBTL alone on these cells. The resulting formulation reduced the P. aeruginosa strains’ adhesion to A549 comparably with mBTL, suggesting their potential anti-adhesive effect. Given the virulence suppressing action, cytocompatibility, and enhanced anti-biofilm effect of mBTL-CANPs, and the advantage of alginate-based NPs as an antimicrobial delivery system these nanoparticles have great potential in the prophylaxis and treatment of infection caused by Pseudomonas aeruginosa. Pseudomonas aeruginosa is a Gram-negative bacterium that causes pneumonia, infection of the bloodstream, urinary tract, burn wounds, and surgical sites and plays a crucial role in cystic fibrosis. Previous reports have found that P. aeruginosa was resistant to many antibiotics [P. aeruginosa is positioned at the top of the World Health Organization’s priority pathogen list  for novel antibiotic development against MDR pathogens [P. aeruginosa strains [Infections caused by multidrug-resistant pathogens kill over 700,000 people worldwide each year, with the number expected to rise to 10 million by 2050 [ibiotics ,4, and,  strains ,7,8.P. aeruginosa virulence factor production involving biofilm development and pyocyanin production via countering signal molecules called autoinducers (AIs) [P. aeruginosa, including two LuxI/R-type coordination (lasI/R and rhlI/R) and a Pseudomonas quinolone signal system (PQS) [P. aeruginosa, and numerous other infections have paved the way for a novel therapeutic target [P. aeruginosa [P. aeruginosa QS receptors lasR and rhlR [P. aeruginosa infection model [Quorum sensing (QS) is a cell–cell communication mechanism that regulates rs (AIs) . There aem (PQS) . The outc target ,12,13. Ic target ,15. Therruginosa . In thisP. aeruginosa [P. aeruginosa and different QS mutants. Furthermore, the cytotoxicity of these NPs on A549 and LL 29 cell lines and their ability to interrupt the adhesion of P. aeruginosa strains to A549 cells were also studied.Alginate is a natural polysaccharide made up of b-D-mannuronic acid (M block) and a-L-guluronic acid (G block) ,18. Algiruginosa . In our ruginosa . TherefoP. aeruginosa PAO1 wild-type and mutants were obtained from the library of PAO1 transposon from the University of Washington Genome Centre  [The strains of WA, USA) . The bacThe effects of plain and mBTL-loaded CANPs on bacterial growth were determined according to the Clinical and Laboratory Standard Institute (CLSI) protocols and as described previously ,23. The P. aeruginosa biofilm development was investigated as formerly reported with slight modification [P. aeruginosa in LB broth was adjusted to 0.5 McFarland standard. Then, 100 µL was added to 100 µL of LB containing different concentrations of mBTL-loaded CANPs ranging from 1 mg/mL to 0.125 in a 96-well microtiter plate (Thomas science), and it was incubated at 37 °C overnight. Bacterial broth suspension was used as a positive control well, while the medium only was used as a negative control well. Planktonic cells were decanted, then the plates were washed twice with 0.9% NaCl and upturned to dry at room temperature for 1 h. Later, 150 µL of crystal violet solution  was added to the wells in order to dye the adherent bacteria for 15 min. Then, the excess stains in the wells were removed, and they were washed 3 times with 0.9% NaCl. The bound CV was then solubilized in 200 µL of ethanol–acetone (80:20 v/v). Finally, the absorbance of the stained adherent bacteria was measured at 595 nm using a microplate reader . The effect of mBTL-loaded CANPs on fication . An overP. aeruginosa pyocyanin production was investigated. The supernatant of the P. aeruginosa strains that were cultured overnight in the presence of mBTL-loaded CANPs was collected and mixed with chloroform. The chloroform layer containing pyocyanin was collected and extracted with 0.2 M HCL, vortexed, and centrifuged for 2 min at 8000 rpm. The absorbance of the pink HCl layer was quantified at 520 nm using a plate reader (BioTek).The effect of mBTL-loaded CANPs against As previously reported , AlamarBCell Viability (%) = (Fluorescence of cells treated with CNPs)/(Fluorescence of non-treated cells) × 1004 cells/well and incubated until they reached a confluency of about 85%. Then, the medium was replaced with serum-free medium for 2 h. After that, the medium was changed to medium containing 0.5 mg/mL of mBTL and mBTL-loaded CANPs, respectively. P. aeruginosa was added to the A549 cells at a multiplicity of infection (MOI) of 100 (bacteria per eukaryotic cell) and incubated for 2 h at 37 °C and 5% of CO2. After incubation, the cells were washed three times with PBS before being treated with 0.1% saponin for 5 min, followed by serial dilution and spread on LB plates and incubated overnight at 37 °C and 5% CO2. Then, the numbers of adhered bacteria (colony forming units) were counted the next day.The adhesion analysis was performed using A549 cells as described in a previous study with minor modifications . A549 cet-test and ANOVA tests were used when appropriate. A p-value of <0.05 is considered significant.The data are presented as the mean ± standard deviation (SD). Student’s P. aeruginosa WT and various QS phenotypic mutants were investigated at concentrations from 1 mg/mL to 0.125 mg/mL. The results of the screening antimicrobial effect determined by the MIC50 were obtained by the broth dilution method. The analysis of the bacterial growth showed a dose-dependent killing of the mBTL , the mBTL significantly reduced the biofilm formation (p < 0.05) of PA01, ΔlasR, ΔlasI, ΔIasA, and ΔrhIR by 79.16%, 67.69%, 58.53%, 76.16%, and 54.39%, respectively. The same concentration of mBTL loaded in CANPs was able to decrease the biofilm formation of PA01, ΔlasR, ΔlasI, ΔIasA, and ΔrhIR by 69.92%, 76.79%, 60.84%, 61.56%, and 89.22%, respectively. Conversely to mBTL alone, loaded CANPs reduced the biofilm formation of a double mutant (ΔlasR/ΔrhIR) by 44.92%. Remarkably, to overcome the effect of mBTL on cell viability and detect the effect only on biofilm, lower concentrations (0.125 and 0.25 mg/mL) were also studied, showing also significant reductions (p < 0.05) in the biofilm formation  inhibited the pyocyanin production of PA01 by 70.48%. To a lesser extent, mBTL decreased pyocyanin levels by 54.14%, 60.84%, 41.04%, and 45.77% in ΔlasR, ΔlasI, ΔIasA, and ΔrhIR respectively (50. The results show consistent and significant (p < 0.05) inhibition of pyocyanin production for only the ΔlasR strain compared to the control cells.In this study, the ectively . The mBTThe results of the Alamar Blue assay revealed that exposing A549 to concentrations of mBTL of up to 0.0156 mg/mL for 24 and 48 h had significant cytotoxic effects . In LL 2P. aeruginosa wild-type and different QS mutants to the surface of A549 lung epithelial cells was evaluated using the plate count method , lasI (PA1432), IasA (PA1871), rhIR (PA3477), and a lasR and rhIR double mutant (DA6) were used. The growth of the strains challenged with increasing concentrations of mBTL was measured, and the results show dose-dependent inhibition of bacterial viability. This corroborates the findings described by O’Loughlina, C.T. et al. [50 from ˃1 mg/mL to 0.5 mg/mL for wild-type isolate, lasR, lasI, and lasA. Of note, the loaded NPs increased the sensitivity of the double mutant to mBTL at a higher concentration of 1 mg/mL. This might be due to the enhanced uptake of mBTL when loaded in CANPs which require further future studies to understand the exact mechanism.Based on a previous successful formulation of mBTL-loaded CANPs in our lab , these N. et al. , in whic. et al. ,27. InteP. aeruginosa strains. In agreement with a previous study [50 concentration reduced the biofilm formation of a rhIR null mutant, which was unexpected. Thus, the biofilm inhibition seen by Bassler and colleagues could be due to mBTL inducing rhamnolipids, as suggested by other researchers [50 concentration). Surprisingly, the effect of mBTL-loaded NPs was stronger on ΔrhIR than mBTL alone  and obtained complete inhibition at a sub-MIC50 concentration. Contrary to mBTL alone, which showed no inhibition, loaded CANPs inhibited biofilm development by 44.92% in the double mutant (lasR/rhIR) at MIC50 and obtained a more significant reduction at a sub-MIC50 concentration. Such an enhancement of the mBTL-loaded CANPs in the biofilm formation of ΔrhIR and the double mutant (lasR/rhIR) could have been caused by alginate NPs, as alginates were previously reported to disrupt biofilm formation and maturation [P. aeruginosa biofilm formation, and therapeutic targeting alginate is a potential antibiofilm, as described previously [To support the findings that mBTL acts as a QSI, QS virulence-dependent phenotypes as biofilm