Histological Evolution of BK Virus-Associated Nephropathy: Importance of Integrating Clinical and Pathological Findings.

Long-term clinicopathological studies of BK-associated nephropathy (PyVAN) are not available. We studied 206 biopsies (71 patients), followed 3.09 ± 1.46 years after immunosuppression reduction. The biopsy features (% immunostain for PyV large T ag + staining and inflammation ± acute rejection) were correlated with viral load dynamics and serum creatinine to define the clinicopathological status (PyVCPS). Incidence of acute rejection was 28% in the second biopsy and 50% subsequently (25% mixed T cell-mediated allograft rejection (TCMR) + antibody-mediated allograft rejection (AMR); rejection overall affected 38% of patients (>50% AMR). Graft loss was 15.4% (0.8-5.3 years after PyVAN); 76% had complete viral clearance (mean 28 weeks). The only predictors of graft loss were acute rejection (TCMR p = 0.008, any type p = 0.07), and increased "t" and "ci" in the second biopsy (p = 0.006 and 0.048). Higher peak viremia correlated with poorer viral clearance (p = 0.002). Presumptive and proven PyVAN had similar presentation, evolution, and outcome. Late PyVAN (>2 years, 9.8%) justifies BK viremia evaluation at any point with graft dysfunction and/or biopsy evaluation. This study describes the histological evolution of PyVAN and corresponding clinicopathological correlations. Although the pathological features overall reflect the viral and immunological interactions, the PyVAN course remains difficult to predict based on any single feature. Appropriate clinical management requires repeat biopsies and determination of the PyVCPS at relevant time points, for corresponding personalized immunosuppression adjustment.

Real Time Central Assessment of Kidney Transplant Indication Biopsies by Microarrays: The INTERCOMEX Study.

The authors conducted a prospective trial to assess the feasibility of real time central molecular assessment of kidney transplant biopsy samples from 10 North American or European centers. Biopsy samples taken 1 day to 34 years posttransplantation were stabilized in RNAlater, sent via courier overnight at ambient temperature to the central laboratory, and processed (29 h workflow) using microarrays to assess T cell- and antibody-mediated rejection (TCMR and ABMR, respectively). Of 538 biopsy samples submitted, 519 (96%) were sufficient for microarray analysis (average length, 3 mm). Automated reports were generated without knowledge of histology and HLA antibody, with diagnoses assigned based on Molecular Microscope Diagnostic System (MMDx) classifier algorithms and signed out by one observer. Agreement between MMDx and histology (balanced accuracy) was 77% for TCMR, 77% for ABMR, and 76% for no rejection. A classification tree derived to provide automated sign-outs predicted the observer sign-outs with >90% accuracy. In 451 biopsy samples where feedback was obtained, clinicians indicated that MMDx more frequently agreed with clinical judgment (87%) than did histology (80%) (p = 0.0042). In 81% of feedback forms, clinicians reported that MMDx increased confidence in management compared with conventional assessment alone. The authors conclude that real time central molecular assessment is feasible and offers a useful new dimension in biopsy interpretation. ClinicalTrials.gov NCT#01299168.

Primary Epstein-Barr virus infection, seroconversion, and post-transplant lymphoproliferative disorder in seronegative renal allograft recipients: a prospective cohort study.

BACKGROUND: Epstein-Barr virus (EBV)-seronegative renal transplant recipients are at risk of post-transplant lymphoproliferative disorder (PTLD). We compared primary EBV infection, seroconversion, and PTLD in EBV-seronegative patients who received renal allograft from seropositive or seronegative donors (D+/R- and D-/R-, respectively). METHODS: We prospectively followed 25 D+/R- and 8 D-/R- recipients. We followed patients from January 1999 to June 2009 with clinical visits, monthly EBV polymerase chain reaction tests, and serologic tests for a period of 1 year after kidney transplantation and on an individual basis thereafter. RESULTS: Three patients (9%) developed PTLD including 2 early-onset (<12 months) and 1 late-onset (>12 months) disease. In D+/R- and D-/R- patients, the frequencies of PTLD (8% vs. 12.5%, P = 0.7), EBV seroconversion (64% vs. 50%, P = 0.4), and EBV viremia (40% vs. 25%, P = 0.6) were not significantly different. Clinical, serologic, and virologic surveillance as well as reduction in immunosuppression after evidence of primary EBV infection resulted in a PTLD rate of 9%, despite a seroconversion rate of 60.6%. Rate of graft loss after reduction in immunosuppression was 10% (2 of 20), which was not significantly different from 13 patients without EBV seroconversion (no graft loss, P = 0.5). Rates of viremia, seroconversion, and PTLD in D+/R- and D-/R- patients appear to be similar. CONCLUSIONS: The incidence of PTLD in renal transplants ranges from 0.5% to 2.9%. Our data show a significantly higher rate in EBV-seronegative renal allograft recipients, suggesting the need for close surveillance. Our data also suggest that donors for EBV-seronegative recipients may be accepted irrespective of positive or negative serostatus, with ongoing surveillance important in either circumstance.

