Immunologic dysfunction, mediated via monocyte activity, has been implicated in the development of HIV-associated neurocognitive disorder (HAND). We hypothesized that transcriptome changes in peripheral blood monocytes relate to neurocognitive functioning in HIV+ individuals, and that such alterations could be useful as biomarkers of worsening HAND.
mRNA was isolated from the monocytes of 86 HIV+ adults and analyzed with the Illumina HT-12 v4 Expression BeadChip. Neurocognitive functioning, HAND diagnosis, and other clinical and virologic variables were determined. Data were analyzed using standard expression analysis and weighted gene co-expression network analysis (WGCNA).
Neurocognitive functioning was correlated with multiple gene transcripts in the standard expression analysis. WGCNA identified two nominally significant co-expression modules associated with neurocognitive functioning, which were enriched with genes involved in mitotic processes and translational elongation.
Multiple modified gene transcripts involved in inflammation, cytoprotection, and neurodegeneration were correlated with neurocognitive functioning. The associations were not strong enough to justify their use as biomarkers of HAND; however, the associations of two co-expression modules with neurocognitive functioning warrants further exploration.
HIV-associated neurocognitive disorder; NeuroAIDS; monocyte; IL6R; KEAP1; LRP12; CSNK1A1; WGCNA
The success of combination antiretroviral therapy (cART) in transforming the lives of HIV-infected individuals with access to these drugs is tempered by the increasing threat of HIV-associated neurocognitive disorders (HAND) to their overall health and quality of life. Intensive investigations over the past two decades have underscored the role of host immune responses, inflammation, and monocyte-derived macrophages in HAND, but the precise pathogenic mechanisms underlying HAND remain only partially delineated. Complicating research efforts and therapeutic drug development are the sheer complexity of HAND phenotypes, diagnostic imprecision, and the growing intersection of chronic immune activation with aging-related comorbidities. Yet, genetic studies still offer a powerful means of advancing individualized care for HIV-infected individuals at risk. There is an urgent need for 1) longitudinal studies using consistent phenotypic definitions of HAND in HIV-infected subpopulations at very high risk of being adversely impacted, such as children, 2) tissue studies that correlate neuropathological changes in multiple brain regions with genomic markers in affected individuals and with changes at the RNA, epigenomic, and/or protein levels, and 3) genetic association studies using more sensitive subphenotypes of HAND. The NIH Brain Initiative and Human Connectome Project, coupled with rapidly evolving systems biology and machine learning approaches for analyzing high-throughput genetic, transcriptomic and epigenetic data, hold promise for identifying actionable biological processes and gene networks that underlie HAND. This review summarizes the current state of understanding of host genetic factors predisposing to HAND in light of past challenges and suggests some priorities for future research to advance the understanding and clinical management of HAND in the cART era.
Combination antiretroviral therapy (cART); Central nervous system; CNS; HIV; HIV-associated neurocognitive disorders; HAND
The apolipoprotein E (APOE) ε4 allele enhances cerebral accumulation of β-amyloid (Aβ) and is a major risk factor for sporadic Alzheimer’s disease (AD). We hypothesized that HIV-associated neurocognitive disorders (HAND) would be associated with the APOE ε4 genotype and cerebral Aβ deposition.
Clinico-pathological study of HIV-infected adults from four prospective cohorts in the U.S. National NeuroAIDS Tissue Consortium.
We used multivariable logistic regressions to model outcomes (Aβ plaques [immunohistochemistry] and HAND [standard criteria]) on predictors (APOE ε4 [allelic discrimination assay], older age [≥ 50 years], Aβ plaques, and their two-way interactions) and co-morbid factors.
Isocortical Aβ deposits generally occurred as diffuse plaques and mild to moderate amyloid angiopathy. Isocortical phospho-Tau-immunoreactive neurofibrillary lesions were sparse. The APOE ε4 and older age were independently associated with the presence of Aβ plaques (adjusted odds ratio [OR] 10.16 and 5.77 [95% confidence interval (CI) 2.89–35.76 and 1.91–17.48], P=0.0003 and 0.0019, respectively, n=96). The probability of HAND was increased in the presence of Aβ plaques among APOE ε4 carriers (adjusted OR 30.00 [95% CI 1.41–638.63], P=0.029, n=15), but not in non-ε4 carriers (n=57).