and pyocyanin were examined using different us study , mBTL reus study . This fuus study , mBTL atearchers . Remarkaturation . Howevereviously . Howevereviously . These Geviously .P. aeruginosa PA14 in response to mBTL with an MIC50 of 8 µM. However, the effect of mBTL on the pyocyanin production of PA01 observed in our study was at a lower concentration than the one reported by others [50 concentration. The increased inhibition of pyocyanin of the ΔlasR/ΔrhIR double mutant in response to the mBTL-loaded CANPs when compared to mBTL alone (72.59% versus 5.52%) suggests that mBTL-NPs attenuated the pyocyanin production by a mechanism other than the lasR/rhIR gene. Interestingly, this pattern was similar to that of biofilm formation, as described earlier. Conversely, the mBTL-loaded CANPs at an MIC50 concentration exhibited no effect on the pyocyanin levels of the ΔIasA and ΔrhIR mutants. This was expected for the rhIR mutant, given the role of this gene, as mBTL functions in vivo by inhibiting quorum sensing via rhlR, as observed by Bassler and co-workers [50 and sub-MIC50 concentrations, which seem to have been mediated mostly through rhIR. The mBTL-loaded CANPs did not affect the pyocyanin production of ΔIasA, while mBTL alone did; this may have been caused by the masking effect of NPs. The level of pyocyanin production observed in our study was high in wild-type and limited in other mutants, as expected and as reported previously by Bassler and co-workers . In agrey others ,28. Grat-workers . It was -workers . This exAs shown in this study, the mBTL-loaded CANPs exhibited higher antimicrobial and comparable anti-virulence activities against the tested isolates when compared to mBTL alone. These outcomes are in line with an earlier study where modified alginate NPs loaded with ciprofloxacin and QSI 3-amino-7-chloro-2-nonylquinazolin-4 (3H) -one (ACNQ) cleared the infection in a 3D ex vivo skin model . AnotherP. aeruginosa strains, as observed earlier. In agreement with our study, the formulation of ISMM in sodium alginate/gum acacia NPs found to reduce the cytotoxicity and hemolysis associated with the trypanocidal drug alone [In the present study, the cytotoxicity of mBTL and mBTL-loaded CANPs was evaluated in A549 and LL 29 cells. The mBTL alone was found to be toxic to A549 and LL 29 even at lower concentrations of up to 0.0156 and 0.0312 mg/mL, respectively. Interestingly, the mBTL-loaded CANPs did not inhibit the cell viability of A549 and LL 29 cells up to a concentration of 0.5 mg/mL. Similar to our findings, a previous study showed that empty sodium alginate/gum Arabic NPs had no inhibitory effect on different cancer cell proliferation, including A549, at different concentrations reaching 100 µg/mL . These rug alone . The cytP. aeruginosa QS system [Pseudomonas aeruginosa to epithelial cells. The data of our results show that mBTL-loaded CANPs reduced P. aeruginosa adhesion to A549 cells, suggesting their potential in the treatment and prophylaxis of bacterial infection.To investigate the potential anti-adhesive action of mBTL and mBTL-loaded CANPs, the adhesion ability of bacterial isolates in the presence of mBTL and mBTL-loaded NPs to human lung A549 cells was studied in the present study. As presented in the adhesion assays results, the mBTL alone reduced the adhesion of wild-type PA01, lasR, lasI, rhIR, and lasA in a dose-dependent pattern and inhibited the adhesion of double mutants to A549 cells. Upon the loading of mBTL into CANPs, the number of bacteria adherent to A549 cells was reduced in a pattern comparable with that of mBTL. Of note, the mBTL inhibited the adhesion of the ΔlasR ΔrhlR double mutant to A549. This mutant was reported to be less virulent owing to its lack of LasR and RhlR genes, the two principal transcriptional regulators of the S system . In thisP. aeruginosa infection is recommended. Considering the promising observed improved in vitro anti-pneumococcal activity of mBTL loaded into CANPs, further in vivo research would provide a higher level of clinical relevance. Further investigation of the formulation on an animal infection model to support the potential use of mBTL-loaded CANPs for the prophylaxis and treatment of P. aeruginosa, requires a new strategy, such as the use of non-antibiotics such as QSI, to combat the infection without inducing resistance. In this study, the prepared mBTL-CANPs showed higher antibacterial activity when compared with mBTL alone against tested bacterial isolates. The findings also show that mBTL-CANPs exhibited anti-virulence activity, including the reduction of biofilm formation and production of pyocyanin against wild-type P. aeruginosa and QS-deficient mutants. The formulated mBTL-CANPs at MIC50 were cytocompatible in the A549 and LL 29 cell lines and decreased the cytotoxic effects shown with mBTL alone on these cells. In a pattern comparable with mBTL alone, the NPs synthesized reduced the P. aeruginosa strains’ adhesion to A549, implying their potential anti-adhesive effect. Considering the mBTL-CANPs’ enhanced anti-pseudomonal effect, cytocompatibility, and anti-virulence suppressing action, as well as the advantage of alginate-based NPs as an antimicrobial delivery system, the formulated nanoparticles have significant potential for the prophylaxis and treatment of P. aeruginosa infection.The development of antimicrobial resistance among pathogenic bacteria, including


Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) represent an unmet clinical need whose prognosis is still dismal. Alterations of immune response play a prominent role in AML/MDS pathogenesis, revealing novel options for immunotherapy. Among immune system regulators, CD47, immune checkpoints, and toll-like receptor 2 (TLR2) are major targets. Magrolimab antagonizes CD47, which is overexpressed by AML and MDS cells, thus inducing macrophage phagocytosis with clinical activity in AML/MDS. Sabatolimab, an inhibitor of T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3), which disrupts its binding to galectin-9, has shown promising results in AML/MDS, enhancing the effector functions of lymphocytes and triggering tumor cell death. Several other surface molecules, namely CD33, CD123, CD45, and CD70, can be targeted with monoclonal antibodies (mAbs) that exert different mechanisms of action and include naked and conjugated antibodies, bispecific T-cell engagers, trispecific killer engagers, and fusion proteins linked to toxins. These novel mAbs are currently under investigation for use as monotherapy or in combination with hypomethylating agents, BCL2 inhibitors, and chemotherapy in various clinical trials at different phases of development. Here, we review the main molecular targets and modes of action of novel mAb-based immunotherapies, which can represent the future of AML and higher risk MDS treatment. Over the past decade, the advances in targeted and large-scale next-generation sequencing (NGS) have helped to elucidate the dynamic genomic landscape in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), allowing for a refinement of prognostic stratification and targeted treatment ,2,3. HowThe dysregulation of the immune system may impact on the pathogenesis of AML and MDS by altering the fine balance between smoldering inflammation, adaptive immunity, and somatic mutations in promoting or suppressing the malignant clone . The bonImmune system regulators are emerging as major targets for immunotherapy in hematological malignancies. Immune system regulators that have been tested for AML and MDS therapy include CD47, immune checkpoints, and TLR2 . + cells, such as erythrocytes and platelets, may be less protected against phagocytosis, leading to hemagglutination, acute anemia, and thrombocytopenia .,46.45,46ytopenia . Immune checkpoints are regulators of key processes in the immune system as they modulate the signaling pathways responsible for immunological tolerance by preventing the immune-mediated destruction of cells . Thus, tCTLA-4, also known as CD152, is a co-receptor of the T-cell receptor (TCR) belonging to the immunoglobulin (Ig) superfamily. It is expressed on CD4+ and CD8+ T lymphocytes, and is responsible for inhibitory immune regulation . The CTLPD-1, also known as CD279, is a co-inhibitory molecule belonging to the Ig superfamily, which is expressed on activated T cells, B cells, and myeloid cells. Engagement of PD-1 with its ligand PD-L1 (or CD274), expressed on the surface of tumor cells and MDSCs, leads to the attenuation of TCR-mediated signaling ,55. ThisTIM3 is a co-inhibitory receptor expressed on CD4+ T helper 1 and CD8+ T cytotoxic cells that acts as a negative regulator of these lymphocyte populations through the interaction with its ligand galectin-9, triggering