Developing Bioactive Dental Resins for Restorative Dentistry.

Despite its reputation as the most widely used restorative dental material currently, resin-based materials have acknowledged shortcomings. As most systematic survival studies of resin composites and dental adhesives indicate, secondary caries is the foremost reason for resin-based restoration failure and life span reduction. In subjects with high caries risk, the microbial community dominated by acidogenic and acid-tolerant bacteria triggers acid-induced deterioration of the bonding interface and/or bulk material and mineral loss around the restorations. In addition, resin-based materials undergo biodegradation in the oral cavity. As a result, the past decades have seen exponential growth in developing restorative dental materials for antimicrobial applications addressing secondary caries prevention and progression. Currently, the main challenge of bioactive resin development is the identification of efficient and safe anticaries agents that are detrimental free to final material properties and show satisfactory long-term performance and favorable clinical translation. This review centers on the continuous efforts to formulate novel bioactive resins employing 1 or multiple agents to enhance the antibiofilm efficacy or achieve multiple functionalities, such as remineralization and antimicrobial activity antidegradation. We present a comprehensive synthesis of the constraints and challenges encountered in the formulation process, the clinical performance-related prerequisites, the materials' intended applicability, and the current advancements in clinical implementation. Moreover, we identify crucial vulnerabilities that arise during the development of dental materials, including particle aggregation, alterations in color, susceptibility to hydrolysis, and loss of physicomechanical core properties of the targeted materials.

Zoonotic bacteria, antimicrobial use and antimicrobial resistance in ornamental fish: a systematic review of the existing research and survey of aquaculture-allied professionals.

Using systematic review methodology, global research reporting the frequency of zoonotic bacterial pathogens, antimicrobial use (AMU) and antimicrobial resistance (AMR) in ornamental fish, and human illness due to exposure to ornamental fish, was examined. A survey was performed to elicit opinions of aquaculture-allied personnel on the frequency of AMU and AMR in ornamental fish. The most commonly reported sporadic human infections were associated with Mycobacterium marinum, while Salmonella Paratyphi B var. Java was implicated in all reported outbreaks. Aeromonas spp. were most frequently investigated (n=10 studies) in 25 studies surveying ornamental fish from various sources. High levels of resistance were reported to amoxicillin, penicillin, tetracycline and oxytetracycline, which was also in agreement with the survey respondents' views. Studies on AMU were not found in our review. Survey respondents reported frequent use of quinolones, followed by tetracyclines, nitrofurans, and aminoglycosides. Recommendations for future surveillance and public education efforts are presented.

Association of serum uric acid with graft survival after kidney transplantation: a time-varying analysis.

The association of serum uric acid (UA) with kidney transplant outcomes is uncertain. We examined the predictive value of UA during the first year posttransplant as a time-varying factor for graft survival after adjustment for time-dependent and independent confounding factors. Four hundred and eighty-eight renal allograft recipients transplanted from January 2004 to June 2006 and followed for 41.1 ± 17.7 months were included. Data on UA, estimated glomerular filtration rate (eGFR), tacrolimus level, mycophenolate mofetil (MMF) and prednisone doses, use of allopurinol, angiotensin-converting enzyme-inhibitor/angiotensin-receptor-blocker (ACEi/ARB) and diuretics at 1, 3, 6, 9 and 12 months were collected. Primary endpoint of the study was graft loss, defined as graft failure and death. Cox proportional hazard models and generalized estimating equations were used for analysis. UA level was associated with eGFR, gender, retransplantation, decease-donor organ, delayed graft function, diuretics, ACEi/ARB and MMF dose. After adjustment for these confounders, UA was independently associated with increased risk of graft loss (HR: 1.15, p = 0.003; 95% CI: 1.05-1.27). Interestingly, UA interacted with eGFR (HR: 0.996, p < 0.05; 95% CI: 0.993-0.999 for interaction term). Here, we report a significant association between serum UA during first year posttransplant and graft loss, after adjustment for corresponding values of time-varying variables including eGFR, immunosuppressive drug regimen and other confounding factors. Its negative impact seems to be worse with lower eGFR.