The APOE ε4 and older age increased the likelihood of cerebral Aβ plaque deposition in HIV-infected adults. Generally Aβ plaques in HIV brains were immunohistologically different from those in symptomatic AD brains. Nonetheless, Aβ plaques were associated with HAND among APOE ε4 carriers. The detection of APOE ε4 genotype and cerebral Aβ deposition biomarkers may be useful in identifying living HAND subjects who could benefit from Aβ-targeted therapies.
Apolipoprotein E; β-amyloid; HIV dementia; neurofibrillary pathology; phospho-Tau
Over the past two decades, there has been a significant amount of research investigating the risks and benefits of hormone replacement therapy (HRT) with regards to neurodegenerative disease. Here, we review basic science studies, randomized clinical trials, and epidemiological studies, and discuss the putative neuroprotective effects of HRT in the context of Alzheimer's disease, Parkinson's disease, frontotemporal dementia, and HIV-associated neurocognitive disorder. Findings to date suggest a reduced risk of Alzheimer's disease and improved cognitive functioning of postmenopausal women who use 17β-estradiol. With regards to Parkinson's disease, there is consistent evidence from basic science studies for a neuroprotective effect of 17β-estradiol; however, results of clinical and epidemiological studies are inconclusive at this time, and there is a paucity of research examining the association between HRT and Parkinson's-related neurocognitive impairment. Even less understood are the effects of HRT on risk for frontotemporal dementia and HIV-associated neurocognitive disorder. Limits to the existing research are discussed, along with proposed future directions for the investigation of HRT and neurodegenerative diseases.
The neuropathogenesis of HIV-associated neurocognitive disorders (HAND) is unclear. Candidate gene studies have implicated genetic susceptibility loci within immune-related genes; however, these have not been reliably validated. Here we employed genome-wide association (GWA) methods to discover novel genetic susceptibility loci associated with HAND, and validate susceptibility loci implicated in prior candidate gene studies.
Data from 1287 participants enrolled in the Multicenter AIDS Cohort Study between 1985 and 2010 were used. Genotyping was conducted with Illumina 1M, 1MDuo, or 550K platform. Linear mixed models determined subject-specific slopes for change over time in processing speed and executive functioning, considering all visits including baseline and the most recent study visit. Covariates modeled as fixed effects included: time since the first visit, depression severity, nadir CD4+ T-cell count, Hepatitis C co-infection, substance use, and antiretroviral medication regimen. Prevalence of HIV-associated dementia (HAD) and neurocognitive impairment (NCI) was also examined as neurocognitive phenotypes in a case-control analysis.
No genetic susceptibility loci were associated with decline in processing speed or executive functioning among almost 2.5 million single nucleotide polymorphisms (SNPs) directly genotyped or imputed. No association between the SNPs and HAD or NCI were found. Previously reported associations between specific genetic susceptibility loci, HIV-associated neurocognitive impairment and HAD were not validated.
In this first GWAS of HAND, no novel or previously identified genetic susceptibility loci were associated with any of the phenotypes examined. Due to the relatively small sample size, future collaborative efforts that incorporate this dataset may still yield important findings.
HIV; NeuroAIDS; HIV-associated neurocognitive disorder; genome-wide association; HIV-associated dementia
Human Immunodeficiency Virus-1 (HIV) infection frequently results in neurocognitive impairment. While the cause remains unclear, recent gene expression studies have identified genes whose transcription is dysregulated in individuals with HIV-association neurocognitive disorder (HAND). However, the methods for interpretation of such data have lagged behind the technical advances allowing the decoding genetic material. Here, we employ systems biology methods novel to the field of NeuroAIDS to further interrogate extant transcriptome data derived from brains of HIV + patients in order to further elucidate the neuropathogenesis of HAND. Additionally, we compare these data to those derived from brains of individuals with Alzheimer’s disease (AD) in order to identify common pathways of neuropathogenesis.
In Study 1, using data from three brain regions in 6 HIV-seronegative and 15 HIV + cases, we first employed weighted gene co-expression network analysis (WGCNA) to further explore transcriptome networks specific to HAND with HIV-encephalitis (HIVE) and HAND without HIVE. We then used a symptomatic approach, employing standard expression analysis and WGCNA to identify networks associated with neurocognitive impairment (NCI), regardless of HIVE or HAND diagnosis. Finally, we examined the association between the CNS penetration effectiveness (CPE) of antiretroviral regimens and brain transcriptome. In Study 2, we identified common gene networks associated with NCI in both HIV and AD by correlating gene expression with pre-mortem neurocognitive functioning.