cell death ,57,58. TTLR2, also known as CD282, is a member of the Toll-like receptor family and is expressed on the surface of various cells, including HSCs and hematopoietic progenitor cells (HSPCs), and plays a fundamental role in pathogen recognition and in the activation of innate immunity . OverexpOther surface molecules of AML and MDS cells that are currently being explored as therapeutic targets with mAbs include CD33, CD123, CD45, and CD70.CD33 is a sialic acid-binding Ig-like lectin (Siglec) expressed as a transmembrane protein on the surface of malignant AML blasts and MDSCs of MDS, but not on HSCs. These features render CD33 an ideal target for immunotherapy by different modalities . The binCD123 represents the alpha-chain of the IL-3 receptor (IL-3Rα) expressed on myeloid pluripotent progenitor cells . Its intProtein tyrosine phosphatase receptor type C, also known as CD45, is a transmembrane protein present in various isoforms on almost all differentiated hematopoietic cells . CD45 isAlthough CD70 is mainly a lymphoid lineage marker, it is also expressed on myeloid leukemic blasts, with an absent or low-level expression in normal BM cells . The intSeveral clinical trials with mAbs (naked and conjugated) are currently ongoing in the frontline, relapsed/refractory (R/R), post allogeneic HSCT, and minimal residual disease (MRD)/maintenance setting, with the aim of improving the outcomes of AML patients. These mAbs may target immune regulatory molecules (CD47 and immune checkpoints) and other membrane antigens  .TP53 mutant patients achieved an objective response, suggesting the efficacy of magrolimab plus AZA in poor prognosis and refractory patients [Targeting CD47 in AML is currently being explored using mAbs or fusion proteins. The rationale for using magrolimab (Hu5F9-G4), a humanized anti-CD47 IgG4, stems from the overexpression of CD47 on AML cells and its association with an adverse prognosis ,73,74,75patients . In contpatients . These rpatients . This mopatients . Several immune checkpoint inhibitors are currently under investigation in AML, alone or in combination with standard therapies. Data from phase I studies suggest a limited efficacy of these mAbs when used as monotherapy and a potential synergistic effect when combined with HMAs . A phase213Bi) or actinium-225 (225Ac) [213Bi-lintuzumab and 225Ac-lintuzumab may have an antileukemic effect, being able to induce remissions after low-dose cytarabine cytoreduction in untreated AML patients [225Ac-lintuzumab monotherapy may induce remissions in 69% of AML receiving two fractions of 74 kBq/kg, and in 22% of patients receiving two 55.5-kBq/kg fractions [In 2000, the FDA approved gemtuzumab ozogamicin (GO), an immunoconjugate drug targeting CD33, for elderly (≥60 years) CD33+ relapsed AML unfit for chemotherapy ,78. In 2 (225Ac) . Initialpatients . A recenractions . Finallyractions . Phase Iractions ,83. Challenging CD123 with bispecific mAbs is currently under investigation. APVO436, a CD123XCD3 BiTE, was evaluated in a phase Ib study (NCT03647800) that demonstrated adequate safety in R/R AML patients . Vibecot131I, was studied in combination with a reduced-intensity conditioning (RIC) regimen of fludarabine (FLU) plus total body irradiation (TBI) in R/R AML patients over 50 years in a phase I clinical trial (NCT00008177) in order to estimate the maximum tolerated dose [90Y-BC8, an anti-CD45 monoclonal antibody conjugated with 90Y, was proven to be well tolerated in a phase I trial (NCT01300572) in combination with FLU/TBI in R/R AML ineligible for myeloablative HSCT. The trial showed an OS at 1.8 years of 53%, which prompts additional clinical trials using radioimmunotherapy as part of the conditioning regimen for allogeneic HSCT [131I, 90Y does not require isolation of the patient, providing a potential advantage in the management and quality of life for AML patients.Iomab-B, an anti-CD45 antibody conjugated to ted dose . Among tted dose . 90Y-BC8eic HSCT . In contBased on preclinical results, a phase I/II trial (NCT03030612) evaluated a single dose of cusatuzumab ARGX-110), an anti-CD70 mAb, monotherapy followed by AZA in untreated AML older patients . AZA ind, an antiVarious clinical trials with immune-based therapies are currently ongoing in previously untreated, R/R, HR, and lower risk (LR) MDS, aiming to fulfil the unmet clinical needs of the disease. Here, we focus on therapeutic strategies targeting CD47, immune checkpoints, TLR2, CD123, and CD33 . TP53 mutation achieved an objective response [Magrolimab is one of the most innovative drugs for MDS treatment . An ongoresponse . These eTP53 mutation) and the five R/R subjects evaluable for response documented three mCR and two cytogenetic responses. Two out of four patients who were transfusion dependent became transfusion independent [A novel treatment approach involves Evorpacept, studied in the phase I of the ASPEN-02 multicentric phase I/II trial (NCT04417517), evaluating the safety and tolerability of its association with AZA in patients with untreated or R/R HR MDS . The iniependent . These rependent .Although studies involving ipilimumab monotherapy have demonstrated a limited efficacy, more encouraging results are emerging from an ongoing phase II study (NCT02530463) analyzing treatment with ipilimumab and/or nivolumab with or without AZA in MDS ,87. The TP53 mutations on both alleles, who remained in SD and was transfusion independent at 34 months of treatment [TP53-mutated HSCs of MDS patients [Additional positive results have been obtained in an ongoing phase II study (NCT03094637) evaluating the safety and efficacy of pembrolizumab, a humanized mAb targeting PD-1, combined with AZA in intermediate-1 or HR MDS . For thereatment . One pospatients . Howeverpatients . These rTP53 mutation, had a better response than the average response observed in the whole population: the ORR was 71.4% and mDOR 12.6 months [TIM-3 is a recently investigated immune checkpoint, which is being studied together with its pathway inhibitor sabatolimab . An ongo6 months . Followi6 months ,89.Early results of two phase I/II trials (NCT02363491 and NCT03337451) with tomaralimab (OPN-305), a fully humanized IgG4 monoclonal antibody against TLR2, suggest its safety and efficacy profile in HMA-failure and transfusion-dependent LR MDS patients ,42,62,64Recent studies have analyzed anti-CD33 BiTEs and trispecific killer engagers (TriKEs) in MDS. AMV564, a CD33XCD3 BiTE, was evaluated in a preclinical study, which showed its in vitro ability to reduce the MDSC count and to increase the anti-PD1 antibody activity . GTB-355TP53 mutated [Although the monospecific anti-CD123 mAb talacotuzumab demonstrated a poor efficacy with an important toxicity profile in MDS, recent clinical trials assessing CD123 target therapy are showing positive indications for safety and efficacy, despite being limited by low patient numbers . Prelimi mutated . ®) targeting CD123 on MDS cells and CD16a on NK cells, with a higher stability compared with conventional Fc-optimized IgG1 antibodies [Finally, two preclinical studies provide the rationale for potential novel strategies ,93. The tibodies . The secCurrently, only a minority of AML patients become long-term survivors using the standard treatments that are approved. The identification of several genetic targets has allowed for the design of small molecule inhibitors, best exemplified by FLT3 inhibitors, that have improved the outcome in patients carrying these molecular predictors ,95. HoweIn the context of MDS, HMA represents the only approved treatment strategy for HR patients. However, a response is limited to a fraction of cases and survival is still inadequate. In addition, the effect of HMA is treatment dependent and patients who discontinue treatment eventually lose their response, progress, and die. The availability of innovative immunotherapy therapies will allow for designing combinations that may possibly target MDS stem cells, thus increasing the response and prolonging survival.The integration of several immunotherapy strategies in AML and MDS treatment still requires large randomized clinical trials to assess the true benefit and safety of these novel medicines. In addition, the precise positioning and sequencing of the different mAbs that are under investigation for AML and MDS needs to be defined. Finally, and perhaps most importantly, in view of a precision medicine approach for AML and MDS immunotherapy, the studies that are ongoing should also aim at the identification of molecular predictors of treatment response to a given mAb. This, in turn, will allow for a biologically rational choice of a specific immunotherapy strategy for the individual patient with AML or MDS.


