GATM) is more frequent in dialysis-independent CKD patients  but not dialysis-dependent kidney failure patients (n = 73) compared to healthy controls (n = 309). The allele C of intronic SNP rs4293393 (UMOD) is more frequent in healthy controls (adjusted p = 0.042) than in CKD patients. The Allele T of intronic SNP rs9895661 (BCAS3) is associated with decreased eGFRcys (adjusted p = 0.001) and eGFRcrea (adjusted p = 0.017). Our results provide further evidence of a genetic difference between dialysis-independent CKD and dialysis-dependent kidney failure, and add the GATM gene locus to the list of loci associated only with dialysis-independent CKD. GATM risk allele carriers in the dialysis-independent group may have a genetic susceptibility to higher creatinine production rather than increased serum creatinine due to kidney malfunction, and therefore, do not progress to dialysis-dependent kidney failure. When using eGFRcrea for CKD diagnosis, physicians might benefit from information about creatinine-increasing loci.The ten most statistically significant estimated glomerular filtration rate (eGFRcrea)-associated loci from genome-wide association studies (GWAs) are tested for associations with chronic kidney disease (CKD) in 208 patients, including dialysis-independent CKD and dialysis-dependent end-stage renal disease (kidney failure). The allele A of intergenic SNP rs2453533 (near  In high-income countries, 2–3% of annual healthcare budgets are spent on 0.03% of the total population—patients whose disease progresses to end-stage renal disease/failure  requiring replacement therapy . Currente months ) values e months . Initiate months . Consense months . Therefoe months  is also 2 [Genome-wide association studies (GWAs) search for associations between genetic loci and CKD-defining traits (CKD-dt)  includin2 . The lat2 . Some of2  suggesteWe enrolled 208 Slovenian CKD patients, including 73 dialysis-dependent kidney failure patients receiving hemodialysis and 135 dialysis-independent patients treated in the outpatient nephrology clinic of the University Medical Centre Maribor (UMCM), Slovenia. As the control group, 309 healthy Slovenian blood donors from the UMCM were available. The inclusion criteria for patients were a CKD diagnosis of any CKD stage ranging from one to five according to the KDOQI Clinical Practice Guidelines . Serum cWe recruited the individuals in this study during routine control visits to the outpatient nephrology clinic  and dialysis center  of the UMCM between 10.2.2015 and 19.5.2016. With the clinical diagnosis of CKD, regardless of the severity of kidney function impairmentOf local ancestral backgroundAble to understand and sign informed consentWe included patients:With active malignant disease Aged below 18 yearsWith missing medical recordsWe excluded patients:Participant data acquisition and processing were performed at the UMCM. Laboratory analyses were performed at the Center for Human Molecular Genetics and Pharmacogenomics, Faculty of Medicine, University of Maribor, Slovenia.Ethics approval and consent to participate: All patients and healthy individuals agreed to participate prior to inclusion in the study with signed informed consent. The study was in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) and approved by the Republic of Slovenia National Medical Ethics Committee, Ministry of Health, with the reference number 110/02/15.The characteristics of the included individuals and their clinical parameters are presented in We isolated the DNA from 9 mL peripheral blood. The peripheral blood lymphocytes were collected using density cell separation media. Subsequently, DNA was isolated from lymphocytes using a monophase solution containing phenol and guanidine thiocyanate. All nucleic acid samples were quantified and diluted to equal concentrations using a BioTek Synergy2 Microplate Reader .2 values are shown in  followed by restriction fragment length polymorphism (RFLP). We selected the primers and restriction enzymes using the freely available software GeneRunner (Hastings Software Inc.). The PCR conditions were as follows: initial denaturation 95 °C for 10 min followed by 35 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s. PCR products were incubated with the restriction enzyme ApoI  at 37 °C overnight. Digested products were resolved in 2% agarose gel.Genotyping of rs2453533-UMOD/PDILT, rs1047891-CPS1 and rs989566-BCAS3 was performed using PCR followed by high-resolution melting (HRM), using the LightCycler 480 instrument for both steps . Primers were designed using Primer3web  and checked for potential primer dimer formation using OligoAnalyzer . We used 10-µL reaction volumes with 30 ng of DNA and LightCycler 480 High-Resolution Melting Master . The concentration of MgCl2 was optimized for each individual primer and was in the range of 2–3 mM. Cycling conditions were as follows: initial denaturation at 95 °C for 10 min, 40 cycles of denaturation , annealing  and extension . After amplification, the samples were heated to 95 °C for 1 min and rapidly cooled to 40 °C for 1 min at the rate of 1 °C/s, to induce heteroduplex formation before melting. Melting curve data were obtained from continuous fluorescence readings from 55 to 90 °C with a thermal transition rate of 0.02 °C/s. We based the genotype readings on negative first-derivate melting curves and comparison to reference samples . We analyzed the data using the LightCycler 480 instrument software via two methods: gene scanning and Tm-calling. Genotyping of rs4293393-Genotypes for other selected SNPs were extracted from our genotype data bank. All genotypes were checked for Hardy-Weinberg equilibrium, and there were no deviations from it for any SNP in the control group. The reference alleles are presented as the effect allele defined by Wuttke et al. where the SNPs were identical, and as alleles in LD with the effect allele as defined by Wuttke et al. where the SNPs were not identical. All patient and control samples were processed together in the same workflows in the case of all respective genotyping methods to avoid batch effects.We reported numeric parameters as mean ± standard deviation (SD). We performed four case-control association analyses: all CKD patients versus controls, dialysis-independent patients versus controls, dialysis-dependent kidney failure patients versus controls and dialysis-dependent kidney failure versus dialysis-independent CKD patients. The case-control association analyses were performed using Fisher’s exact test. eGFRcrea and eGFRcys genetic association analysis was performed using multiple linear regression. Clinical parameter measurements were not available for control individuals; therefore, control individuals are excluded from this analysis. The statistical analysis was performed using SPSS software, version 26 . We used the Bonferroni correction for multiple testing with 12 comparisons; the corrected p  was set to 0.004.Using multiple linear regression, an α of 0.004 (Bonferroni correction for 12 SNPs–α = 0.05 / 12), a large effect size and 19 predictors, along with a sample size of at least 110 participants are needed to detect a significant association with a power of at least 80%.Using Fisher’s exact test, an α of 0.004 (Bonferroni correction for 12 SNPs–α = 0.05 / 12), an odds ratio of approximately three or difference in proportions of approximately 0.25, a sample size of at least 70 cases and at least 200 controls are needed to detect a significant association with a power of at least 80% .p = 1.0 × 10−6). The 309 control individuals had a mean age of 42 years with an SD of 14.55 years, and there were 40% males and 60% females in the control group. The controls, like the patients, were of Slovenian origin. All control individuals were disease-free at the time of recruitment in the study. No other demographic information was available for the control individuals. The patient demographics and clinical phenotypes are shown in There were no statistically significant differences in the distributions of age, sex, body mass index (BMI), systolic blood pressure, comorbidities or extent of edema between the dialysis-independent CKD and dialysis-dependent kidney failure groups. The dialysis-dependent kidney failure patients had higher albuminuria levels  CKD patients (41%) compared to healthy controls . Interestingly, the frequency of allele A was not significantly increased in dialysis-dependent kidney failure patients requiring hemodialysis. Additionally, we found a lower frequency of allele C of SNP rs4293393-UMOD/PDILT in CKD patients (15%) than in healthy controls . Other associations with unadjusted p values lower than 0.05 did not remain significant after Bonferroni correction for multiple testing. We found no statistically significant differences between the genotypes of CKD patients and controls for the other investigated SNPs. Associations with adjusted p values < 0.05 are presented in We found a higher frequency of allele A of SNP rs2453533--BCAS3. The results are presented in We tested the average eGFRcrea and eGFRcys against all dialysis-independent CKD patient genotypes. The eGFRcrea and eGFRcys were lower in carriers of the T allele of rs9895661GATM is significantly more frequent in dialysis-independent CKD but not dialysis-dependent kidney failure patients. Böger et al. [GATM) gene locus to the loci associated with dialysis-independent CKD but not dialysis-dependent kidney failure patients. It is not unusual for an association to be present in dialysis-independent CKD, but not dialysis-dependent kidney failure patients. In a previous larger study, 11 of the 16 investigated eGFR-associated loci were also associated with CKD initiation risk, but only two with dialysis-dependent kidney failure. Differences in the genetic architectures of CKD and dialysis-dependent kidney failure resulting in differential mechanisms contributing to kidney disease initiation, incidence and progression, as well as premature cardiovascular mortality in CKD patients prior to dialysis-dependent kidney failure onset and the impact of non-genetic factors, have been suggested as possible explanations by Böger et al. [2, and there is often no comparison between dialysis-independent CKD and dialysis-dependent kidney failure patients, measures of kidney disease progression, biopsy results or other markers of kidney pathology. Genetic association studies comparing dialysis-independent CKD and dialysis-dependent kidney failure as separate groups are still rare [GATM was increased in the dialysis-independent CKD group compared to the healthy control group; however, the frequency of the allele was not significantly increased in a comparable dialysis-dependent kidney failure group. Allele A of rs2453533-GATM has been linked to decreased eGFRcrea (increased serum creatinine) on a population level, but no effect on eGFRcys has been identified [GATM with CKD-related clinical phenotypes. As eGFRcrea measurement is a principal part of the CKD diagnostic workup, patients with genetically increased serum creatinine levels