Study 1: WGCNA largely corroborated findings from standard differential gene expression analyses, but also identified possible meta-networks composed of multiple gene ontology categories and oligodendrocyte dysfunction. Differential expression analysis identified hub genes highly correlated with NCI, including genes implicated in gliosis, inflammation, and dopaminergic tone. Enrichment analysis identified gene ontology categories that varied across the three brain regions, the most notable being downregulation of genes involved in mitochondrial functioning. Finally, WGCNA identified dysregulated networks associated with NCI, including oligodendrocyte and mitochondrial functioning. Study 2: Common gene networks dysregulated in relation to NCI in AD and HIV included mitochondrial genes, whereas upregulation of various cancer-related genes was found.
While under-powered, this study identified possible biologically-relevant networks correlated with NCI in HIV, and common networks shared with AD, opening new avenues for inquiry in the investigation of HAND neuropathogenesis. These results suggest that further interrogation of existing transcriptome data using systems biology methods can yield important information.
HIV encephalitis; HIV-associated dementia; HIV-associated neurocognitive disorder; Weighted gene coexpression network analysis; WGCNA; CNS penetration effectiveness; National neuroAIDS tissue consortium; Coexpression module
The National NeuroAIDS Tissue Consortium (NNTC) performed a brain gene expression array to elucidate pathophysiologies of Human Immunodeficiency Virus type 1 (HIV-1)-associated neurocognitive disorders.
Twenty-four human subjects in four groups were examined A) Uninfected controls; B) HIV-1 infected subjects with no substantial neurocognitive impairment (NCI); C) Infected with substantial NCI without HIV encephalitis (HIVE); D) Infected with substantial NCI and HIVE. RNA from neocortex, white matter, and neostriatum was processed with the Affymetrix® array platform.
With HIVE the HIV-1 RNA load in brain tissue was three log10 units higher than other groups and over 1,900 gene probes were regulated. Interferon response genes (IFRGs), antigen presentation, complement components and CD163 antigen were strongly upregulated. In frontal neocortex downregulated neuronal pathways strongly dominated in HIVE, including GABA receptors, glutamate signaling, synaptic potentiation, axon guidance, clathrin-mediated endocytosis and 14-3-3 protein. Expression was completely different in neuropsychologically impaired subjects without HIVE. They had low brain HIV-1 loads, weak brain immune responses, lacked neuronally expressed changes in neocortex and exhibited upregulation of endothelial cell type transcripts. HIV-1-infected subjects with normal neuropsychological test results had upregulation of neuronal transcripts involved in synaptic transmission of neostriatal circuits.
Two patterns of brain gene expression suggest that more than one pathophysiological process occurs in HIV-1-associated neurocognitive impairment. Expression in HIVE suggests that lowering brain HIV-1 replication might improve NCI, whereas NCI without HIVE may not respond in kind; array results suggest that modulation of transvascular signaling is a potentially promising approach. Striking brain regional differences highlighted the likely importance of circuit level disturbances in HIV/AIDS. In subjects without impairment regulation of genes that drive neostriatal synaptic plasticity reflects adaptation. The array provides an infusion of public resources including brain samples, clinicopathological data and correlative gene expression data for further exploration (http://www.nntc.org/gene-array-project).
Progression of HIV/AIDS is frequently associated with frontal/subcortical dysfunction and mean reaction time (RT) slowing. Beyond group means, within-subject variability of RT has been found to be particularly sensitive to frontal/subcortical dysfunction in other populations. However, the possible relevance of RT variability to HIV/AIDS patients remains unknown. This study evaluated the relationships between RT variability and indicators such as neurocognitive, behavioral, and immunological status. A total of 46 HIV-positive adults on antiretroviral medication regimens were included in this study. Overall performance of this sample was poorer than normative means on measures of RT latency, RT variability, and traditional neurocognitive domains. Results demonstrated that the measures of RT variability were associated with global cognition, medication adherence rates, and peak immunological dysfunction, above and beyond the effects of RT latency. These preliminary findings suggest that measures of RT variability may provide enhanced sensitivity to neurocognitive disease burden in HIV/AIDS relative to more traditional measures of mean RT or cognitive function.