Plasmodium falciparum strains. Calxinin treatment resulted in diminished maturation of parasite sexual precursor cells (gametocytes) accompanied by distorted parasite morphology. Further, in vitro liver-stage testing with a mouse model showed reduced parasite load at an IC50 of 79 nM. A single dose (10 mg/kg) of Calxinin resulted in a 30% reduction in parasitemia in mice infected with a chloroquine-resistant strain of the rodent parasite P. berghei. The ex vivo ookinete inhibitory concentration within mosquito gut IC50 was 150 nM. Cellular in vitro toxicity assays in the primary and immortalized human cell lines did not show cytotoxicity. A computational protein target identification pipeline identified a putative P. falciparum membrane protein (Pf3D7_1313500) involved in parasite calcium (Ca2+) homeostasis as a potential Calxinin target. This highly conserved protein is related to the family of transient receptor potential cation channels (TRP-ML). Target validation experiments showed that exposure of parasitized RBCs (pRBCs) to Calxinin induces a rapid release of intracellular Ca2+ from pRBCs; leaving de-calcinated parasites trapped in RBCs. Overall, we demonstrated that Calxinin is a promising antimalarial lead compound with a novel mechanism of action and with potential therapeutic, prophylactic, and transmission-blocking properties against parasites resistant to current antimalarials.Malaria elimination urgently needs novel antimalarial therapies that transcend resistance, toxicity, and high costs. Our multicentric international collaborative team focuses on developing multistage antimalarials that exhibit novel mechanisms of action. Here, we describe the design, synthesis, and evaluation of a novel multistage antimalarial compound, ‘Calxinin’. A compound that consists of hydroxyethylamine (HEA) and trifluoromethyl-benzyl-piperazine. Calxinin exhibits potent inhibitory activity in the nanomolar range against the asexual blood stages of drug-sensitive (3D7), multidrug-resistant (Dd2), artemisinin-resistant (IPC4912), and fresh Kenyan field isolated  Plasmodium parasites 25.8589 = -30.25 [1% (1g/100 mL) in chloroform]. 1H NMR : δ 7.55 , 7.43 , 7.28 , 7.20 , 7.19 , 3.60 , 3.53 , 2.92—2.83 , 2.66 , 2.64—2.56 , 2.45 , 2.40—2.33 . 13C NMR : δ 142.55, 139.20, 129.59, 129.38, 129.27, 128.61, 126.41, 125.69, 125.26, 125.23, 122.99, 77.48, 77.17, 76.85, 68.80, 62.45, 61.20, 55.41, 53.27, 41.47. 19F NMR : δ–62.5 . ESI (HR-MS) m/z: (M+) calcd for C22H28F3N3O 408.2218; found 408.2267.Yield: 86%. MP. 93-95Pf strains such as 3D7 (drug-sensitive) , D6 MRA-285 (chloroquine (CQ) sensitive, BEI Resources), Dd2 , and IPC4912  were acquired from the Malaria Research and Reference Reagent Resource Center (MR4) , new BEI resources  and plotted against the counts  and the data were analyzed by nonlinear regression (sigmoid dose-response/variable slope equation) to yield the mean 50% growth inhibitory concentration (IC50).An SYBRarasites ,23. Briearasites ,20. DrugPf parasites used in this study were obtained using a protocol entitled, “Epidemiology of malaria and drug sensitivity patterns in Kenya” under ethical approval of Walter Reed Army Institute of Research Institutional Review Board (study protocol number WRAIR 2454) and KEMRI Scientific and Ethics Review Unit (study protocol number KEMRI 3628). Patients aged six months and above presenting at outpatient departments with symptoms of malaria and/or testing positive for uncomplicated malaria by rapid diagnostic test , Zephyr Biomedicals, Verna Goa, India) were recruited into the study after providing written informed consent or assent. The clinician then performed the diagnosis by assessing symptoms such as conjunctival pallor, lymphadenopathy, and splenomegaly. The final diagnosis results were based on clinical evaluation confirmed by mRDT and/or microscopy. All Pf-positive cases were treated with artemether–lumefantrine (AL) Coartem® as per the Kenya Ministry of Health’s recommended case management guidelines for uncomplicated malaria. The dose of AL was based on weight and the initial dose administration was directly observed and the remaining doses were dispensed to the participant to take at home. Each patient was given the remainder of the full dose of Coartem® and advised to take the next dose after eight hours with a follow-up with the remaining doses at 12 hourly intervals until dose completion. The participants were encouraged to return to the hospital should symptoms persist.This study was conducted under the approval of the Kenya Medical Research Institute (KEMRI), Scientific and Ethics Review Unit (SERU), and Walter Reed Army Institute of Research (WRAIR) Institutional Review Boards, protocol numbers WRAIR#2257, KEMRI/SERU/CCR/0018/3126 entitled “Assessment of in vitro antiplasmodial activities of compound libraries for malaria drug discovery and development”. Written informed consent was obtained from a parent/legal guardian, and assent was obtained from individuals aged 13–17 years following the Kenyan law. The fresh field Pf isolates obtained from natural infections were tested within 6 h, post-phlebotomy, without culture adaptation (immediate ex vivo) for susceptibility to Calxinin and reference antimalarial drugs. Blood samples with greater than 1.0% parasitemia were adjusted to 1.0% parasitemia at 2% hematocrit. Those samples, at less than 1.0% parasitemia, were tested unadjusted at 2% hematocrit. Culture media for the Pf field isolates testing was prepared as described Akala et al. 2011 —[Mean OD of control cells]/[Mean OD of control cells] × 100. Viability counts were then plotted against corresponding drug concentrations to yield cytotoxicity  were transfected with 10 μg of α1H plasmid using Lipofectamine , according to the manufacturer’s protocol , against cell viability counts (ODs) to yield the CC50. An SI value  ≥ 2.0 (~20-fold) was used as the cut-off for low cytotoxicity to PBMCs. After Calxinin established a low cytotoxicity, according to the aforementioned criteria, it was advanced to high-throughput screening with the IC50 cut-off value set at <2 µM [All the in vitro screening of infected cells was performed in triplicate . Parasitescribed ,26,27. Cat <2 µM ,63. CompPf infected RBCs (iRBC) were assessed by using the spots function of Imaris image analysis software . Fluo-4 spots were semi-automatically detected and classified as positive or negative using an XY diameter equal to 4 µm and with background correction. The fluorescence intensity of the Flou-4+ spots was measured as the intensity of the voxel at the center of the spot. All images were analyzed using identical image analysis parameters. Data were exported to Prism software (GraphPad) for statistical analysis. Differences between the control and Calxinin-treated cells were assessed using unpaired t-tests. The number of Fluo-4+ spots over time was analyzed via nonlinear regression. An α level of 0.05 was used for statistical tests.Images were acquired with Zen software (Carl Zeiss Microscopy) and analyzed offline with Imaris Software . The number and fluorescence intensity of Fluo-4 positive (Fluo-4+) t-test, with p-values < 0.05 considered statistically significant.All experiments were performed in triplicate or more as stated. Statistical analyses were performed using GraphPad Prism . For each experiment, means ±standard deviations (SD) were calculated. Statistically significant differences were identified using Student’s 1H, 13C, and 19F) spectroscopic techniques and high-resolution mass spectrometry (HR-MS) (Data not shown). To the best of our knowledge, this is the first report of the combination of a ((trifluoromethyl)benzyl)piperazine with HEA. In continuation of our previous work on antimalarial agents, we functionalized benzyl piperazine with the trifluoromethyl group to study the effect of this substitution on the antimalarial activity of the compound. The use of organofluorinated molecules offers valuable avenues for the design of new potential drug candidates against malaria. The incorporation of fluorine substituents, particularly trifluoromethyl groups, into organic molecules has led to their high potency against many diseases, including malaria. The noteworthy characteristics of fluorine include high electron affinity, lipophilicity, and bioavailability, extending