The mRNA landscape at yeast translation initiation sites.

SUMMARY: Although translation initiation has been well studied, many questions remain in elucidating its mechanisms. An ongoing challenge is to understand how ribosomes choose a translation initiation site (TIS). To gain new insights, we analyzed large sets of TISs with the aim of identifying common characteristics that are potentially of functional importance. Nucleotide sequence context has previously been demonstrated to play an important role in the ribosome's selection of a TIS, and mRNA secondary structure is also emerging as a contributing factor. Here, we analyze mRNA secondary structure using the folding predictions of the RNAfold algorithm. We present a method for analyzing these results using a rank-ordering approach to assess the overall degree of predicted secondary structure in a given region of mRNA. In addition, we used a modified version of the algorithm that makes use of only a subset of the standard version's output to incorporate base-pairing polarity constraints suggested by the ribosome scanning process. These methods were employed to study the TISs of 1735 genes in Saccharomyces cerevisiae. Trends in base composition and base-pairing probabilities suggest that efficient translation initiation and high protein expression are aided by reduced secondary structure upstream and downstream of the TIS. However, the downstream reduction is not observed for sets of TISs with nucleotide sequence contexts unfavorable for translation initiation, consistent with previous suggestions that secondary structure downstream of the ribosome can facilitate TIS recognition.

Revealing non-analytic kinematic shifts in smooth goal-directed behaviour.

How do biological agents plan and organise a smooth accurate path to shift from one smooth mode of behaviour to another as part of graceful movement that is both plastic and controlled? This paper addresses the question in conducting a novel shape analysis of approach and adjustment phases in rapid voluntary target aiming and 2-D reaching hand actions. A number of mode changing experiments are reported that investigate these actions under a range of goals and conditions. After a typically roughly aimed approach, regular projective adjustment is observed that has height and velocity kinematic profiles that are scaled copies of one another. This empirical property is encapsulated as a novel self-similar shift function. The mathematics shows that the biological shifts consist of continual deviation from their full Taylor series everywhere throughout their interval, which is a deep form of plasticity not described before. The experimental results find the same approach and adjustment strategy to occur with behavioural trajectories over the full and varied range of tested goals and conditions. The trajectory shapes have a large degree of predictability through using the shift function to handle extensive variation in the trajectories' adjustment across individual behaviours and subjects. We provide connections between the behavioural features and results and various neural studies to show how the methodology may be exploited. The conclusion is that a roughly aimed approach followed by a specific highly plastic shift adjustment can provide a regular basis for fast and accurate goal-directed motion in a simple and generalisable way.

Bifunctional Composites for Biofilms Modulation on Cervical Restorations.

Cervical composites treating root carious and noncarious cervical lesions usually extend subgingivally. The subgingival margins of composites present poor plaque control, enhanced biofilm accumulation, and cause gingival irritation. A potential material to restore such lesions should combine agents that interfere with bacterial biofilm development and respond to acidic conditions. Here, we explore the use of new bioresponsive bifunctional dental composites against mature microcosm biofilms derived from subgingival plaque samples. The designed formulations contain 2 bioactive agents: dimethylaminohexadecyl methacrylate (DMAHDM) at 3 to 5 wt.% and 20 wt.% nanosized amorphous calcium phosphate (NACP) in a base resin. Composites with no DMAHDM and NACP were used as controls. The newly formulated 5% DMAHDM-20% NACP composite was analyzed by micro-Raman spectroscopy and transmission electron microscopy. The wettability and surface-free energy were also assessed. The inhibitory effect on the in vitro biofilm growth and the 16S rRNA gene sequencing of survival bacterial colonies derived from the composites were analyzed. Whole-biofilm metabolic activity, polysaccharide production, and live/dead images of the biofilm grown over the composites complement the microbiological assays. Overall, the designed formulations had higher contact angles with water and lower surface-free energy compared to the commercial control. The DMAHDM-NACP composites significantly inhibited the growth of total microorganisms, Porphyromonas gingivalis, Prevotella intermedia/nigrescens, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum by 3 to 5-log (P < 0.001). For the colony isolates from control composites, the composition was typically dominated by the genera Veillonella, Fusobacterium, Streptococcus, Eikenella, and Leptotrichia, while Fusobacterium and Veillonella dominated the 5% DMAHDM-20% NACP composites. The DMAHDM-NACP composites contributed to over 80% of reduction in metabolic and polysaccharide activity. The suppression effect on plaque biofilms suggested that DMAHDM-NACP composites might be used as a bioactive material for cervical restorations. These results may propose an exciting path to prevent biofilm growth and improve dental composite restorations' life span.