AIDS; Cognitive ability; Medication adherence; Immunological status; Continuous Performance Test; CPT-II
The human immunodeficiency virus type-1 (HIV-1) gp160 (gp120-gp41 complex) trimer envelope (ENV) protein is a potential vaccine candidate for HIV/AIDS.
HIV-1 vaccine development has been problematic and charge polarity as well as sequence variation across clades may relate to the difficulties. Further obstacles
are caused by sequence variation between blood and brain-derived sequences, since the brain is a separate compartment for HIV-1 infection. We utilize a threedimensional
residue measure of solvent exposure, accessible surface area (ASA), which shows that major segments of gp120 and gp41 known structures are
solvent exposed across clades. We demonstrate a large percent sequence polarity for solvent exposed residues in gp120 and gp41. The range of sequence polarity
varies across clades, blood, and brain from different geographical locations. Regression analysis shows that blood and brain gp120 and gp41 percent sequence
polarity range correlate with mean Shannon entropy. These results point to the use of protein modifications to enhance HIV-1 ENV vaccines across multiple
clades, blood, and brain. It should be noted that we do not address the issue of protein glycosylation here; however, this is an important issue for vaccine design
HIV-1 - human immunodeficiency virus type 1,
AIDS - acquired immunodeficiency syndrome,
ENV - envelope,
gp160 - 160,000d glycoprotein,
gp120 - 120,000d glycoprotein,
gp41 - 41,000d glycoprotein,
LANL - Los Alamos National Laboratories,
PDB - Protein Data Bank,
HVTN - STEP HIV vaccine trial,
AA - amino acids,
MSA - multiple sequence alignment,
ASA - accessible surface area,
SNPs- single nucleotide polymorphisms,
HAART - Highly Active Antiretroviral Therapy,
CCR5 - C-C chemokine receptor type 5,
CNS - central nervous system,
HIVE - HIV encephalitis,
P - polarity,
NP - non-polarity,
CTL - cytotoxic T lymphocyte,
NIAID - National Institute of Allergy and Infectious Diseases.
HIV-1; clades; blood; brain; accessible surface area; compositional polarity; Shannon entropy; gp120; gp41; gp120-gp41 complex; gp160; ENV; trimer; vaccine
The prevalence of Human immunodeficiency virus (HIV)-associated dementia (HAD) has continued to rise even as incidence has fallen. Several host genetic variants have been identified that modify risk for HAD. However, the findings have not been replicated consistently and most studies did not consider the multitude of factors that might themselves confer risk for HAD. In the current study, we sought to replicate the findings of previous studies in a neurologically and behaviorally well-characterized cohort.
The sample consisted of 143 HIV+ individuals enrolled in the National NeuroAIDS Tissue Consortium (NNTC). Based on consensus diagnosis, 117 were considered neurologically normal upon study entry, and 26 had HAD. Seven single-nucleotide polymorphisms (SNPs) were genotyped within seven genes (CCL2, CCL3, CCL5, interleukin-1α [IL-1α], IL-10, stromal cell-derived factor 1, and tumor necrosis factor-α). Logistic regression analysis was used to predict group membership (normal vs HAD), with predictor variables including length of infection, age, current drug dependence, current depression, and genotype.
The two groups were statistically similar with regards to demographic characteristics, current drug use, and disease factors. The HAD group had significantly greater number of individuals with current depression. Only one SNP, rs1130371 within the gene for CCL3, was entered into the analysis as the others showed symmetric distribution between groups. Logistic regression indicated that current depression and CCL3 genotype were significant predictors of HAD. Depression conferred a fivefold greater risk of HAD, while the TT genotype for CCL3 SNP (rs1130371) was associated with twofold risk for HAD.
Depression and CCL3 genotype predicted HAD. The fact that SNPs previously found to be associated with HAD were not in our analysis, and that rs1130371 is in high linkage disequilibrium with neighboring genes indicates that more dense genotyping in significantly larger cohorts is required to further characterize the relationship between genotype and risk for HAD.