could be diagnosed with CKD as false positives. In this case, we would not expect carriers of A of rs2453533-GATM to progress to dialysis-dependent kidney failure since they have no significant underlying kidney disease, and this would explain the lack of association in the dialysis-dependent kidney failure group. The influence of allele A of rs2453533-GATM on serum creatinine levels requires further investigation. GATM encodes a mitochondrial enzyme that belongs to the amidinotransferase family and is involved in creatine biosynthesis, whereby it catalyzes the transfer of a guanidino group from L-arginine to glycine, resulting in guanidinoacetic acid, the immediate precursor of creatine. Since rs2453533 is located in the non-coding region near the GATM gene, it most likely influences GATM gene expression, resulting in higher creatine and consequently creatinine production rates. Indeed, the increased expression of GATM in skeletal muscle was associated with the A allele of rs2453533 in an eQTL analysis [GATM is most highly expressed and most of the body’s creatine is produced. Our study warrants such studies. In addition, the traditional notion that creatine is almost exclusively produced in major organs such as the kidney, liver and pancreas, and then utilized in muscle, has been challenged most recently, suggesting that skeletal muscle, and some other tissues where GATM is significantly expressed, could have their own active creatine-producing machinery [In the present study, we demonstrate for the first time that the risk allele A of rs2453533-r et al.  previousr et al. . Generalr et al. ,16,17,19ill rare ,20,21, eentified . Accordianalysis . UnfortuGATM has a role in CKD pathology. After all, our results associate this long-known eGFRcrea-associated locus with clinically manifest CKD. GATM is highly expressed in the kidney tubule and mutation in the GATM gene causes a recessive disorder of creatine deficiency and the autosomal dominant Renal Fanconi syndrome, which includes kidney failure [GATM cause fibrillary deposition of GATM inside the mitochondria and lead to elongated, abnormal and dysfunctional mitochondria [GATM aggregates. Overexpression of mutant GATM and subsequent aggregation in mitochondria leads to oxidative damage, inflammation, fibrosis and increased cell death [GATM knockout mice were viable and no aminoaciduria or glucosuria was observed, indicating that a lack of GATM did not directly affect the function of proximal tubules. Rather, Reichold et al. suggest that an excess of mutant GATM proteins in the mitochondria triggers a pathologic cascade that eventually leads to the signs and symptoms in the patients with Fanconi syndrome due to a chronic inflammatory response as a reaction to impaired mitochondrial degradation [GATM, the first enzyme in creatine biosynthesis, is higher in the kidney ) than in most other human tissues [GATM or involvement in kidney dysfunction is lacking, and to our knowledge, this locus was not previously replicated in any case-control study of clinically diagnosed CKD patients compared to healthy controls. To summarize, GATM has a known role in CKD pathology in the case of a known missense gain-of-function mutation, while loss of function in knockout mouse experiments shows no influence on kidney function. It is likely that in contrast with missense mutations causing GATM structural changes, mutations causing slight changes in GATM expression have no influence on kidney function. However, the functional role of allele A of rs2453533-GATM on kidney pathology and its relationship to creatine and creatinine production still require further investigation.However, we are not able to completely exclude the possibility that allele A of rs2453533- failure . Fully pchondria . Kidney ll death . On the radation . Creatinradation . The turradation . The nor tissues . PTCs ha tissues . EvidencUMOD/PDILT and CKD. The association was significant after adjustment only in the combined group of all CKD patients. This is consistent with Böger et al. [UMOD/PDILT and rs11864909-UMOD/PDILT are located relatively close to each other (~326 kbp), but outside of reliable LD. In both cases, the frequency of the minor allele is lower in affected individuals. Our findings are consistent with previous studies of the better-known rs4293393-UMOD/PDILT, the minor allele C, which decreases the odds of developing CKD [UMOD/PDILT correlates with CKD. A previous much larger study by Böger et al. has described CKD risk variants that correlate with dialysis-independent CKD but not dialysis-dependent kidney failure, including in UMOD [UMOD was associated with a reduced risk of incident CKD [Our case-control analysis also identified an association between a lower frequency of allele C of SNP rs4293393-r et al. , who ideping CKD . In our  in UMOD . Accordident CKD  and was dent CKD . In the dent CKD . BCAS3 and decreased eGFRcrea. However, several newer population-based studies, including Wuttke et al. [Köttgen et al.  and Okade et al.  and anote et al.  describee et al. . In our The advantages of our study were good patient group comparability , availability of clinical parameters and relatively long retrospective observation periods. Sample size represents a major limitation of our study. This is especially true for the dialysis-dependent kidney failure group, which was the smallest (n = 73). In addition, clinical parameter measurements were only available for patients, and in the case of eGFRs, only for dialysis-independent CKD patients . The dialysis-dependent kidney failure and dialysis-independent CKD patient groups were matched by age; however, the control group individuals were on average younger than the patients (see Results section). Assuming about a 10% lifetime risk of CKD incidence in the general population (mainly in later life), this is also a limitation of our case/control analysis. However, since genotypes generally do not change throughout life, and we can assume that the majority of our control group will not develop CKD in the coming few years, we have considered this an adequate approximation. A limitation of our analysis when studying the correlations between polymorphisms in/near the analyzed genes with eGFR in the group of dialysis-independent CKD patients  is that GATM in a Slovenian clinically diagnosed CKD patient cohort and demonstrated for the first time that the frequency of the risk A allele of rs2453533-GATM is increased in dialysis-independent CKD but not in dialysis-dependent kidney failure patients. Because GATM is an enzyme in the creatinine production chain, and it could only be associated with eGFRcrea but not eGFRcys in population-based GWAs, it was previously assumed that its polymorphisms likely have no influence on kidney function. Our results could be interpreted as showing a higher likelihood of individuals with genetically increased serum creatinine levels but no significant underlying kidney disease being diagnosed with CKD as false positives. The association was notably absent in a comparable dialysis-dependent kidney failure group. This would mean that physicians might benefit from having access to genetic information on these individuals when interpreting serum creatinine measurements. To avoid false-positive CKD diagnoses, eGFRcrea should be interpreted with caution, and other diagnostic criteria including eGFRcys should be preferred in the carriers of creatinine-increasing variants. However, our replication of this SNP in a case-control study between a clinically diagnosed CKD patient group and healthy controls might warrant further study of this polymorphism’s influence on kidney function, and the investigation of clinical features and structural changes on larger patient samples. A decrease in GATM showed no effect in knockout mouse studies, while a known GATM missense mutation causes excessively expressed mutant GATM proteins to deposit in mitochondria and interfere with their function. Interpreting general population-based eGFRcrea associations in relation to CKD pathology can often prove challenging. By focusing on clinical phenotypes related to disease manifestation and progression rather than just eGFR variation in the healthy population, researchers in the field of CKD genetics should be able to discover markers with stronger connections to CKD pathology. In the meantime, clinicians should keep in mind that certain polymorphisms may affect the levels of serum creatinine in the absence of kidney disease, and interpret eGFRcrea results with caution.We independently replicated GWAs’ results for rs2453533-


Objective: To identify factors related to the conversion of robot-assisted partial nephrectomy (RPN) to robot-assisted radical nephrectomy (RRN) based on data collected by a statewide database in Michigan.Methods: Using the Michigan Urological Surgery Improvement Collaborative-Kidney mass: Identifying and Defining Necessary Evaluation and therapY (MUSIC-KIDNEY) database we identified 574 patients for whom RPN was planned. Patient and tumor characteristics were obtained including body mass index (BMI), Charlson comorbidity index (CCI), RENAL nephrometry score, tumor size, and pathologic staging. Treating centers were subdivided by annualized case volume and academic status. Bivariate and multivariate analyses were performed to assess the impact of these factors on the risk of conversion to RRN from RPN.Results: The conversion rate of RPN to RN was 5.75% (33/574). The difference in RENAL nephrometry score, tumor stage, and size reached statistical significance on bivariate analysis (p<0.001). The tumor stage also reached statistical significance on multivariate analysis . The conversion rate was lower among high-volume versus low-volume practices; however, statistical significance was not reached .Conclusions: Patient factors such as tumor size and renal nephrometry score are likely related to the conversion of RPN to RRN decisions. The data shows that Michigan urologists appear to appropriately assess intra-operative findings and convert to RRN in cases of more advanced kidney tumors. Lower volume centers appear to trend towards a higher conversion rate. Continued quality improvement tracking analysis may further clarify this relationship. Contemporary literature supports partial nephrectomy as a preferred approach to treating low stage, localized kidney cancer ,2. NephrAny partial nephrectomy may be converted to a radical nephrectomy intra-operatively if a surgeon is concerned about safety or oncologic risk. This is also true regarding robot-assisted partial nephrectomy. Recent literature suggests that the rate of conversion from RPN to robot-assisted radical nephrectomy (RRN) ranges from 0.7-5% -13. MostThe Michigan Urological Surgery Improvement Collaborative-Kidney mass: Identifying and Defining Necessary Evaluation and therapY (MUSIC-KIDNEY) conception and data collection methods have been previously outlined by the collaborative in 2019 . The MUSFor the purposes of this study, we included all patients diagnosed with clinical stage cT1a or cT1b renal mass (measuring less than 4 cm or 4-7cm respectively) and for whom RPN was planned captured by the MUSIC database . The priFor each patient, the following variables were extracted and included for analysis: age, body mass index (BMI), Charlson comorbidity index (CCI), tumor size , tumor complexity using RENAL nephrometry score , and prePatient-level and practice-level characteristics were compared between patients converted from RPN to RRN and those receiving successful RPN, using Chi-squared test for categorical variables and Wilcoxon rank-sum test for continuous measures. Practice-level variation in the proportion of patients converting to RRN was examined. Pathological outcomes between patients undergoing RRN vs RPN were also compared through the Chi-squared test or Fisher’s exact test. A mixed-effects logistic regression model was performed to identify factors associated with converting to RRN. The model included predictors such as patient age, BMI, comorbidity, and tumor size/staging, and random intercepts for each surgeon to account for within-surgeon correlation. All the analyses were performed using SAS 9.4 , and statistical significance was set at p=0.05.Between July 2016 and Jan 2021, a total of 574 patients were diagnosed with T1 renal masses and scheduled for an RPN in the MUSIC registry. The rate of conversion from RPN to RRN was 5.75% (33/574). No open conversions were documented. Table A total of 11 practices contributed data to the study sample. The conversion rate among these practices ranges from 0% to 40%  of RRN specimens, which was significantly more than those found on RPN specimens (6.3% p<0.001). The rate of pT4 disease was 6% vs 1.5% for converted vs RPN cases, respectively (p=0.11) A significantly higher rate of vascular invasion was observed among converted cases compared to RPN patients (21.2% vs.5.2%p=0.002). The direct adrenal invasion was found in 3% (1/33) of the converted cases whereas no RPN cases had adrenal invasion (p=0.06).The MUSIC-KIDNEY collaborative database provides a system for statewide tracking and analysis of renal surgery performed in Michigan. This has offered the opportunity to evaluate perioperative outcomes of high and low-volume institutions. The findings reported here are similar to previous studies with regard to the overall RPN to RRN conversion rate. There is not a clear consensus on which perioperative factors appear to impact this rate the most. Petros et al. and Kara et al. showed tumor size, stage, and complexity to be significantly associated with conversion ,11. ArorAlthough the clinical importance of the significant difference in creatinine reported here is questionable, preoperative assessment of renal function is important when considering treatment options and counseling patients found to have renal masses. As the prevalence of diabetes mellitus and hypertension and associated kidney disease continue to rise in the United States, pressure to preserve nephrons in these patients leads clinicians to perform NSS whenever possible since radical nephrectomy has been shown to independently predict new onset and progression of kidney disease ,18. AlsoClinical registries have an important role in urologic research and quality improvement initiatives by providing data on granular outcome measures and patient-reported outcomes that may be lacking in claims-based data . AlthougMost of the contemporary urologic literature in the United States is based on data from high volume and/or major academic centers, especially regarding operative outcomes. Although centralization of care to these centers can be more easily tracked, representation of outcomes from lower volume and/or community-based centers is lacking. There is limited data available comparing outcome differences between these types of centers. A literature review of over four thousand patients showed excellent outcome measures of RPN with no meaningful differences between high and low volume centers . In our This study is limited by its retrospective nature and individual records may be inconsistent in the granularity of detail available for abstraction. Although we were able to demonstrate a statistically significant difference in RENAL nephrometry score between groups, some records were incomplete regarding this datapoint. The records revealed some information about perioperative factors that may have played a role in the decision to convert; however, explicit documentation from the surgeon was not consistently available. Therefore, our data is limited regarding some outcome measures of interest. Although the total cohort size reported here is well over 500 cases, subgroup analysis is limited by the relatively low total number of converted cases. The data may not be powered to show statistically significant differences for some subgroup analyses. It appears to trend towards a higher conversion rate for low-volume centers. The MUSIC database will continue to compile the outcomes of these cases and future analysis may elucidate this difference.Patients with small renal masses considering minimally invasive surgical management with RPN should be counseled on the low risk of conversion to RRN. The data reported here suggest the risk of conversion is significantly higher in patients with larger, complicated tumors. There appears to be an increased rate of conversion to RRN at lower volume centers. Continued data collection and investigation are needed to clarify this relationship.


Background: Ischemia with non-obstructive coronary arteries (INOCA) is part of the ischemic heart disease spectrum, and is particularly observed in women. INOCA has various mechanisms, such as coronary vasospasm and coronary microvascular dysfunction (CMD). A decreased coronary flow reserve (CFR) and-or increased myocardial resistance (MR) are commonly used to diagnose CMD. However, CFR and MR do not describe all pathophysiological mechanisms underlying CMD. Increased myocardial oxygen consumption (MVO2) normally increases myocardial blood volume (MBV), independently from myocardial blood flow (MBF). In addition insulin enhances MBV in healthy skeletal muscle, and this effect is impaired in INOCA-related conditions such as diabetes and obesity. Therefore, we propose that MBV is reduced in INOCA patients.Aim: To assess whether myocardial blood volume (MBV) is decreased in INOCA patients, at baseline, during hyperinsulinemia and during stress.Design: The MICORDIS-study is a single-center observational cross-sectional cohort study (identifier NTR7515). The primary outcome is MBV, compared between INOCA patients and matched healthy controls. The patient group will undergo coronary function testing using a Doppler guidewire, intracoronary adenosine and acetylcholine to measure CFR and coronary vasospasm. Both the patient- and the control group will undergo myocardial contrast echocardiography (MCE) to determine MBV at baseline, during hyperinsulinemia and during stress. Subsequently, cardiac magnetic resonance (CMR) will be evaluated as a new and noninvasive diagnostic tool for CMD in INOCA patients. Microvascular endothelial function is a determinant of MBV and will be evaluated by non-invasive microvascular function testing using EndoPAT and by measuring NO production in circulating endothelial cells (ECFCs). Chronic ischemic heart disease (IHD) has a complex multifactorial origin and may present with a variety of symptoms related to chest discomfort, referred to as angina pectoris or angina-like symptoms. The current diagnostic process for angina is dominated by the evaluation of atherosclerotic obstructive coronary artery disease (CAD) as its potential cause. However, a substantial proportion of patients with angina do not have relevant obstructive CAD on computed tomography or invasive coronary angiography (CAG) , 2. ThisThe pathophysiology underlying INOCA is complex and frequently multifactorial, but commonly starts with abnormal vasomotor responses of the coronary circulation, typically involving an abnormal vasoconstrictor response, abnormal vasodilator response, or a combination of these entities. Abnormal vasoconstriction refers to a variety of vasospastic patterns, including focal or diffuse epicardial vasospasm, microvascular vasospasm, or a combination of these. These disease patterns are, amongst others, associated with endothelial dysfunction and vasomotor smooth muscle cell hyperreactivity. Abnormal vasodilatation in INOCA, frequently referred to as coronary microvascular dysfunction (CMD), refers to the presence of impaired vasodilator reserve of the coronary microcirculation, due to abnormally high microvascular resistance at maximal vasodilatation , or exaggerated vasodilatation already in resting conditions , or a combination of both –12. ThesCurrently, diagnosis of CMD in routine ICFT protocol focuses mainly on reduced CFR both in presence or absence of abnormal MR as the cause of a reduced oxygen supply provoking ischemic complaints. CFR reflects myocardial blood flow (MBF). However, an unexplored feature of microvascular integrity is the myocardial blood volume (MBV) [%]. The coronary microcirculation determines 90% of the variation in MBF and contains most of the MBV. MBV is the fraction of tissue consisting of blood, and about 50% of the myocardial microcirculation simultaneously contains blood in resting conditions , 20.Reduced MBV, for example by rarefaction can reduce microvascular delivery of oxygen, and MBF and MBV are independently regulated. As such, MBV may play a role in CMD that is independent from the parameters identified using ICFT. Both MBF and MBV are linked to myocardial oxygen consumption (MVO2), and both independently increase when MVO2 increases . MBV canThese microvascular effects of insulin are impaired in insulin-resistance, diabetes mellitus, obesity and hypertension, all common comorbidities in INOCA patients . MoreoveMyocardial contrast echocardiography (MCE) allows real-time, non-invasive quantitative assessment of myocardial perfusion, using continuous infusion of gas-filled microbubbles , 28. MBVThe MICORDIS study aims to provide insight into the role of MBV in INOCA, using a variety of stressors to test the regulation of MBV, and using several conventional and new imaging techniques to determine MBV. The main objective of the MICORDIS study is to investigate whether MBV is reduced in INOCA patients.https://www.trialregister.nl/, unique identifier NTR7515). The trial will be performed at the Amsterdam University Medical Centers (UMC), location VU medical center. The study has been approved by the institutional ethics committee on human research of the Amsterdam UMC and carried out in compliance with the Declaration of Helsinki and in accordance with the Medical Research Involving Human Subjects Act (WMO).The MICORDIS trial is a single-center observational cross-sectional cohort study, comparing MBV in INOCA patients with a group of matched healthy controls  >0.80. Signs and symptoms of ischemia are defined as; (1) stable chronic  symptoms suggesting IHD, such as chest discomfort with classic  