The detrimental effect of poor early graft function after laparoscopic live donor nephrectomy on graft outcomes.

We undertook this study to assess the rate of poor early graft function (EGF) after laparoscopic live donor nephrectomy (lapNx) and to determine whether poor EGF is associated with diminished long-term graft survival. The study population consisted of 946 consecutive lapNx donors/recipient pairs at our center. Poor EGF was defined as receiving hemodialysis on postoperative day (POD) 1 through POD 7 (delayed graft function [DGF]) or serum creatinine >/= 3.0 mg/dL at POD 5 without need for hemodialysis (slow graft function [SGF]). The incidence of poor EGF was 16.3% (DGF 5.8%, SGF 10.5%), and it was stable in chronologic tertiles. Poor EGF was independently associated with worse death-censored graft survival (adjusted hazard ratio (HR) 2.15, 95% confidence interval (CI) 1.34-3.47, p = 0.001), worse overall graft survival (HR 1.62, 95% CI 1.10-2.37, p = 0.014), worse acute rejection-free survival (HR 2.75, 95% CI 1.92-3.94, p < 0.001) and worse 1-year renal function (p = 0.002). Even SGF independently predicted worse renal allograft survival (HR 2.54, 95% CI 1.44-4.44, p = 0.001). Risk factors for poor DGF included advanced donor age, high recipient BMI, sirolimus use and prolonged warm ischemia time. In conclusion, poor EGF following lapNx has a deleterious effect on long-term graft function and survival.

Open-field behavior in mice: evidence for a major gene effect mediated by the visual system.

In segregating F(2), F(3), and F(4) generations, albino mice had lower activity and higher defecation scores than pigmented animals when tested in a brightly lighted open field. These differences persisted when members of an F(5) generation were tested under white light, but largely disappeared under red light. Thus it was concluded that there is a major gene effect on the quantitative traits of open-field activity and defecation which is mediated by the visual system and that albino mice are more photophobic than pigmented mice under conditions of bright illumination.

Incorporating time postinoculation into a dose-response model of Yersinia pestis in mice.

AIMS: To develop a time-dependent dose-response model for describing the survival of animals exposed to Yersinia pestis. METHODS AND RESULTS: Candidate time-dependent dose-response models were fitted to a survival data set for mice intraperitoneally exposed to graded doses of Y. pestis using the maximum likelihood estimation method. An exponential dose-response model with the model parameter modified by an inverse-power dependency of time postinoculation provided a statistically adequate fit to the experimental survival data. This modified model was verified by comparison with prior studies. CONCLUSIONS: The incorporated time dependency quantifies the expected temporal effect of in vivo bacteria growth in the dose-response relationship. The modified model describes the development of animal infectious response over time and represents observed responses accurately. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to incorporate time in a dose-response model for Y. pestis infection. The outcome may be used for the improved understanding of in vivo bacterial dynamics, improved postexposure decision making or as a component to better assist epidemiological investigations.

The dual impact of rehabilitation admission and race/ethnicity on discharges against medical advice.