HIV-associated dementia; NeuroAIDS; CCL3; cytokine; depression; HIV
Because of the multifactorial nature of neuropsychological tests, attention remains poorly defined from a neuropsychological perspective, and conclusions made regarding attention across studies may be limited due to the different nature of the measures used. Thus, a more definitive schema for this neurocognitive domain is needed. We assessed the applicability of Mirsky and Duncan's (2001) neuropsychological model of attention to a cohort of 104 HIV+ adults. Our analysis resulted in a five-factor structure similar to that of previous studies, which explained 74.5% of the variance. However, based on the psychometric characteristics of the measures comprising each factor, we offer an alternative interpretation of the factors. Findings also indicate that one factor, which is generally not assessed in clinical neuropsychology settings, may be more predictive of real-world behaviors (such as medication adherence) than those composed of traditional measures. Suggestions for further research in this important area are discussed.
Central nervous system opportunistic infections (CNS-OI) are a significant cause of morbidity and mortality in AIDS. While current interventions are increasingly successful in treating CNS-OI, little information exists regarding long-term behavioral outcomes among survivors. In this exploratory study we examined neurocognitive data among three groups of adults with different AIDS-related CNS-OI: 15 with past cryptococcal meningitis (CM), 8 with toxoplasmosis encephalitis (TE), and 8 with progressive multifocal leukoencephalopathy (PML). A group of 61 individuals with AIDS, but without CNS-OI, was used as a comparison group. A battery of standardized neuropsychological tests assessing a variety of cognitive domains was administered upon entry. Results indicate that individuals with a history of CNS-OI were most impaired on measures of cognitive and psychomotor speed relative to the HIV+ comparison group. Among the CNS-OI groups, individuals with history of TE had the most severe and varied deficits. The results are discussed in relation to what is known about the neuropathological consequences of the various CNS-OIs. While this is the first systematic group study of residual CNS-OI effects on neurocognitive function, future studies employing more participants, perhaps focusing on specific CNS-OIs, will further characterize the long-term outcomes in AIDS-related CNS-OI.
Opportunistic infection; AIDS; Neuropsychological functioning; Toxoplasmosis encephalitis; Progressive multifocal leukoencephalopathy; Cryptococcal meningitis
Due to similar routes of viral transmission, many individuals infected with the human immunodeficiency virus (HIV) are also infected with the hepatitis C virus (HCV). Each virus can cause cognitive compromise among mono-infected individuals; evidence is accumulating that HIV/HCV co-infection may have a particularly deleterious impact on cognition. We present neuropsychological data obtained from 118 HIV+ adults with advanced HIV disease, 35 of whom were co-infected with HCV, who completed a comprehensive neurocognitive evaluation. Rates of global cognitive impairment were higher among co-infected patients than among those with HIV alone (63% vs. 43%). Within the specific domains of learning and memory, co-infected individuals were significantly more likely to be impaired than were the HIV mono-infected participants. Finally, we discuss implications of these findings and potential future directions for research in this area.
HIV infection; HCV infection; HIV/HCV co-infection; neurocognition
This longitudinal study examined the impact of drug use and abuse on medication adherence among 150 HIV-infected individuals, 102 who tested urinalysis positive for recent illicit drug use. Medication adherence was tracked over a 6-month period using an electronic monitoring device (MEMS caps). Over the 6-month study drug-positive participants demonstrated significantly worse medication adherence than did drug-negative participants (63 vs. 79%, respectively). Logistic regression revealed that drug use was associated with over a fourfold greater risk of adherence failure. Stimulant users were at greatest risk for poor adherence. Based upon within-participants analyses comparing 3-day adherence rates when actively using versus not using drugs, this appears to be more a function of state rather than trait. These data suggest that it is the acute effects of intoxication, rather than stable features that may be characteristic of the drug-using populace, which leads to difficulties with medication adherence.
HIV infection; AIDS; Medication adherence; Drug use; Methamphetamine; Cocaine
Objective methods for determining clinically relevant neurocognitive change are useful for clinicians and researchers, but the utility of such methods requires validation studies in order to assess their accuracy among target populations. We examined the generalizability of regression equations and reliable change indexes (RCI) derived from a healthy sample to two HIV-infected samples, one similar in demographic makeup to the normative group and the other dissimilar. Measures administered at baseline and follow-up included the Trail Making Test, Controlled Oral Word Association Test (COWAT), Grooved Pegboard, and Digit Span. Frequencies of decline, improvement, or stability were determined for each measure. Among the demographically similar clinical cohort, elevated rates of decline among more immunologically impaired participants were indicated by simple regression method on measures of psychomotor speed and attention, while RCI addressing practice effects (RCI-PE) indicated improvement on most measures regardless of immunostatus. Conversely, among the demographically dissimilar cohort, simple regression indicated high rates of decline across all measures, while RCI-PE indicated elevated rates of decline on psychomotor and attention measures. Thus, the accuracy of the two methods examined for determining clinically significant change among HIV+ cohorts differs depending upon their similarity with the normative sample.