and/or atypical features regarding location, intensity and provoking factors, and 2) with or without objective evidence for myocardial ischemia on ECG or a cardiac imaging study , nuclear imaging or CMR) at rest or during stress induced by exercise, mental or pharmacological triggers. After informed consent is obtained, eligible candidates meeting the selection-criteria listed in Study candidates will be identified at the outpatient cardiology clinic by active screening, while the control group is recruited by posters and advertisements in local media. Eligible subjects receive an information letter containing information about background, timeframes and risks of the study. Since subjects receive specific instructions regarding their eating habits and medication adjustments may be required prior to their first visit, informed consent is obtained before the first visit. The control group is matched to the patients with respect to sex and age, and are screened for exclusion criteria using TTE, electrocardiography (ECG), and laboratory tests to exclude severe heart disease and impaired renal function.de novo blood vessel formation in vivo  is advanced in the left anterior descending coronary artery (LAD) to perform ICFT . Flow veAfter repeated nitroglycerin administration, and weaning of its initial effects, ICFT is completed by invasive coronary pressure and flow measurements during resting conditions and during maximal vasodilatation to calculate fractional flow reserve (FFR), coronary flow reserve (CFR), and microvascular resistance (MR). Hyperemia is induced by an intracoronary bolus of 150 μg adenosine applied manually and reflects maximal, endothelium-independent vasodilation of the coronary arterioles . FFR is Minimal MR is calculated as the ratio of distal coronary pressure to distal coronary flow and during maximal coronary vasodilatation and reflects the intrinsic resistance of the coronary microvasculature blood flow as a marker of structural microvascular alterations . Minimal*MFV . The product of these parameters is MBF, using the equation MBF = MBV*MFV . Microbu*MFV .MCE is performed using the Philips iE33  ultrasound machine and is performed at baseline, after 2 h of hyperinsulinemia and during intravenous infusion of dobutamine. Following the first MCE-measurement at baseline, hyperinsulinemia is induced using the hyperinsulinemic-euglycemic (HE) clamp technique, developed by DeFronzo et al. . The goa* 0,04 * 60 . The M-value represents the metabolic insulin-resistance , 46.The final MCE-measurement is performed during administration of dobutamine. Dobutamine is administered in incremental doses, and MCE is performed at a target dose of 40 μg/kg/min dobutamine, or at a lower dose if a diagnostic endpoint is reached or in case of undesired effects .All subjects undergo CMR using a 3 Tesla-scanner  after abstaining from caffeine and xanthine for 24 h. Myocardial perfusion imaging is performed during stress and rest, using a dual sequence technique optimized for quantification of absolute myocardial blood flow. For this purpose, three short axis images  will be acquired during first-pass perfusion imaging, both during adenosine stress (adenosine infusion 140 μg/kg/min) and at rest, during administration of an intravenous bolus of contrast gadolinium (Dotarem 0.075 mmol/kg). Stress perfusion CMR-derived MPRI will be calculated .T1-mapping is applied to assess MBV and extracellular volume (ECV). For this purpose, short-axis images at mid-ventricular level and 4-chamber long axis images are acquired using the MOdified Look-Locker Inversion (MOLLI) sequence with a 5S(3s)3s sampling scheme, during rest and adenosine injection (adenosine infusion 140 μg/kg/min) and after administration of gadolinium , 48.All CMR images are collected centrally and are analyzed using Circle .The EndoPAT  is a device used to non-invasively assess microvascular function. By using the EndoPAT insight is given in the functioning of the endothelial and smooth muscle cells of the patients as well as the healthy controls, since this latter group does not undergo CFT. The EndoPAT is non-operator-dependent, and records post-ischemic reactive hyperemia known as the PAT  signal using plethysmographic probes. These probes are placed on the index finger of both hands. First a baseline flow-signal is obtained for 5 min. Subsequently, flow-mediated changes are evoked by occluding blood flow through the brachial artery using an inflatable blood-pressure cuff. After 5 min of inflation the cuff is deflated, which induces reactive hyperemia. During this hyperemic period, a second flow measurement is performed .The EndoPAT displays the pulse amplitude response to hyperemia, and automatically calculates the Reactive Hyperemia Index (RHI) as the post-to pre-occlusion PAT-ratio on the index finger of the occluded arm, divided by the corresponding ratio from the contralateral, control arm , 50. TheThe primary objective of the MICORDIS study is a difference in MBV in INOCA patients compared to healthy controls, evaluated at baseline, during hyperinsulinemia and during increased myocardial contraction. Secondary objectives of the study are (1) sex differences in MBV, MBF, ΔMBV, baseline characteristics, cardiovascular risk factors, depression, stress level, blood parameters and physiological indexes in patient group, (2) differences in baseline characteristics, cardiovascular risk factors, depression, stress level and physiological indexes between patient and control group, (3) comparison of MBV, MBF and ΔMBV at baseline and after HE-clamp and dobutamine of the patient and control group, (4) MBV, MBF and ΔMBV compared between insulin resistant subjects and non-insulin resistant subjects, (5) correlation between MBV (+other related variables) measured with MCE and MBV measured with CMR, (6) difference in peripheral endothelial function between INOCA patients and healthy controls, and (7) differences in protein expression and phosphorylation of microvascular endothelial cells compared between patient and control group.t-test. To account for a projected dropout of 15%, the study population consist of 28 INOCA patients  and 28 healthy controls . The primary analyses of the study consist of a comparison of MBV at baseline, during hyperinsulinemia and during increased MVO2, and will be corrected for multiple testing using the Bonferroni method. Based on the distribution of continuous outcomes, independent samples t-test or Mann–Whitney U test are used as appropriate.The expected effect size was estimated based on a study of Rinkevich et al. . RinkeviTo summarize, the MICORDIS study is a single-center, observational cross-sectional cohort trial to study the change in MBV in INOCA patients at baseline, during hyperinsulinemia and hyperemia. This is the first study that incorporates a range of ICFT and advanced non-invasive imaging methods to assess MBV and associated pathophysiological mechanisms in INOCA patients. This study may provide novel insight into the pathophysiological and diagnostic process of microvascular coronary dysfunction, which are of great importance in a comprehensive and effective treatment of INOCA patients.Currently including patients. Currently enrolled eight patients and three matched healthy-controls.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Medisch Ethische Toetsingscommissie VUmc OZW O8A-08 | De Boelelaan 1109 |1081 HV Amsterdam. The patients/participants provided their written informed consent to participate in this study.YA and EE were involved in the conception and design of the study. CV drafted the first version of the manuscript and has performed perfusion analysis on the images. All authors read, revised the manuscript for critically intellectual content, and approved the manuscript. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.Funding was received from the Amsterdam UMC Foundation, Netherlands Heart Foundation and Netherlands Heart Institute.MG is consultant for Circle CVI . The remAll claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Exaggerated blood pressure response to exercise (EEBP = SBP ≥ 190 mmHg for women and ≥210 mmHg for men) during cardiopulmonary exercise test (CPET) is a predictor of cardiovascular risk. Sympathetic hyperactivation and decreased baroreflex sensitivity (BRS) seem to be involved in the progression of metabolic syndrome (MetS) to cardiovascular disease.To test the hypotheses: (1) MetS patients within normal clinical blood pressure (BP) may present EEBP response to maximal exercise and (2) increased muscle sympathetic nerve activity (MSNA) and reduced BRS are associated with this impairment.n = 27, 59.3% males, 46.1 ± 7.2 years) and a control group without MetS . We evaluated BRS for increases (BRS+) and decreases (BRS−) in spontaneous BP and HR fluctuations, MSNA (microneurography), BP from ambulatory blood pressure monitoring (ABPM), and auscultatory BP during CPET.We selected MetS (ATP III) patients with normal BP  and diastolic BP  during CPET than C. Additionally, we found that MetS patients with normal BP had lower spontaneous BRS−  and higher levels of MSNA  compared to C. Interestingly, 10 out of 27 MetS_NT (37%) showed EEBP (MetS_NT+), whereas 2 out of 19 C (10.5%) presented (P = 0.044). The subgroup of MetS_NT with EEBP  had similar MSNA (P = 0.437), but lower BRS+ (P = 0.039) and BRS− (P = 0.039) compared with the subgroup without EEBP . Either office BP or BP from ABPM was similar between subgroups MetS_NT+ and MetS_NT−, regardless of EEBP response. In the MetS_NT+ subgroup, there was an association of peak SBP with BRS− , triglycerides with peak SBP during CPET , and of triglycerides with BRS− .Normotensive MetS (MetS_NT) had higher body mass index and impairment in all MetS risk factors when compared to the C group. MetS_NT had higher peak systolic BP (SBP) (195 ± 17 vs. 177 ± 24 mmHg, Normotensive MetS patients already presented higher peak systolic and diastolic BP during maximal exercise, in addition to sympathetic hyperactivation and decreased baroreflex sensitivity. The EEBP in MetS_NT with apparent well-controlled BP may indicate a potential depressed neural baroreflex function, predisposing these patients to increased cardiovascular risk. Metabolic syndrome (MetS) is the overlap of cardiovascular risk factors   and has In hypertensive patients, arterial baroreflex sensitivity (BRS) is reduced, and this impairment has been found to be associated with increased sympathetic drive and higher BP . RegardlThe BRS is an important sophisticated autonomic mechanism in the regulation of the cardiovascular system, designated to buffer beat-to-beat fluctuations in arterial BP. Arterial baroreceptors are mechanosensitive nerves, located in the adventitia of the carotid sinus and aortic arch. An increase in BP causes vascular distension, and the baroreceptor deformation exerts reflex bradycardia and sympathoinhibition, which results in a vasodilation reflex. On the other hand, the baroreflex deactivation causes tachycardia reflex and elicits sympathetic-mediated vasoconstriction