the half-life of the drugs ,64. ThesPf3D7 strain, using an SYBR Green assay, showed highly potent antiparasitic activity with a mean IC50 value of 88.0 (±1.1) nM. Calxinin activities against various Plasmodium strainsm and different developmental stages are summarized in The initial assessment of the antimalarial activity of Calxinin on asynchronous cultures of the chloroquine-sensitive Pf strain IPC 4912 from BEI Resources with an IC50 of 93.0 nM , which is higher than the IC50 for freshly collected field isolates.Following confirmation of Calxinin’s activities against lab strains in vitro, blood stages of the mouse, liver stages, and mosquito stages, we tested its efficacy against freshly collected field isolates . aptation . Rich wiaptation  and highaptation  The drugaptation ,69. The 20 years . For exaPf 3D7, 41.0 ± 3.1% inhibition) and morphologically distorted parasites when compared to the DMSO-treated control  as described in the Methods. This exposure resulted in a decreased maturation of stage III–V gametocytes  on day 2 post-infection significantly reduced the parasite load. Parasitemias of treated mice were 0.21% on day 3, 2.45% on day 7, and 12.3% on day 10. In comparison, the untreated control group exhibited a parasitemia of >19% by day 10  dissolved in 1% DMSO. Subsequently, the parasite load was quantified by qPCR in the control and Calxinin-treated cells using parasite-specific 18S rRNA primers. Approximately 60% and 88% reduction of the parasite load was observed in cells treated with 0.1 µM and 1 µM of Calxinin, respectively. IC50 values were determined to be 79 nM  in culture  were treated with incremental concentrations of Calxinin for 48 h. The recovery time following 10-µg/mL and 20-µg/mL exposure were 7 and 10 days, respectively. Parasites treated with 30 µg/mL were rendered nonviable and, hence, unrecoverable. Our experiment failed to select resistant isolates as the cultures frequently crashed at even low Calxinin concentrations, resulting in ‘sluggish’ growth rates. Parasite cultures that were subjected to multiple cycles of exposure showed no change in IC50 for Calxinin, dihydroartemisinin (DHA), or chloroquine (CQ). The Calxinin target in plasmodium may have a high functional cost for viable mutants selection under culture conditions. To identify the potential target(s) of Calxinin in Plasmodium, we opted for a computational approach by using an in silico pipeline to predict target(s) and elucidate the mechanism of action of Calxinin.Parasite cultures , the proteins that exhibited the most significant pore homology with T-type and N-type channels of humans were PF3D7_0212500 (32.03% pore homology) and 1313500 (19.28% pore homology). The amino acid residues of all constructed models were within the allowed regions of the Ramachandran plot; (>90%). The C- score of all models was < −3.00, the expected TM Score was <0.5, RMSD values were within 11–15, and z-scores were less than -5.00. The docking scores for Calxinin were highest within the PF3D7_1313500 pore region  simulations for 100 nanoseconds were performed for the 2R,3S enantiomer  top docking poses with the PF3D7_1313500 pore region B–D. Calx2+ concentration of Pf and/or the infected RBCs, either Calxinin  or an equivalent volume of DMSO was added to the culture media and the Pf-infected RBCs. The culture was imaged once per second for 2 min. Fluo-4 AM is a cell-permeant dye that is used to measure intracellular Ca2+ concentration . Given that the Calxinin-induced increase in intracellular Ca2+ concentration reached a plateau at ~60 s, subsequent live cell images were acquired 60 s after the addition of Calxinin. In these experiments, Calxinin treatment increased fluorescence in Pf -infected RBCs by greater than three-fold and decreased the variability in the intensity  treatment produced a significant decrease in intracellular Ca2+ concentration in parasites in the ring, early and late trophozoite, and schizont stages. In contrast, neither DMSO or Calxinin (500 nM) affected the intracellular Ca2+ concentration of uninfected RBCs , HEK293 cells, and liver (Huh 7.1 and HepG2) cells were exposed to various Calxinin concentrations. Exposure of HEK293, Huh7.1, and HepG2 cells to Calxinin did not result in any toxicity. Interestingly, we observed that Calxinin seemingly enhanced the growth of PBMC (% PBMC viability >100% relative to the vehicle control) at the same mean IC2+ channels  were subjected to various Calxinin concentrations, ranging from 200 nM to 1 µM . There was a 20% reduction in amplitude with Sipatrigine (positive control) at 10 µM (IC50~30 µM data not shown). Either Calxinin or DMSO (vehicular/negative) was added directly into the experimental chamber after seal formation and basal currents were recorded. h = −90 mV) and reached a peak at −50 mV, with a peak current density of –48.05 ± 2.23 pA/p.f. As shown, the addition of Calxinin at any concentration did not produce significant changes in the dependence of potential or in the maximum peak current density, which was −46.22 ± 3.89 pA/p.f for 1 µM Calxinin .Although the sequence homology data from the computational analysis indicated that Calxinin most likely would have no affinity for human T-type calcium channels, we performed experiments to determine whether Calxinin cross-reacts with human calcium channels. HEK-293 cells stably expressing voltage T-type CaThe impact of Calxinin on hemostatic parameters in human blood was assessed following supplementation to plasma and whole blood collected from healthy individuals. Clotting assays to assess the extrinsic pathway , intrinsic pathway , and common pathway  of blood coagulation were performed following supplementation with Calxinin at concentrations of up to 20 µg/mL. Our results revealed that clotting times in the presence of Calxinin were not different from vehicle-supplemented samples –C. In vi2+ from parasites in infected red blood cells. The compound was developed from the HEA and piperazine backbone [Combating drug resistance requires the continued development of new antimalarials that target multiple essential metabolic pathways and, ideally, several developmental stages of the parasite . Existinbackbone ,14,19,20backbone ,76. The backbone  and othebackbone ,78,79. Fbackbone ,80,81.Conventional antimalarial treatments are being developed primarily on their antiplasmodial activity against parasites in cell culture and often with an incomplete understanding of the molecular mechanism of action . RecentlPf isolates from different geographical regions of the world. In particular, C-terminal amino acid side chain residues that we predict to be critical for predicted Calxinin binding are 100% conserved. Orthologs in several pathogenic parasite genera show high conservation of the predicted Calxinin-binding residues, suggesting a fundamental significance in terms of function and possibly regulation in apicomplexan and kinetoplastid parasites. These in silico predictions were the basis of examining the calcium homeostasis in parasites. The results from live imaging and electron microscopy experiments strongly indicate calcium homeostasis in Pf is affected by Calxinin. Calxinin does not seem to have any direct effect on homologous proteins in the human host. This is expected, as none of the putative calcium channel/pump proteins shares significant homology with the host mammalian homologs Ca2+ storage in human cells differs from that in parasites . Yet, Ca2+ signaling and homeostasis are just as critical for the Pf life cycle. This includes key steps in host cell invasion, egress from host cells, and genomic regulation [2+ regulates apicoplast metabolism, which plays a key role in parasite growth [Our in silico analysis and MD simulations indicated an uncharacterized putative cation channel of the malaria parasite (TRP-ML) as a potential target for Calxinin. We have based our predictions on SAR reports of similar calcium channel blockers ,89. The gulation ,91. Furte growth . Specifie growth . The MD 2+ homeostasis of Plasmodium-infected RBCs by Calxinin. Increased intracellular calcium alters the cytoskeleton and causes cell injury by facilitating swelling and/or affecting membrane permeability [Pf. The ART-resistant profile did not affect the parasiticidal breakpoints of Calxinin. This could