Although most agree that poor adherence to antiretrovirals is a common problem, relatively few factors have been shown to consistently predict treatment failure. In this study, a theoretical framework encompassing demographic characteristics, health beliefs/attitudes, treatment self-efficacy, and neurocognitive status was examined in relationship to highly active antiretroviral therapy adherence.
Prospective, cross-sectional observational design.
Main Outcome Measures
Neuropsychological test performance, health beliefs and attitudes, and medication adherence tracked over a 1-month period using electronic monitoring technology (Medication Event Monitoring System caps).
The rate of poor adherence was twice as high among younger participants than with older participants (68% and 33%, respectively). Results of binary logistic regression revealed that low self-efficacy and lack of perceived treatment utility predicted poor adherence among younger individuals, whereas decreased levels of neurocognitive functioning remained the sole predictor of poor adherence among older participants.
These data support components of the health beliefs model in predicting medication adherence among younger HIV-positive individuals. However, risk of adherence failure in those ages 50 years and older appears most related to neurocognitive status.
HIV; HAART; adherence; health beliefs; cognitive function
The association between sensation seeking and visual selective attention was examined in 31 adults with the Human Immunodeficiency Virus (HIV). Sensation seeking was measured with Zuckerman’s Sensation Seeking Scale Form V (SSS-V). Selective attention was assessed with a perceptual span task, where a target letter-character must be identified in a quickly presented array of nontarget letter-characters. As predicted, sensation seeking was strongly associated (R2 = .229) with perceptual span performance in the array size 12 condition, where selective attention demands were greatest, but not in the easier conditions. The Disinhibition, Boredom Susceptibility, and Experience Seeking subscales of the SSS-V were associated with span performance. It is argued that personality factors such as sensation seeking may play a significant role in selective attention and related cognitive abilities in HIV positive adults. Furthermore, sensation seeking differences might explain certain inconsistencies in the HIV neuropsychology literature.
HIV; AIDS; Cognition; Personality; Performance; Individual differences
Decision making was assessed using a laboratory gambling task in 67 adults with the Human Immunodeficiency Virus (HIV+) and in 19 HIV-seronegative (HIV−) control participants. Neurocognitive test performance across several domains was also analyzed to examine potential cognitive mechanisms of gambling task performance. As predicted, the HIV+ group performed worse on the gambling task, indicating greater risky decision making. Specifically, the HIV+ group selected more cards from the “risky” or disadvantageous deck that included relatively large payoffs but infrequent large penalties. The control group also selected such risky cards but quickly learned to avoid them. Exploratory analyses also indicated that in the HIV+ group, but not in the control group, gambling task performance was correlated with Stroop Interference performance and long delay free recall on the California Verbal Learning Test, suggesting the role of inhibitory processes and verbal memory in the poorer gambling task performance in HIV. These findings indicate the usefulness of the gambling task as a laboratory tool to examine risky decision making and cognition in the HIV population.
HIV; decision making; gambling task; cognition
We describe current research that applies epigenetics to a novel understanding of the immuno-neuropathogenesis of HIV-1 viral infection and NeuroAIDS. We propose the hypothesis that HIV-1 alters the structure-function relationship of chromatin,
coding DNA and non-coding DNA, including RNA transcribed from these regions resulting in pathogenesis in AIDS, drug abuse, and NeuroAIDS. We discuss the general implications of molecular epigenetics with special emphasis on drug abuse, bar-codes,
pyknons, and miRNAs for translational and clinical research. We discuss the application of the recent recursive algorithm of biology to this field and propose to synthesize the Genomic and Epigenomic views into a holistic approach of HoloGenomics.
epigenetics; hologenomics; coding and noncoding DNA; HIV-1; AIDS; NeuroAIDS; molecular medicine paradigm-shift; translation-clinic