reflex. The main role of baroreflex is to maintain BP at physiological levels . In humaSince exercise induces physiological stress, the analysis of HR and BP responses during maximal progressive exercise may be used as a simple tool to test the integrity of autonomic control to adjust cardiac output. In this context, in a previous study we observed that MetS patients with the comorbidity obstructive sleep apnea (OSA) had impaired HR recovery after maximal exercise, and this seemed to be partly due to the sympathetic hyperactivity in these patients .There are a large number of studies suggesting that the response (EEBP) during cardiopulmonary exercise test (CPET) that occurs in individuals with normal resting BP is predictive of risk for new-onset hypertension  and cardAlbeit the clinical relevance of EEBP is related to an increased risk of onset hypertension , the undIn this prospective study, we recruited 72 newly diagnosed and unmedicated MetS patients from the Outpatient Unit of the Heart Institute (InCor), University of São Paulo Medical School, aged between 40 and 60 years, non-smokers, sedentary, with no history of alcohol consumption, and with no evidence of cardiopulmonary or skeletal muscle disorders. From the initial sample, the ones invited to participate in this study were only those 27 MetS (37.5%) without hypertension based on the office BP (SBP ≤ 139 and DBP ≤ 89 mmHg) , and witThe study was approved by the Scientific Commission of the Instituto do Coracao (InCor), and by the Ethics in Research Commission of the Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de São Paulo (#1222/05). All participants signed a written informed consent form.All evaluations were carried out in about 2–3 weeks on subsequent visits. The subjects were instructed to abstain from caffeine and physical activity for the 48 h leading up to the evaluations. Initially, venous blood was collected after 12 h of overnight fasting to measure total serum cholesterol, triglycerides, HDL cholesterol (enzymatic method), and plasma glucose (standard glucose oxidase method). After a light meal, all subjects underwent three standard BP measurements, and assessment of body weight, height, body mass index (BMI), and waist circumference (WC). Then, the 24-h ambulatory blood pressure monitoring (ABPM) was placed. The next day, ABPM was removed and the autonomic evaluation was performed in a quiet room with controlled temperature (22°C). In a lying position, the patient’s leg was positioned for microneurography, and a microelectrode was placed on the peroneal nerve. After instrumentation and a 15-min rest period, the biological signals of MSNA, heart rate (HR) on the electrocardiogram (ECG), and BP on a beat-to-beat basis were recorded for 10 min at rest in a lying position using a software program . When we did not succeed in assessing MSNA, a second attempt was made on another date. On another visit, a CPET was performed.Also, individuals underwent a night polysomnography to assess the apnea/hypopnea index (AHI).Metabolic syndrome patients were diagnosed according to Adult Treatment Panel III (ATP-III) , which rIn the present study, we selected MetS patients without hypertension .Systolic BP and DBP were measured following the recommended procedure for routine office BP measurement and with appropriate cuff size .The 24-h BP measurements were recorded using a continuous blood pressure monitor . The device was placed on the non-dominant upper limb of the patient early in the morning and was removed 24 h later. The device was programed to record BP readings every 10 min during wake (daytime) and every 20 min during sleep (nighttime). To validate the ABPM, at least 70% of expected measurements should be conducted in 24 h recording . DaytimeMuscle Sympathetic Nerve Activity was directly measured through a multiunit postganglionic efferent from the peroneal nerve using the microneurography technique, which consists of the impaction of a tungsten microelectrode to the peroneal nerve, as previously described .During 10 min at rest in a lying position, mean BP was continuously and non-invasively monitored by Finometer, a finger photoplethysmography device  on a beat-to-beat basis  at a frequency of 500 Hz. Simultaneously, HR was continuously monitored through the lead II of the ECG. The respiratory rate was monitored with a piezoelectric thoracic belt  placed around the upper abdomen.R–R interval (reflex bradycardia) or by a progressive decrease in SBP and shortening of the R–R interval (reflex tachycardia). The consecutive and simultaneous increases in SBP and increases in R–R intervals represent spontaneous activation of baroreceptors (BRS+), and consecutive and simultaneous decreases in SBP and decreases in R–R intervals represent spontaneous deactivation of baroreceptors (BRS−)  in milliseconds (ms). After plotting the values side by side, we observed each consecutive sequence of the SBP (from 1 mmHg) and R–R interval (from 3 ms), and a slope by a linear regression was generated. Finally, all slopes were averaged, resulting in an index of the sensitivity of arterial baroreflex modulation of heart rate (BRS). The cuff used was adapted according to the size of the patient’s finger and arm  in the time domain. This method consists of the identification of three or more sequential beats, characterized by either a progressive rise in SBP and enlargement of the s (BRS−) . Basical and arm .2) and carbon dioxide production (CO2) were determined by gas exchange on a breath-by-breath basis in a computerized system . Peak VO2 was defined as the maximum VO2 reached at the end of the exercise  using a ramp protocol. The workload increments of 10 or 15 W were added every minute at constant cadence (60–70 rpm) until exhaustion. Oxygen uptake  test was used to assess categorical data differences. Comparisons between MetS_NT and C groups as well as between normotensive MetS with EEBP (MetS_NT+) and without EEBP (MetS_NT−) during CPET were carried out using Student’s t-test. The bivariate correlation (Pearson correlation) was conducted to test the association of peak SBP response during maximal CPET with baroreflex sensitivity (BRS−), association of triglycerides with peak SBP response during maximal CPET and association of triglycerides and BRS−. P < 0.05 values were considered statistically significant.Statistical analysis was carried out using SPSS software . The data are presented as mean and standard deviation for parametric measurements and median and [interquartile range] for non-parametric measurements. The Kolmogorov–Smirnov and Levine tests were used to assess the normality and homogeneity of distribution of each variable studied. A Chi-square (χ2) were similar between groups. As expected, MetS_NT patients had higher weight and BMI and all MetS risk factors were impaired compared to C (red to C . Regardired to C  and highred to C  when comred to C .P = 0.044), with only two in 19 individuals (two men), i.e., 10.5% presented EEBP response (response .n = 10) and MetS_NT−  regarding physical characteristics, office BP, BP of the ABPM recordings , and BP response during CPET. The subgroups MetS_NT+ and MetS_NT− were similar in sex distribution, age, weight, BMI, and peak VO2 (P = 0.127).In peak VO2  and AHI Despite similar office BP (SBP and DBP), similar 24-h, daytime, and nighttime averages of SBP and DBP measured by the ABPM, these 10 MetS_NT+ who presented EEBP during CPET had higher SBP and DBP levels at peak of maximal exercise and higher SBP levels at the first minute of recovery .The MetS_NT+ subgroup had lower BRS+ and BRS− when compared to the MetS_NT− subgroup , whereasThe univariate linear regression between peak SBP and BRS−, BRS+, and MSNA and between SBP at the first min of recovery and BRS−, BRS+, and MSNA in the MetS_NT subgroups with (MetS_NT+) and without (MetS_NT−) EEBP response during maximal CPET is shown in Interestingly, only in the MetS_NT+ subgroup, we found a strong negative correlation between peak SBP and BRS− . SimilarP” value in the univariate linear regression. In multivariate linear regression with peak SBP, no variable was found to be significant. On the other hand, with BRS− the triglycerides remained significant.In order to determine potential metabolic alterations related to impaired peak SBP during CPET, we analyzed the univariate and multivariate linear regression of all metabolic risk factors with peak SBP during CPET and with BRS− in the MetS_NT+ subgroup . The uni2, and AHI. In addition, they already presented sympathetic hyperactivation and decreased BRS−. Interestingly, some of these normotensive MetS patients (37%) had an EEBP response during maximal exercise (peak SBP ≥ 210 mmHg for men and ≥190 mmHg for women), in spite of similar office BP and similar averages of SBP and DBP at ABPM  when compared with normotensive MetS patients without EEBP response. Moreover, this prevalence of EEBP is higher in MetS_NT compared to C. We would like to point out that our MetS_NT subgroups, with or without EEBP response, were similar in age, sex, and functional capacity, factors that are known to impact EEBP , there was a strong negative association of peak SBP with BRS−, lending strength to the hypothesis that this autonomic reflex is an important pathophysiological mechanism underlying EEBP.It is well-established that adrenergic activation and baroreflex dysfunction are present in all hypertensive states . LikewisThe mechanisms underlying an exacerbated BP response during exercise have yet to be fully understood and seem indeed to be multifactorial. So far, studies have pointed to endothelial dysfunction and changes in the intima-media thickness as the mechanisms underlying the exacerbation of BP during CPET in hypertension , perhapsWhen hypertension is eliminated, controversy remains regarding the pathophysiological change and the EEBP response. In addition, only a few studies have focused on the effects of MetS on BRS . SharmanOur MetS population sample comprised recently diagnosed and unmedicated MetS patients without hypertension, a very common condition in middle-aged individuals who often do not consider themselves to be diseased. Our study brings a new perspective in that it lends prominence to the role of BRS as a key mechanism which may underlie the response of BP to CPET.While elucidating the risk factors that impair autonomic control of BP during exercise in normotensive patients with MetS is indeed complex, given that these factors share pathophysiological pathways and seem to potentiate each other, the association of triglycerides with peak SBP may suggest the potential of this risk factor over some pathophysiological changes involved in hemodynamic control. However, the overlap of metabolic risk factors seems to be the main cause of the autonomic alteration in normotensive MetS patients.In general, the overlap of several risk factors seems to be stronger in predicting the pathophysiological alterations related to attenuation of BRS as well as to EEBP response. Indeed, the overlap of several metabolic risk factors is the