mitigate the evolution of drug resistance as calxinin acts against novel target proteins [Pf strains from malaria-endemic regions of Kenya. This demonstrates that the activity of the compound is not restricted to lab-optimized Plasmodium strains and would be useful in the field without further major optimization.Experimentally supporting our computational predictions are results that show the alteration of the Caeability . The swoproteins . CalxiniCalxinin is active against parasites in the malaria mouse model, which is often used as the first in vivo test of potential antimalarials but also to specifically investigate the malaria mosquito stages. Our data demonstrate that Calxinin also effectively blocks malaria transmission in the mosquito. These results would expand the use of Calxinin for chemoprophylaxis.50 required to kill the parasite is <90 nM, which is 650-fold below the concentration that would kill human cells. This confirms that Calxinin exhibits no significant toxicity in tested human cells. Additionally, no significant toxicity was detected in mice treated with high doses of Calxinin. Further testing of hematological parameters  showed that Calxinin had no effect on blood clot formation and did not alter the agonist-induced aggregation of human platelets. The PBMC proliferation induction by calxinin also needs to be explored further for the mechanism of action and implication on Malaria immune profiles and prognosis.An evaluation of the cytotoxicity of Calxinin was conducted in human primary cells, as well as in lab-established human cell lines. It is important to note that the IC2+ homeostasis in Pf parasites. Using complementary approaches, we show that pRBC treated with Calxinin exhibit a dramatic change in the internal Ca2+ concentration in parasites and, concomitantly, in the host RBCs but not in the uninfected RBCs. The intracellular Ca2+ concentration in pRBCs is higher than in uninfected RBCs [2+ concentrations in pRBCs. Non-infected RBCs did not show any change in Ca2+ concentration. We encountered two major variables for infected RBCs regarding Ca2+ concentrations: the developmental stage of the parasite and the age of the red blood cell, Each can cause varying basal Ca2+ concentrations in a given sample. When an infected red blood cell is exposed to Calxinin, it causes the rapid release of Ca2+ from the parasite into the host RBCs. This is only temporary as this excess Ca2+ is pumped out of the RBCs cytosol into the environment to reestablish the low intracellular Ca2+. Our photoconversion TEM data indicate that Calxinin causes a robust change in intracellular Ca2+ in Pf resulting in an irreversible change in calcium levels. This is consistent with the proposed mechanism of action of Calxinin. Interestingly, Ca2+ homeostasis perturbation is expected to negatively affect multiple cellular processes within parasites enhancing their vulnerability to other chemotherapeutics. Voltage-gated channel inhibitors are known to cause oxidative stress in target cells [Although an investigation of the precise molecular mechanisms of Calxinin’s target modulation in parasites was beyond the scope of the work presented here, our results suggest that Calxinin exerts its effect through dysregulation of Cated RBCs ,95,96. Eet cells . Antimalet cells . Therefo2+ in its cytoplasm. As stated above, this produces a subsequent release of Ca2+ from the other intracellular compartments, and this Ca2+ is recaptured by these compartments and/or is extruded. In the continuous presence of Calxinin, the recaptured Ca2+ is released again and those released cations are extruded again. This process produces the eventual emptying of the internal Ca2+ compartments and the translocation of the released Ca2+ to the external environment. This ultimately results in parasites with lower than normal Ca2+ concentrations near the parasite cellular peripheral zone, where acidocalcisomes are pushed by larger organelles [2+ to show a robust Calxinin-induced increase in intracellular Ca2+ levels. The most robust Calxinin-induced increase in intracellular Ca2+ was observed in mid-and late-stage trophozoites infecting comparatively younger RBCs. Notably, the maximum Ca2+ loss was in merozoites/schizonts, which was not documented in live imaging. We interpret this finding as evidence that the Ca2+ surge originates from the parasite’s intracellular compartments The data reported herein, coupled with the computationally predicted target allows us to hypothesize that Calxinin directly affects a putative Ca2+ permeable channel, possibly the putative TRP-ML channel of Plasmodium. In parasitized pRBCs, Calxinin produces an increase in parasite cytoplasmic Ca2+ levels. This spike is masked by the high baseline level of intracellular Ca2+ in the parasites. In infected RBCs that are not near the maximum level of intracellular Ca2+, Calxinin produced a more robust transient increase in RBC intracellular Ca2+ which decreases gradually due to Ca2+ efflux into the host cell  channel blockers, it is highly selective for the parasite and does not show cross-reactivity with human T-type channels. The results from live imaging support the nanomolar efficacy of the compound. We hypothesize that the RBC plasma membrane rapid Ca2+ efflux acts as a one-way valve, as the parasite cannot recover lost Ca2+ quickly due to a low Ca2+ concentration within the RBCs. Thus, the nanomolar concentration of Calxinin can rapidly and irreversibly deprive the parasite of critical intracellular Ca2+. The drug-target relationship may be further exploited to develop highly potent antimalarial agents.Here, we report on a novel antimalarial agent ‘Calxinin’ with a simple structure and desirable chemical properties. Calxinin was effective at nanomolar concentrations against all stages of the malaria life cycle. It was effective against common drug-sensitive and drug-resistant laboratory strains, as well as against Overall, Calxinin is a novel and target-based anti-malarial agent and the data presented herein suggest that it is a promising lead antimalarial compound.


We investigated the mechanisms involved in the ([Ca2+]i) increase in N18TG2 neuroblastoma cells, which endogenously express both receptors. Changes in [Ca2+]i were measured in cells exposed to 0.25 or 2.5 mM eCa2+ by a ratiometric method (Fura-2 fluorescence) and expressed as the difference between baseline and peak responses (ΔF340/380). The increased ([Ca2+]i) in cells exposed to 2.5 mM eCa2+ was blocked by the CaSR antagonist, NPS2143, this inhibition was abrogated upon stimulation with WIN55212-2. WIN55212-2 increased [Ca2+]i at 0.25 and 2.5 mM eCa2+ by 700% and 350%, respectively, but this increase was not replicated by CP55940 or methyl-anandamide. The store-operated calcium entry (SOCE) blocker, MRS1845, attenuated the WIN55212-2-stimulated increase in [Ca2+]i at both levels of eCa2+. Simultaneous perfusion with the CB1 antagonist, SR141716 or NPS2143 decreased the response to WIN55212-2 at 0.25 mM but not 2.5 mM eCa2+. Co-perfusion with the non-CB1/CB2 antagonist O-1918 attenuated the WIN55212-2-stimulated [Ca2+]i increase at both eCa2+ levels. These results are consistent with WIN55212-2-mediated intracellular Ca2+ mobilization from store-operated calcium channel-filled sources that could occur via either the CB1R or an O-1918-sensitive non-CB1R in coordination with the CaSR. Intracellular pathway crosstalk or signaling protein complexes may explain the observed effects.The CB Although initial studies could not detect Ca2+ mobilization in cultured cell models, evidence indicates that the effects of cannabinoid receptors on [Ca2+]i depend on the agonist and the cell type tested. In CHO cells, the synthetic cannabinoid HU210 and its non-CB1-binding isomer HU211 (10 μM) both induced only a non-receptor-mediated increase in [Ca2+]i in untransfected, CB1R-expressing, or CB2R-expressing cells, using a Fura-2 method that readily detected muscarinic receptor-mediated Ca2+ mobilization i in HEK293 cells exogenously expressing CB1R [2+ mobilization occurred in a CB1-dependent manner requiring Gαq activation and release of Ca2+ from thapsigargin-sensitive endoplasmic reticulum (ER) stores [2+]i in neurons and other cell types in response to lipid mediators, including atypical cannabinoids i i in a