INTRODUCTION: Predictors of hospital discharges against medical advice (AMA), including race/ethnicity, have been examined previously. However, the predictive effect of an admission for rehabilitative care has not been examined in an inpatient stroke population, nor has the impact of race/ethnicity on this relationship been reported. METHODS: Live hospital discharges with a primary diagnosis of stroke from 2000-2005 were identified in a longitudinal dataset. The outcome of interest was a discharge AMA. A hierarchical logistic model was estimated to examine the effect of race/ethnicity and rehabilitative care on the outcome while controlling for patient and hospital characteristics. RESULTS: A total of 569 of the 79,561 stroke admissions (0.7%) ended in a discharge AMA. There were 1,565 admissions for rehabilitative care and 32% of patients were non-Caucasian. Among Caucasians, adjusted odds of a discharge AMA were higher for patients with an admission for rehabilitative care (AOR = 3.83, p < 0.0001). Among those not admitted for rehabilitative care, non-Caucasian patients were more likely to leave AMA (AOR = 1.4; p = 0.0005). CONCLUSIONS: This study identifies dependence between race/ethnicity, rehabilitative care, and discharges AMA. More research is needed to understand the implications of differential rates of discharges AMA among race/ethnic groups and across patient care settings.

Nicastrin, a presenilin-interacting protein, contains an aminopeptidase/transferrin receptor superfamily domain.

Nicastrin, a protein implicated in Alzheimer's disease, has a domain that is found in the aminopeptidase/transferrin receptor superfamily. In nicastrin, this domain might possess catalytic activity (as observed with aminopeptidases) or it could serve merely as a binding domain (with analogy to the transferrin receptors) for the beta-amyloid precursor protein.

Mammalian target of rapamycin inhibitor dyslipidemia in kidney transplant recipients.

The incidence, pathogenesis, consequences and treatment of mammalian target of rapamycin (mTOR) inhibitor dyslipidemia are not well described. We conducted a systematic review of randomized controlled trials reporting cholesterol and triglycerides in mTOR versus non-mTOR inhibitor immunosuppressive treatment regimens in kidney transplant recipients. All but one of 17 trials reported higher levels of cholesterol and triglycerides, or an increased prevalence of treatment with lipid-lowering agents. Approximately 60% of mTOR inhibitor-treated patients received lipid-lowering agents (2-fold higher than controls). There appeared to be little difference between dyslipidemias caused by sirolimus (14 trials) versus everolimus (3 trials). It was difficult to determine the extent to which declines in lipids over time posttransplant were due to lipid-lowering therapy, changes in doses and/or discontinuations of mTOR inhibitors. From the four trials that measured lipoproteins, it appeared that at least some of the increase in total cholesterol with mTOR inhibitors was due to increased low-density lipoprotein cholesterol. What direct or indirect effects mTOR inhibitors have on atherosclerotic cardiovascular disease in kidney transplant patients are unknown. However, in the absence of the necessary clinical trials, dyslipidemia should be managed, as it would be in nontransplant patients at high risk for cardiovascular disease.

Optimizing early de novo immunosuppression with sirolimus.

In the past, renal transplant deterioration was approached as a problem of "chronic rejection," which implied that deterioration of kidney function occurred because of past injuries that programmed the kidney for loss of function. New evidence suggests that deterioration occurs because of a new or recent source of injury. If a graft is losing function, the cause of deterioration needs to be identified. Some causes are treatable; those that are not should be diagnosed and further studied. To this end, we need to approach deterioration (loss of function or proteinuria) of renal transplant function by identifying distinct entities or components of the problem. Early and late kidney graft survival has improved considerably, owing to advances in clinical care, particularly immunosuppression. Many of the kidney transplants functioning today should serve their new owners for their life expectancy. The main cause of late kidney function deterioration remains allograft nephropathy. Often this reflects a failure of the immunosuppressive prescription. Subclinical rejection, chronic nephrotoxicity, recurrent disease, infections, or diabetes may contribute to this process. Optimal early and late immunosuppression is required to provide efficacy without attendant risk for graft dysfunction owing to nephrotoxicity. Because 1-year serum creatinine levels often provide an indication of long-term graft function, early evaluation of subtle degrees of graft dysfunction should prompt a graft biopsy to identify treatable causes.

Targeted localized degradation of Paired protein in Drosophila development.