probable trigger to higher values of peak systolic BP during exercise, as shown by a previous study conducted by We have previously observed that OSA, a common comorbidity in patients with MetS, leads to a further impairment in autonomic control . HoweverAlthough several methods have been developed to study baroreflex function in humans, most of these techniques are of limited value for a daily practice in the clinical setting . Given tIn conclusion, the present study offers a new insight on the mechanisms underlying EEBP response to exercise and more particularly on the key role of BRS impairment in the increased BP response during exercise in normotensive patients with MetS. Our findings emphasize the importance of identifying EEBP response to exercise in MetS patients, even in those within the normal range of clinical BP, since exaggerated BP during CPET was associated with the baroreflex in MetS patients, which put these patients at a greater cardiovascular risk. Thus, an awareness of EEBP during maximal CPET in normotensive MetS patients should be part of any clinical interventions.There are some limitations in our study that merit discussion. Previous studies addressing the mechanisms underlying EEBP have been carried out with healthy volunteers. The association of risk factors (at least 3) in our sample of normotensive patients with MetS can cause interpretation bias, since any of the factors, according to their prevalence, may be involved in the EEBP during CPET .We would like to point out that it has already been demonstrated that women have distinct pathophysiological pathways from men , as wellIn this study, the office BP was measured in a single visit. According to the “Brazilian Guidelines of Hypertension–2020” , true hyRegarding the method used for determining the BRS, the venous vasoactive drug infusion technique has been largely used. However, the lack of selectivity in the response has been considered as one important limitation of the use of vasoactive drugs . Thus, wAssessing auscultatory BP during treadmill maximal exercise is sometimes difficult. We took great care to have reliable peak BP measurement, using a stationary electromagnetic brake cycle ergometer.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Scientific Commission of the Instituto do Coracao (InCor), and by the Ethics in Research Commission of the Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de São Paulo (#1222/05). The patients/participants provided their written informed consent to participate in this study.ACD-M, SR, FXC, and ICT: conception and design of the work, data acquisition, analysis, interpretation of data, drafting of the work, and critical revision for important intellectual content. ET-D, ER, JCC, MJNNA, AMFWB, and MUPBR: data acquisition, interpretation of data for the work, and critical revision for important intellectual content. All authors read and approved the final version to be published, and all authors agreed with all aspects of the work in order to ensure the accuracy and integrity of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Hypertension is among the most commonly diagnosed non-communicable diseases in Africa, and studies have demonstrated a high prevalence of hypertension among individuals with HIV. Despite high prevalence, there has been limited attention on the clinical outcomes of hypertension treatment in this population.We sought to characterize rates of and factors associated with blood pressure control over one year among individuals on antiretroviral therapy (ART) and antihypertensive medications.We performed a prospective observational cohort study at an HIV clinic in Malawi. We defined uncontrolled hypertension as a systolic blood pressure ≥140 mm Hg and/or diastolic blood pressure ≥90 mm Hg at two or more follow-up visits during the year, while controlled hypertension was defined as <140 mm Hg systolic and <90 mm Hg diastolic at all visits, or at all but one visit. We calculated an antihypertensive non-adherence score based on self-report of missed doses at each visit (higher score = worse adherence) and used rank sum and chi-square tests to compare sociodemographic and clinical factors (including adherence) associated with blood pressure control over the year.At study entry, 158 participants (23.5%) were on antihypertensive medication; participants had a median age of 51.0 years, were 66.5% female, and had a median of 6.9 years on ART. 19.0% (n = 30) achieved blood pressure control over the year of follow-up. Self-reported non-adherence to hypertension medications was the only factor significantly associated with uncontrolled blood pressure. The average non-adherence score for those with controlled blood pressure was 0.22, and for those with uncontrolled blood pressure was 0.61 (p = 0.009).Adults living with HIV and hypertension in our cohort had low rates of blood pressure control over one year associated with self-reported non-adherence to antihypertensive medications. Given the high prevalence and incidence of hypertension, interventions to improve blood pressure control are needed to prevent associated long-term cardio- and cerebrovascular morbidity and mortality. Non-communicable diseases (NCDs) are the leading cause of death worldwide and have become increasingly prevalent in low and middle-income countries (LMICs) . Over 801Countries in sub-Saharan Africa with a high burden of NCDs also have the highest prevalence of HIV globally, which is notable because HIV infection and antiretroviral therapy (ART) are associated with increased risk of developing hypertension, dyslipidemia, diabetes, and cardiovascular disease through a combination of chronic inflammation and immune activation associated with HIV itself, toxicities of treatment, and unhealthy lifestyle changes . While t78Hypertension is among the most commonly diagnosed NCDs in sub-Saharan Africa, and studies have demonstrated a high prevalence of hypertension among individuals with HIV 11%–46%) %–46% 1113. Measu. Measu13Malawi is a small country in Africa with a robust national HIV treatment program, which incorporated screening and treatment for hypertension into its national HIV guidelines in 2016 [We performed a prospective observational cohort study at Partners in Hope Medical Center, a faith-based non-governmental organization located in Lilongwe, Malawi. The clinic provides free HIV care in the urban setting of Lilongwe, Malawi to an active cohort of over 5,000 patients on ART. During the time of the study, hypertension screening was provided free of charge, and medications for hypertension could be purchased at Partners in Hope  or acquired for free at government-operated health facilities. Hypertension management follows a specific algorithm outlined by the Malawi Standard Treatment Guidelines: first line therapy with a thiazide diuretic  and sequential additions of medications if blood pressure control is not achieved  . LifestyIndividuals were approached while waiting to receive routine HIV care and asked if they would be interested in participating in a study about hypertension. Those who expressed interest were taken to a private room to hear more information about the study, provide oral consent for screening, and if eligible, provided written informed consent to participate in the study. Individuals were eligible if they were ≥18 years of age, had been receiving ART from Partners in Hope for at least one year at the time of study entry, planned to remain a patient at Partners in Hope for at least the next one year, and did not have any clinical condition that required urgent medical attention on the day of study entry (such that they were deemed stable to have a study visit on that day).At the entry visit, participants completed a survey about HIV clinical history and adherence to ART, lifestyle , and, if on medications for hypertension, antihypertensive medication adherence, side effects, and barriers to taking antihypertensive medications . Participant charts were reviewed at entry to confirm current hypertension medications and to document HIV disease characteristics .All participants had blood pressure measured and recorded at least twice during the course of the entry visit: one time at the patient registration area (part of routine care at the HIV clinic) and one time by the study nurse (at the beginning of the study visit). For those with an elevated blood pressure  on either of the first two measurements, a third blood pressure was obtained at the end of the study visit. All measured blood pressures were recorded in the study record. Blood pressure equipment was calibrated weekly and refresher trainings on proper measurement of blood pressure were provided to staff at the beginning of the study and then quarterly during study follow-up.All participants were followed for one year. At every routine ART visit at Partners in Hope (approximately every three months as per standard of care at the time under study), the participant had blood pressure measured as described above, and completed a brief survey about adherence to ART, lifestyle , and if on antihypertensive medication, adherence, side effects, and barriers to taking these medications. The participant chart was reviewed for antihypertensive medications.The target sample size for the overall cohort was 700 with the assumption that approximately 30% would have hypertension based on regional data , and tha14The study was reviewed and approved by the Malawi National Health Sciences Research Committee and the University of California, Los Angeles Institutional Review Board.Given a lack of consistent medical record documentation related to a diagnosis of hypertension and challenges with the ability of individuals to self-report a previous diagnosis of hypertension, participants were defined as hypertensive based on records of active prescriptions for antihypertensive medication at the entry visit. For classification of severity of hypertension, we used the 2015 Malawi National Guidelines, as follows: mild hypertension as a systolic blood pressure 140–159 mm Hg and/or diastolic blood pressure 90–99 mm Hg; moderate hypertension as a systolic blood pressure 160–179 mm Hg and/or diastolic blood pressure 100–109 mm Hg; and severe hypertension as a systolic blood pressure ≥180 mm Hg and/or diastolic ≥110 mm Hg [Main individual-level factors explored for associations with blood pressure control and incident hypertension were sociodemographic factors , clinical parameters , health behaviors and non-adherence score. The non-adherence score was calculated based on respondents’ self-reported average weekly adherence to antihypertensive medication since their last visit: miss less than once per week, miss once per week, miss two or three times per week, or miss more than three times per week. These responses were assigned between zero and three points, corresponding to these response options. The non-adherence score was each respondent’s mean number of points across all rounds of follow-up visits, with a higher value indicating poorer adherence to antihypertensive