response dependent on Gαi/o and modulated by Gαq [2+]i also seems dependent on PKC activity and localization i . Thus, ofunction . The CaSfunction . CaSR activation . In N18Td by Gαq . Modulatlization . It appe via Gαi .2+ mobilization as observed by Lauckner and colleagues [2+] influence associated with the Sugiura procedure [2+]i and the role of CaSR activation on those responses. [Ca2+]i regulation has relevant physiological significance, for example, in muscle  (eCa2+) and the responses in intracellular [Ca2+] ([Ca2+]i) were measured.Mouse N18TG2 neuroblastoma cells were cultured as described , maintai340 and F380 were measured for one min. KHB containing 0.25 mM Ca2+ (Low eCa2+) was passed through the imaging chamber for 5 min after which eCa2+ was changed to 2.5 mM (High eCa2+) using a perfusion valve control system  and cells perfused for additional 10 min. This procedure was repeated in the presence of cannabinoid receptor agonists  in KHB containing low or high eCa2+. Cells were pre-incubated for 15 min for the treatments with antagonists, and the corresponding antagonists were added to the low and high eCa2+ solutions. Only one treatment was carried out on each coverslip used.Coverslips were transferred to an imagining chamber on an inverted Olympus BBX51WI microscope equipped with a 40× objective, a xenon arc lamp , and a manual stage, and a cooled charge-couple device (CCD) camera (Hamamatsu Orka II). For ratiometric imaging, the microscope was computer-controlled by HCImage software . Cells on the field were manually marked for analysis, and F2+ to a working concentration of 5 μM as described i responses were determined using the ratiometric method (ratio between F340 and F380) i transiently by 11%. When eCa2+ was changed to 2.5 mM, [Ca2+]i increased by 304% over basal  and by 350% over basal in 2.5 mM eCa2+   . These findings might suggest that the WIN55212-2 can influence [Ca2+]i under “basal” CaSR conditions, but the WIN55212-2 stimulus was not influenced by NPS2143-inhibited CaSR. An alternative interpretation is that WIN55212-2 provided a mechanism to protect the CaSR from inhibition by the negative allosteric modulator.NPS2143 is a CaSR negative allosteric modulator that binds to the 7-transmembrane domain of the CaSR to inhibit the Ca pathway . We usedreceptor . High eC2+]i in neuronal cells, the non-classical cannabinoid full agonist, CP55940, and endocannabinoid partial agonist, methanandamide (Me-AEA) were used at concentrations previously shown to inhibit cAMP accumulation [2+]i relative to basal values at either 0.25 mM  or at 2.5 mM eCa2+ . These findings support the WIN55212-2 selectivity, implicating a non-CB1 and non-CB2 mechanism for this response.To check the selectivity of WIN55212-2-induced increase in  sensor stromal interacting molecule (STIM) and its association and activation of Ca2+ release-activated Ca2+ channels (CRAC) comprised of Orai1, Orai2, and Orai3 proteins. Cells also utilize non-selective cation channels as store-operated channels (SOCs), comprised of both Orai1 and transient receptor potential canonical channel 1 (TRPC1) channel subunits. Current reviews describe these processes in detail i, we employed the Orai1 inhibitor N-propargyl-nitrendipine (MRS1845), which has a reported IC50 = 1.7 μM to block capacitative Ca2+ influx in HL60 cells [2+ replacement via TRPC1 at higher concentrations [2+]i, in both eCa2+ conditions (0.25 mM or 2.5 mM Ca2+) were attenuated by incubation with MRS1845 (10 μM) . These results are consistent with a requirement for continuous refilling of the intracellular Ca2+ stores in the ER as the source of the mobilized Ca2+.To evaluate the role of SOCE in WIN55212-2-stimulated increases in i in 0.25 mM eCa2+ was partially blocked by simultaneous perfusion with the CB1R antagonist SR141716 (1 μM) . Under conditions of 2.5 mM eCa2+, SR141716 does not affect the WIN55212-2-dependent increase in [Ca2+]i . Simultaneous perfusion with the nonCB1/CB2 receptor antagonist O-1918 (10 μM), attenuated WIN55212-2-promoted increases in [Ca2+]i at both eCa2+ levels  activation, synthesis of diacylglycerol (DAG), and inositol triphosphate (IP3) with further activation of ER IP3 receptors promoting Ca2+ release i after GPCR activation i, it is conceivable that WIN55212–2-dependent CB1R activation increases [Ca2+]i by augmenting CaSR-Gαq-dependent activation of PLC with the participation of CB1R-mediated Gi/o βγ release in neuroblastoma cells.Our results of an eCanstrated . The eff release . PLC canc domain . Gαq andtivation . Recentlproposed . Regardi2+ mobilization. The other is that we observed a role for CaSR in WIN55212-2/CB1R-dependent increases in [Ca2+]i at low eCa2+ and not at high eCa2+. These results suggest the possibility of different Gαq-PLC-Gi/o βγ complexes being formed depending on eCa2+ and the cellular proximity of the receptors and the G proteins to which they are pre-coupled , and the lysophospholipid, lysophosphatidylinositol (LPI). GPR55 utilizes Gαq or Gα12/13 for signal transduction [2+ mobilization and mitogen-activated protein kinase (MAPK) phosphorylation [2+]i i through a mechanism involving Gαq, PLC, and IP3 receptors i . Activat2+]i by GPR55 WIN55212-2 binding sites in neuroblastoma-glioma hybrid NG108-15 cells [1 knock-out mouse which showed that anandamide and WIN55212-2 could promote G protein activation ([35S]GTPγS binding) i in N18TG2 cells via CB1R-dependent and independent mechanisms. We observed functional interactions between the CB1R and CaSR activation in regulating [Ca2+]i, and these interactions are dependent on eCa2+ with the participation of nonCB1/CB2 receptors in neuroblastoma cells. Future studies should address the effects of LPI, abn-CBD, CBD and RvD2 to determine the role of GPR55 and GPR18 on Ca2+ mobilization influenced by the CaSR. Additionally, studies should address whether aminoalkylindole analogs that act on the putative WIN55212-2 Alkyl Indole “receptor” are involved in the regulation of [Ca2+]i by CaSR. Details regarding the mechanism by which the CaSR interfaces with Class A GPCRs to regulate intracellular calcium stores could be analyzed using thapsigargin-dependent control of [Ca2+]i i  and othe


We hypothesized that IGF-1 enhances mitochondrial energy production through a Ca2+-dependent mechanism to ensure adaptive cardiomyocyte growth. We found that stimulation with IGF-1 resulted in increased mitochondrial Ca2+ uptake in neonatal rat ventricular myocytes and human embryonic stem cell-derived cardiomyocytes, estimated by fluorescence microscopy and indirectly by a reduction in the pyruvate dehydrogenase phosphorylation. We showed that IGF-1 modulated the expression of mitochondrial Ca2+ uniporter (MCU) complex subunits and increased the mitochondrial membrane potential; consistent with higher MCU-mediated Ca2+ transport. Finally, we showed that IGF-1 improved mitochondrial respiration through a mechanism dependent on MCU-mediated Ca2+ transport. In conclusion, IGF-1-induced mitochondrial Ca2+ uptake is required to boost oxidative metabolism during cardiomyocyte adaptive growth.A physiological increase in cardiac workload results in adaptive cardiac remodeling, characterized by increased oxidative metabolism and improvements in cardiac performance. Insulin-like growth factor-1 (IGF-1) has been identified as a critical regulator of physiological cardiac growth, but its precise role in cardiometabolic adaptations to physiological stress remains unresolved. Mitochondrial calcium (Ca Cardiac hypertrophy is a type of cardiac remodeling characterized by the enlargement of the heart in response to a repetitive or sustained increase in cardiac workload, classified as either physiological or pathological . Physiol2+ mishandling and mitochondrial dysfunction, which are common features of pathological cardiac hypertrophy  cycle activity, thus promoting ATP synthesis  complex, strategically positioned in the vicinity of the junctional sarcoplasmic reticulum-mitochondrial associations   and the MCU regulator 1 (MCUR1), which respectively control the channel gating and the conductance of MCU (Ca demands . This prynthesis . To reacciations . The MCUr (EMRE) . In addie of MCU .2+ mishandling contribute to the pathological cardiac hypertrophy  or 0.1% BSA in CDM3 or maintenance medium (hES-CMs