BACKGROUND: Selective spatial regulation of gene expression lies at the core of pattern formation in the embryo. In the fruit fly Drosophila, localized transcriptional regulation accounts for much of the embryonic pattern. RESULTS: We identified a gene, partner of paired (ppa), whose properties suggest that localized receptors for protein degradation are integrated into regulatory networks of transcription factors to ensure robust spatial regulation of gene expression. We found that the Ppa protein interacts with the Pax transcription factor Paired (Prd) and contains an F-box, a motif found in receptors for ubiquitin-mediated protein degradation. In normal development, Prd functions only in cells in which ppa mRNA expression has been repressed by another segmentation protein, Even-skipped (Eve). When ppa was expressed ectopically in these cells, Prd protein, but not mRNA, levels diminished. When ppa function was removed from cells that express prd mRNA, Prd protein levels increased. CONCLUSIONS: Ppa co-ordinates Prd degradation and is important for expression of Prd to be correctly localized. In the presence of Ppa, Prd protein is targeted for degradation at sites where its mis-expression would disrupt development. In the absence of Ppa, Prd is longer-lived and regulates downstream target genes.

Effects of calcium phosphate nanoparticles on Ca-PO4 composite.

Nano-particles of dicalcium phosphate anhydrous (DCPA) were synthesized for the first time. The objectives of this study were to incorporate DCPA nano-particles into resin for Ca-PO(4) release to combat dental caries, and to investigate the filler level effects. Nano-DCPA and nano-silica-fused silicon nitride whiskers at a 1:1 ratio were used at filler mass fractions of 0-75%. The flexural strengths in MPa (mean +/- SD; n = 6) of DCPA-whisker composites ranged from (106 +/- 39) at 0% fillers to (114 +/- 23) at 75% fillers, similar to (112 +/- 22) of a non-releasing composite (TPH) (p > 0.1). The composite with 75% fillers in a NaCl solution (133 mmol/L, pH = 7.4, 37 degrees C) yielded a Ca concentration of (0.65 +/- 0.02) mmol/L and PO(4) of (2.29 +/- 0.07) mmol/L. Relationships were established between ion-release and DCPA volume fraction V(DCPA): Ca = 4.46 V(DCPA)(1.6,) and = 66.9 V(DCPA)(2.6). Nano-DCPA-whisker PO(4) composites had high strength and released high levels of Ca-PO(4) requisite for remineralization. These new nano-composites could provide the needed combination of stress-bearing and caries-inhibiting capabilities.

Synergistic activation of transcription is mediated by the N-terminal domain of Drosophila fushi tarazu homeoprotein and can occur without DNA binding by the protein.

Synergistic activation of transcription by Drosophila segmentation genes in tissue culture cells provides a model with which to study combinatorial regulation. We examined the synergistic activation of an engrailed-derived promoter by the pair-rule proteins paired (PRD) and fushi tarazu (FTZ). Synergistic activation by PRD requires regions of the homeodomain or adjacent sequences, and that by FTZ requires the first 171 residues. Surprisingly, deletion of the FTZ homeodomain does not reduce the capacity of the protein for synergistic activation, although this mutation abolishes any detectable DNA-binding activity. This finding suggests that FTZ can function through protein-protein interactions with PRD or other components of the homeoprotein transcription complex, adding a new layer of mechanisms that could underlie the functional specificities and combinatorial regulation of homeoproteins.

Targeting mechanisms of hypertensive vascular disease with dual calcium channel and renin-angiotensin system blockade.

Patients with hypertension, particularly those with diabetes mellitus, are at heightened risk for cardiovascular and renal disease. Accumulated evidence indicates that the majority of hypertensive patients at high risk will require more than one antihypertensive agent to reach their blood pressure (BP) target. A reasonable strategy is to use agents with complementary mechanisms of action to enhance BP-lowering efficacy and prevent target organ damage. In experimental models, the combination of a calcium channel blocker (CCB) with an agent that blocks the renin-angiotensin system (RAS), an angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker, improves measures of endothelial function, inflammation, ventricular remodelling and renal function to a greater degree than these classes given as monotherapy. In clinical trials, calcium channel/RAS blockade combination therapy has been shown to provide greater BP reductions and improve renal function in patients with diabetic and nondiabetic renal disease earlier and to a greater extent than monotherapy. In addition, dual calcium channel/RAS blockade increases arterial compliance, arterial distensibility and flow-mediated vasodilation. Expanding upon extensive research on the benefits of calcium channel blockade and RAS blockade for the prevention of vascular events and preclinical and clinical trial evidence suggests added effects of combination therapy by targeting the underlying mechanisms of hypertensive vascular disease.