medication. For health behaviors, current cigarette use was defined as self-report of smoking, regardless of frequency, and alcohol use was based on self-report of active drinking, regardless of type or frequency. Sedentary lifestyle was defined as spending more than half of the day seated during most days per week in the past month. Daily added salt was determined based on participant response to the question: ‘Do you add salt to your food on a daily basis?’Summary statistics were generated to describe characteristics of participants at baseline, overall and stratified by sex. We used rank sum and chi-square tests  to compare factors associated with blood pressure control over the one year among all individuals on treatment for hypertension at baseline, and to evaluate factors associated with hypertension incidence among those without hypertension at baseline. Finally, a multivariate analysis was performed to evaluate blood pressure control among all individuals on treatment for hypertension at baseline. For all statistical analyses, significance was defined as p < 0.050.Since no standard definitions for defining longitudinal hypertension control exist in Malawi, we performed a sensitivity analysis for factors associated with hypertension control using a strict definition, which required blood pressure at all visits <140 mm Hg systolic and <90 mm Hg diastolic versus uncontrolled, defined as any blood pressure during the year ≥140 mm Hg systolic and/or ≥90 mm Hg diastolic.We enrolled 704 adults on ART in the study between November 2015 and October 2017. Thirty-three individuals (4.7%) were excluded from analyses because they were lost from the cohort before two follow-up visits were completed  leaving a total of 671 individuals in the observational cohort study.Table Diabetes was present in 3.0% of the population (n = 20) and rates of tobacco and alcohol use were low . Women were slightly younger than men , were less likely to report smoking  and alcohol use , had higher body mass index , and were less likely to report a sedentary lifestyle . Most participants had three follow-up visits after study entry . The remainder had either two  or four visits  after entry.Figure Of those on antihypertensive medication at entry, 19.0% n = 30) achieved blood pressure control over the year of follow-up. Those with uncontrolled blood pressure were more likely to have medication class switches (54.7% versus 20.7%), medication additions (31.3% versus 10.3%), and discontinuations (12.5% versus 0%) . This was more common among those with blood pressure control  compared to those without control . Only three individuals had a non-adherence score higher than two and all had uncontrolled blood pressure (Table Approximately 20% (n = 102) of participants not known to be hypertensive at study entry (based on prescription data) developed hypertension over one year of follow-up. Of these, 26.5% (n = 27) had a blood pressure < 140/90 mm Hg at entry, and 73.5% (n = 75) had a systolic blood pressure ≥140 mm Hg and/or diastolic blood pressure ≥90 mm Hg. Of those with incident hypertension, 62 (60.8%) were started on antihypertensive medication. Of those not started on antihypertensive medication, 82.5% had mild hypertension at all visits. Blood pressure control could not be assessed among those started on medications due to limited follow-up time within this group.Side effects from antihypertensive medication were reported by 99 of 220 individuals (45.0%) taking these medications over the course of study follow-up and are described in Table Of those on antihypertensive medication at baseline, 74/158 (46.8%) reported one or more barriers to adherence. Among the reported barriers at baseline, the most common were not having enough money to purchase antihypertensive medications , difficulty remembering to take medications daily , lack of funds for transport to clinic for refills , and feeling like the medications were no longer needed due to good health  : the average adherence score was 0.22 for those with controlled blood pressure and 0.61 for those with uncontrolled blood pressure (p = 0.009) Table . AlthougIn a sensitivity analysis in which blood pressure control was defined as <140/90 mm Hg at all visits and uncontrolled as any visit with a blood pressure ≥140 mm Hg and/or ≥90 mm Hg, no statistically significant associations were identified; however, there was a trend towards higher non-adherence scores among those in the uncontrolled group  (Supplementary Table 1).In a sex-stratified analysis of factors associated with blood pressure control (Supplementary Table 2), the average non-adherence score remained strongly associated for men . Although women with uncontrolled hypertension had higher non-adherence scores compared to women with controlled blood pressure (0.62 versus 0.32), this difference did not achieve statistical significance. Men with controlled hypertension were on ART for a median of 10.7 years versus 6.6 years for those with uncontrolled hypertension (p = 0.01); however, there was no significant difference in ART duration for women .Individuals with incident hypertension were slightly older , and had a higher body mass index  compared to those who did not have incident hypertension  within an integrated chronic disease clinic . Hyperte2, and history of an elevated blood pressure in the prior year were all independently associated with uncontrolled hypertension [Risk factors for poor blood pressure control have not been well-characterized in people living with HIV and hypertension in Africa. In the aforementioned SEARCH study, men  and individuals over age 50 years  were less likely to have controlled blood pressure . A case-rtension . Our stuOur study is among the first to longitudinally evaluate adherence to antihypertensive medication therapy and blood pressure control among people living with HIV in Africa. A study from Ghana in people without HIV also found non-adherence to antihypertensive medication to be associated with poorly controlled blood pressure, in addition to male sex, concurrent diabetes, a higher number of antihypertensive medications, and lower glomerular filtration rate . In our The cost of hypertension medications emerged as a consistent barrier for individuals in our cohort. While care for HIV is provided for free in Malawi and similar settings, free medication for NCDs is limited to specific government clinics, which can be overwhelmed with patients and often have medication stock-outs or have limited medications for the management of NCDs. At Partners in Hope, while HIV care (including ART) and hypertension screening and clinical management are provided free-of-charge, medications for NCDs were only available for purchase during the period under study. While patients can travel to a government clinic to obtain free medications, the additional time  and cost of transport may serve as a significant barrier to obtaining refills elsewhere. Previous research by our group showed that despite the costs of medications, integrated care in which medications for hypertension are purchased at the point of care does provide cost savings for people living with HIV due to the efficiencies created by avoiding the time and opportunity costs of seeking free medications elsewhere . Real wo251527We hypothesize several mechanisms for poor blood pressure control in this population that merit further study and may be amenable to intervention. First, it is possible that poor medication quality , which has been documented in African settings including for NCDs 3031, may30Our study sample is small and the number of participants with hypertension was smaller than anticipated, which limited our power to identify factors associated with blood pressure control, as well as to perform a robust multivariate analysis. We did not collect data on the number of individuals approached who declined study participation nor the demographics of this group, and therefore cannot determine whether those enrolled differ from those who did not join the cohort. We collected data on the prevalence of diabetes by self-report and therefore likely underestimate the true prevalence of this condition in our study population. We did not have information about whether hypertension had been formally diagnosed by a provider among those with elevated blood pressure but not on antihypertensive medication at study entry, and we could not rely on self-report due to individuals’ limited knowledge about previous blood pressure measurements. It is unclear whether these individuals had been diagnosed with hypertension and given lifestyle counseling, or whether they were truly incident cases of hypertension. This may have resulted in an underestimation of hypertension prevalence and overestimation of hypertension incidence. We could not evaluate rates of blood pressure control in those with incident hypertension due to limited follow-up time on study. All adherence data was from self-report and could result in over- or under-estimation of actual pill-taking behavior. At the time under study, viral load scale up was just beginning in Malawi with the recommendation for viral load monitoring every two years; therefore, viral load data was available for only a small subset of our cohort and we thus could not explore the relationship between viral suppression and hypertension control. This is a single center experience from an urban clinic and findings may not be generalizable to other settings.Adults living with HIV and hypertension in our cohort had low rates of blood pressure control associated with self-reported non-adherence to antihypertensive medication. Given the high prevalence and incidence of hypertension in Malawi and similar countries in southern Africa, interventions to improve blood pressure control are needed to prevent associated long-term cardio- and cerebrovascular morbidity and mortality. Further research is also needed on strategies that can help individuals overcome barriers to antihypertensive medication adherence, including evaluation of models of integrated care for HIV and NCDs. Ultimately, major gains in rates of hypertension control will be needed to optimize the benefits of ART for improving life expectancy.The additional file for this article can be found as follows:10.5334/gh.1081.s1Supplementary Figure 1.Changes in blood pressure treatment from first study visit to final visit among participants with controlled hypertension (panel A) and participants with uncontrolled hypertension (panel B).10.5334/gh.1081.s2Supplementary Table 1.Factors associated with uncontrolled blood pressure over one year of follow-up in which controlled blood pressure was defined as <140/90 mm Hg at all follow-up visits.10.5334/gh.1081.s3Supplementary Table 2.Factors associated with uncontrolled blood pressure over one year of follow-up, stratified by sex, among individuals diagnosed with hypertension and on antihypertensive medication at baseline.10.5334/gh.1081.s4Supplementary Table 3.Factors associated with incident hypertension, stratified by sex, during one year of follow-up.