or NRVMs respectively) between 15 min and 48 h depending on the experimental setup.2 in Dulbecco modified Eagle medium  supplemented with 10% fetal calf serum (Sigma-Aldrich). At 70% confluence, the cells were transfected with Fugene HD  together with pCMV∆8.91-transfer plasmid, VSV-G-packaging plasmid, and pLKO.1-plasmid expressing the genetically encoded mitochondrial Ca2+ indicator Mitycam provided by Dr. Adam Cohen . The medium was replaced with CDM3 after 24 h and the supernatant containing the viral particles was harvested and filtered with a 0.45 nm filter after 48 h hES-CMs were incubated with CDM3 supplemented with 40% clean viral supernatant for 24 h. The next day, the medium was replaced by CMD3. After 48 h, cells were available for experiments.HEK-293T cells were cultured at 37°C, and 5% CO2+ indicator Mitycam were incubated in Tyrode medium  and mounted at 37°C in the Olympus IX-71 inverted microscope DeltaVision Elite . Mitycam fluorescence (excitation/emission spectra: 498/515) was recorded at baseline (5 min) and during electric stimulation  in the presence of 10 nM isoproterenol (Sigma-Aldrich).Transfected hES-CMs with the Ca2+ uptake was determined using the Ca2+ indicator Rhod2-AM  , and the baseline Rhod2 fluorescence was recorded for 20 s. The medium was replaced by an internal medium with 2 µM free Ca2+, and the signal was recorded for 100 s.Mitochondrial Ca States) . BrieflyFor both cell types (hES-CMs and NRVMs), the images were processed and analyzed in the open-source software Fiji . For imaCardiomyocytes were lysed in RIPA buffer supplemented with protease inhibitors , phosphatase inhibitors (Sigma-Aldrich), sodium orthovanadate (Sigma-Aldrich), and phenylmethylsulphonyl fluoride (Roche). Subsequently, cells were centrifuged at 12,000 g for 10 min at 4°C. The supernatant was recovered, and the isolated protein was quantified using the BCA protein assay (Thermo Fisher Scientific). The protein extract was cooked at 95°C for 5 min in SDS-PAGE loading buffer (Thermo Fisher Scientific) and stored at −20°C.Protein electrophoresis was carried out by standard methods. Briefly, SDS PAGE was loaded with 10 µg of protein extract. Proteins were separated by electrophoresis at 80 mV in running buffer and electro-transferred to a PVDF membrane at 0.45 A for 90 min in transference buffer. Primary antibodies were diluted in blocking buffer  in a 1:1,000 ratio and incubated overnight at 4°C. Membranes were washed in TBS-0.1% tween and incubated in blocking buffer with the secondary antibody  for 1 h in a 1:5,000 ratio. REVERT™ Total protein staining was used as loading control . Western blot detection was carried out in a chemiluminescence detector by standard methods.®  following the manufacturer’s instructions. The mRNA was quantified using a NanoDrop 2000 machine (Thermo Fisher Scientific). The cDNA was synthesized with the Quantitect RT kit  following the manufacturer’s instructions. All primers were designed in Primer-Blast software  and internally validated. qPCR was carried out using the SYBR® Green Master Mix  in the thermocycler CFX384 Touch Real-Time PCR Detection System (Bio-Rad).Cells were lysed with TRIzol+, Invitrogen; excitation/emission spectra: 553/577) in Tyrode medium at 37°C for 30 min. Cardiomyocytes were mounted in the Olympus IX-71 inverted microscope DeltaVision Elite. Baseline fluorescence was recorded for ∼200 s and subsequently, exposed to 10 mM Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) for ∼1,000 s. The noise signal was subtracted. TMRE+ fluorescence was expressed as F—Fmin .Cardiomyocytes were incubated with 20 nM tetramethylrhodamine ethyl ester . Oxygen consumption rate was determined by the Seahorse XF Cell Mito Stress Test programmed in the extracellular metabolic flux analyzer Agilent Seahorse XF96 . 20 min before the assay, CDM3 medium was replaced by Seahorse assay medium XF (Agilent Technologies) supplemented with 10 mM glucose and 1 mM sodium pyruvate at 37°C without CO2 control. The standard stress protocol was performed . hES-CMs were seeded in a clear bottom 96 well plate. CDM3 medium was replaced by the corresponding experimental medium and the luminescence was recorded from 0 to 48 h in the Synergy H1 plate reader . The signal was corrected by the protein content in each well.Cell viability was determined using the Real-Time-Glo™ MT Cell viability assay Kit  following the manufacturer’s instructions. Briefly, the NanoLucNRVMs were seeded in gelatin-coated coverslips in a maintenance medium and stimulated with IGF-1 for 48 h. Subsequently, NRVMs were fixed in PBS + 4% paraformaldehyde for 15 min. Afterward, cells were incubated with rhodamine-phalloidin  and Hoescht  for 1 h at room temperature. Coverslips were mounted in slides with DAKO mounting medium (Agilent Technologies). The images were acquired using the Zeiss LSM 700 laser scanning confocal microscope. The relative cell area was quantified using the software Fiji. 50 to 100 cells were analyzed per condition.t-test or one-way ANOVA was followed by the Tukey post-hoc test for multiple comparisons. For comparing groups without normal distribution was used U Mann-Whitney test or the Kruskal–Wallis test followed by the Dunn post-hoc test for multiple comparisons. A p-value < 0.05 was considered for statistical differences. The software GraphPad Prism 9  was used for data analysis and visualization.All the results were expressed as mean ± SE from at least three independent assays. For values with normal distribution and equal variances was used 2+ handling under conditions of physiological hypertrophy, we studied Ca2+ uptake kinetics of NRVMs in response to IGF-1 stimulation. We first corroborated the activation of IGF-1 receptor-dependent signaling in these cells and observed a time-dependent increase in AKT phosphorylation , confirming the role of the MCUC on the observed effect (2+ concentration activates the pyruvate dehydrogenase (PDH) phosphatase, which dephosphorylates and activates the PDH complex . We observed a drop in Mitycam fluorescence during electric stimulation, which was more pronounced at the end of the protocol in the cells treated with IGF-1  compared to non-treated cells . This is the main driving force for Ca2+ influx through the MCUC  and ATP synthesis   . Therefoechanism . Paupe ethe ∆Ψmt . Interes2+ increments are necessary and sufficient for maintaining the ∆Ψmt and ATP levels of adult cardiomyocytes during situations of high energetic demand (2+ influx promoted by IGF-1 will potentiate metabolism and respiration to sustain the energy demands imposed by the hypertrophic stimulus. As hypothesized, we found that IGF-1 significantly enhanced ATP-linked respiration in a Ca2+-dependent manner (2+ uptake, OCR, and ATP synthesis (Wescott et al. showed that mitochondrial Cac demand . Based ot manner . Other st manner , especiat manner . Interesynthesis . In addiynthesis , which c2+ handling and improves the oxidative metabolism of cardiomyocytes during physiological growth. In addition, we identified changes in the MCUC components and ∆Ψmt as possible regulatory mechanisms of the IGF-1-mediated mitochondrial Ca2+ uptake. These findings shed new light on the regulatory factors of cardiomyocyte remodeling, highlighting mitochondrial Ca2+ handling and MCUC as potential therapeutic targets for the prevention and treatment of pathologies related to energetic imbalance, such as pathological cardiac hypertrophy and heart failure.In summary, this study shows that IGF-1 boosts mitochondrial Ca2+ uptake promoted by IGF-1. Further studies should focus on determining the role of MICU1 and MCUR1 on IGF-1-mediated mitochondrial Ca2+ uptake, using genetic approaches of gain and loss of function of these proteins. An additional limitation is the absence of direct estimations of mitochondrial ATP production, which could demonstrate that IGF-1 is responsible to maintain the energy balance during cardiomyocyte hypertrophy. Despite these limitations, our study provides deeper insight into the regulation of hypertrophy in two different cardiomyocyte models and highlights the role of mitochondrial function in the process.The major limitation of this study is the absence of a causal relationship between the changes observed in MCUC stoichiometry and the increments in mitochondrial Ca