We aimed to develop and validate a new nomogram for predicting the risk of intracranial hemorrhage (ICH) in patients with acute ischemic stroke (AIS) after intravenous thrombolysis (IVT).n = 387) and the testing set . The factors in the predictive nomogram were filtered using multivariable logistic regression analysis. The performance of the nomogram was assessed based on the area under the receiver operating characteristic curve (AUC-ROC), calibration plots, and decision curve analysis (DCA).A retrospective study enrolled 553 patients with AIS treated with IVT. The patients were randomly divided into two cohorts: the training set . The calibration plot demonstrated good agreement in both the training and testing sets. DCA indicated that the nomogram was clinically useful.After multivariable logistic regression analysis, certain factors, such as smoking, National Institutes of Health of Stroke Scale (NIHSS) score, blood urea nitrogen-to-creatinine ratio (BUN/Cr), and neutrophil-to-lymphocyte ratio (NLR), were found to be independent predictors of ICH and were used to construct a nomogram. The AUC-ROC values of the nomogram were 0.887 (95% CI: 0.842–0.933) and 0.776 (95% CI: 0.681–0.872) in the training and testing sets, respectively. The AUC-ROC of the nomogram was higher than that of the Multicenter Stroke Survey (MSS), Glucose, Race, Age, Sex, Systolic blood Pressure, and Severity of stroke (GRASPS), and stroke prognostication using age and NIH Stroke Scale-100 positive index (SPAN-100) scores for predicting ICH in both the training and testing sets (The new nomogram, which included smoking, NIHSS, BUN/Cr, and NLR as variables, had the potential for predicting the risk of ICH in patients with AIS after IVT. Stroke is the second leading cause of death and a major leading contributor to disability worldwide . IntraveSince ICH may lead to poor prognosis, a reliable scoring tool that can be used to identify the risk of post-thrombolysis ICH is essential. Several scoring systems have been applied to predict the risk of ICH after thrombolysis –10. HoweIn this study, we aimed to develop a new nomogram to predict the risk of ICH after IVT therapy and to compare the performance of our model with that of other scoring systems for predicting ICH. The nomogram we developed will help clinicians identify patients with AIS with a higher risk of ICH after IVT therapy.The present study was a retrospective study performed in the stroke center of the Central Hospital of Shaoyang between November 2013 and January 2021. The patients met the following inclusion criteria: 1) more than 18 years of age; 2) diagnosed with AIS confirmed by MRI within 24 h after admission; 3) onset-to-treatment time for thrombolysis of <4.5 h; 4) they or their legal representatives provided written informed consent to participation in the study; and 5) treated with IVT. The exclusion criteria were as follows: 1) treated with endovascular procedures after IVT; and) not treated with IVT or refused IVT treatment; and 3) medical contraindications for IVT.This study was approved by the Ethics Committee of the Central Hospital of Shaoyang. The Ethics Committee approval letter is available in Online Resource 1.We collected demographic, clinical, and laboratory information at admission and during hospitalization. The baseline data included age, sex, current smoking status, current drinking status, NIHSS score on admission, systolic blood pressure (SBP), diastolic blood pressure (DBP), blood glucose level on admission, history of hypertension and diabetes mellitus, atrial fibrillation (AF), international normalized ratio (INR), activated partial thromboplastin time (APTT), PLT, fibrinogen, albumin (ALB), white blood cells (WBC), neutrophil-to-lymphocyte ratio (NLR), high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglyceride (TG), total cholesterol (TC), blood urea nitrogen-to-creatinine ratio (BUN/Cr), and uric acid (UA).All patients received rt-PA treatment within 4.5 h after the onset of stroke. Intravenous rt-PA at 0.9 mg/kg (90 mg maximum) was used; 10% of the total dose was administered as an intravenous bolus, followed by infusion of the remaining dose over 60 min .All patients underwent a CT scan on admission, and each patient underwent another CT scan 24 h after intravenous rt-PA administration. In cases in which the patient's neurological function deteriorated rapidly, a CT scan was immediately performed to assess the presence of ICH. According to the European Cooperative Acute Stroke Study-2 criteria , the disn = 387) and the testing set (n = 166), at a theoretical ratio of 7:3. Categorical variables and continuous variables are expressed as frequencies  and means (SDs) or medians , respectively. The differences in baseline characteristics between the training set and the testing set were assessed using Student's t-test or the non-parametric Mann–Whitney U test for continuous variables and the χ2 or Fisher's exact test for categorical variables. The least absolute shrinkage and selection operator (LASSO) method was used to screen the optimal risk factors related to ICH in the total dataset. The variables with non-zero coefficients in the LASSO regression model were selected for further analysis. In the training cohort, univariable logistic regression analysis was used to predict the probability of ICH. The variables with P-value <0.05 were added to the multivariable logistic regression analysis to screen for independent clinical predictors related to ICH. A nomogram was generated based on these risk factors in multivariable analysis. The area under the receiver operating characteristic curve (AUC-ROC) analysis was performed to assess the predictive accuracy of the nomogram, and calibration curves were drawn to compare the predicted probabilities with the observed probabilities. Decision curve analysis (DCA) was used to evaluate the clinical value of the predictive model.A total of 553 patients were randomly divided into two groups, the training set  score, the Glucose, Race, Age, Sex, Systolic blood Pressure, and Severity of stroke (GRASPS) score, and the stroke prognostication using age and NIH Stroke Scale-100 positive index (SPAN-100) score. The AUC-ROC was used to estimate the accuracy and discrimination of the nomogram and these scoring systems in the training and testing sets. DeLong's test was performed to compare the differences in AUC-ROC between the ICH nomogram and the MSS score, GRASPS score, and SPAN-100 score.http://www.R-project.org/) and SPSS version 27.0 . Two-tailed values of p < 0.05 were considered statistically significant.Statistical analyses were performed using R version 3.6.3 software .The training set consisted of 387 individuals, and the remaining 166 individuals were included in the testing set. The baseline characteristics of the patients in the training and testing sets are shown in Through searching and analysis of the related literature, 24 potential risk factors among the demographic, clinical, and laboratory indicators of the patients were included in the LASSO regression analysis . The varp < 0.05), as shown in A univariable analysis of the training set revealed that smoking, AF, NIHSS, BUN/Cr, and NLR were related to ICH. These factors were therefore used in multivariable logistic regression analysis for screening independent clinical predictors of ICH. Multivariable logistic regression analysis demonstrated that four variables  are independently associated with ICH  were used to build a nomogram for predicting the risk of ICH in AIS patients with IVT . The ratp < 0.05). Similar to the results obtained with the training set, the value of AUC-ROC in our model  was superior to the values obtained using the MSS scores , the GRASPS scores , and the SPAN-100 scores  in the testing set (p < 0.05).To assess the performance of the nomogram in predicting ICH, the likelihood that each patient would experience ICH was also predicted based on his or her MSS scores, GRASPS scores, and SPAN-100 scores. We compared the discrimination of our nomogram with those obtained using MSS scores, GRASPS scores, and SPAN-100 scores using AUC-ROC. As shown in We developed a new nomogram based upon smoking, NIHSS scores, BUN/Cr, and NLR for use in predicting the risk of ICH after IVT therapy. The developed nomogram demonstrated good discrimination and calibration in the training and testing sets. Furthermore, the AUC-ROC curve obtained using our nomogram showed better performance than the curves obtained using the MSS scores, the GRASPS scores, and the SPAN-100 score in both the training and testing sets. In addition, the DCA results suggested that the developed nomogram has a marked net benefit for predicting the risk of ICH.p < 0.05). The better performance of our nomogram may be explained as follows. First, the three scoring systems with which it was compared converted continuous variables to dichotomization/categorization variables; this conversion is statistically inefficient and may decrease the accuracy of prediction  scores and the hemorrhage after thrombolysis (HAT) scores rely on CT images to detect early ischemic changes and hyperdense cerebral artery signs , 7. Howeediction . Second,ediction , 22. In ediction –25.n = 345). In addition, the percentage of ICH based on this nomogram was 14.5%, significantly higher than the percentage of ICH in Asian patients  nomogram was constructed to predict ICH in stroke patients after IVT treatment in Italy . Howeverpatients , 30. TheIn this study, smoking, NIHSS scores, BUN/Cr, and NLR were shown to be independent predictors of ICH in patients with AIS after rt-PA administration. Consistent with previous studies, smoking and NIHSS were common risk factors for predicting ICH. A previous study reported that age ≥68 years, smoking, AF, SP ≥149 mmHg 2 h after rt-PA administration, and NIHSS scores ≥17 before thrombolysis were associated with the risk of ICH after IVT . In WangThe NLR is another new risk factor we found that can contribute to the risk of ICH. Neuroinflammation is related to the entire pathological process of stroke. The NLR is a readily accessible and reproduces new inflammatory biomarker . It was Generally, the nomogram developed in this study, which includes four parameters, is convenient and efficient for use in the management of patients with AIS. First, the predictors used in our predictive nomogram are easily available at almost all medical centers, even those in poor areas. In addition, the nomogram is particularly suitable for use by non-neurologists because CT imaging is excluded. Furthermore, the discrimination and calibration performance of the nomogram was good; therefore, it could be a reliable tool for predicting the risk of ICH in patients with AIS after rt-PA treatment.Our study has some limitations. First, our data came from a single-center retrospective analysis, and this might have limited the statistical power of the results. Second, our model has not been validated in external cohorts. Prospective, multicenter studies will be required in the future to assess the applicability of our nomogram. Third, data on onset-to-treatment time for thrombolysis or administration of oral antiplatelet drugs or anticoagulants, all of which may be risk factors for ICH, were not available in our study.In conclusion, the new nomogram that includes smoking, NIHSS, BUN/Cr, and NLR may predict the risk of ICH after IVT in AIS patients in the Asian population.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of the Central Hospital of Shaoyang. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.A-DX, DL, Z-AW, and X-XH contributed to the study conception and design. Material preparation, data collection were performed by DD, Z-GY, S-YL, J-KZ, Y-FLi, Y-FLiu, Y-SW, T-YZ, and X-LS. Analysis and interpretation of the data were done by Z-AW. The first draft of the manuscript was written by Z-AW, X-XH, A-DX, and DL. All authors read and approved the final manuscript.This work was supported by grants from the National Natural Science Foundation of China , the Science and Technology Planning Project of Guangdong Province, China (2017A020215049 and 2019A050513005), Natural Science Foundation of Guangdong Province (2018A0303130182 and 2020A1515010279), Postdoctoral Research Foundation of China (2018M643370 and 2021M701419), Guangzhou Science and Technology Planning Project (201508020004), Technology and People's Livelihood Major Project of Guangzhou (2014Y2-00505), and the Fundamental Research Funds for the Central Universities (21621102).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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