Quantification of Carbonic Anhydrase Inhibitors and Metabolites in Urine and Hair of Patients and Their Relatives.

Carbonic anhydrase inhibitors (CAIs) are prescription drugs also used in doping to dilute urine samples and tamper with urinalyses. Dorzolamide, brinzolamide, and acetazolamide are prohibited by the World Anti-Doping Agency. Detecting CAIs and their metabolites in biological samples is crucial to documenting misuse in doping. We quantified dorzolamide, brinzolamide, acetazolamide, and their metabolites in the urine and hair of 88 patients under treatment for ocular hypertension or glaucoma. Samples of the patients' relatives were analyzed to assess potential for accidental exposure. After washing, 25 mg hair was incubated with an acidic buffer at 100 °C for 1 h. After cooling and centrifugation, the supernatant was analyzed by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Urine (100 µL) was diluted and centrifuged before UHPLC-MS/MS analysis. Run time was 8 min through a reverse-phase column with a mobile phase gradient. MS/MS analysis was performed in a multiple-reaction monitoring mode after positive electrospray ionization. Median urinary concentration was 245 ng/mL (IQR: 116.2-501 ng/mL) for dorzolamide, 81.1 ng/mL (IQR: 35.9-125.3 ng/mL) for N-deethyl-dorzolamide, 0.77 ng/mL (IQR: 0.64 ng/mL-0.84 ng/mL) for N-acetyl-dorzolamide, 38.9 ng/mL (IQR: 20.4-79.2 ng/mL) for brinzolamide, and 72.8 ng/mL (IQR: 20.7-437.3 ng/mL) for acetazolamide. Median hair concentration was 0.48 ng/mg (IQR: 0.1-0.98 ng/mg) for dorzolamide, 0.07 ng/mg (IQR: 0.06-0.08 ng/mg) for N-deethyl-dorzolamide, 0.40 ng/mL (IQR: 0.13-1.95 ng/mL) for brinzolamide. Acetazolamide was detected in only one hair sample. Dorzolamide and brinzolamide were detected in the urine of three and one relatives, respectively. Cutoff concentrations of urinary dorzolamide and brinzolamide are necessary to preclude false positives due to contamination or passive exposure. We reported the first concentrations of brinzolamide in hair.

Prognostic value of soluble ST2, high-sensitivity cardiac troponin, and NT-proBNP in type 2 diabetes: a 15-year retrospective study.

BACKGROUND: Patients with type 2 diabetes (T2DM) present an increased risk of cardiovascular (CV) disease and excess CV-related mortality. Beyond the established role of brain natriuretic peptide (BNP) and cardiac troponins (cTn), other non-cardiac-specific biomarkers are emerging as predictors of CV outcomes in T2DM. METHODS: Serum levels of soluble suppression of tumorigenesis 2 (sST2), high-sensitivity (hs)-cTnI, and N-terminal (NT)-proBNP were assessed in 568 patients with T2DM and 115 healthy controls (CTR). Their association with all-cause mortality and the development of diabetic complications was tested in T2DM patients over a median follow-up of 16.8 years using Cox models and logistic regressions. RESULTS: sST2 followed an increasing trend from CTR to uncomplicated T2DM patients (T2DM-NC) to patients with at least one complication (T2DM-C), while hs-cTnI was significantly higher in T2DM-C compared to CTR but not to T2DM-NC. A graded association was found between sST2 (HR 2.76 [95% CI 1.20-6.33] for ≥ 32.0 ng/mL and 2.00 [1.02-3.94] for 16.5-32.0 ng/mL compared to < 16.5 ng/mL, C-statistic = 0.729), NT-proBNP (HR 2.04 [1.90-4.55] for ≥ 337 ng/L and 1.48 [1.05-2.10] for 89-337 ng/L compared to < 89 ng/L, C-statistic = 0.741), and 15-year mortality in T2DM, whereas increased mortality was observed in patients with hs-cTnI ≥ 7.8 ng/L (HR 1.63 [1.01-2.62]). A 'cardiac score' based on the combination of sST2, hs-cTnI, and NT-proBNP was significantly associated with all-cause mortality (HR 1.35 [1.19-1.53], C-statistic = 0.739) and development of CV events. CONCLUSIONS: sST2, hs-cTnI, and NT-proBNP are associated with 15-year mortality and onset of CV events in T2DM. The long-term prognostic value of sST2 and its ability to track variables related to insulin resistance and associated metabolic disorders support its implementation into routine clinical practice.

Expression Levels and Clinical Significance of miR-21-5p, miR-let-7a, and miR-34c-5p in Laryngeal Squamous Cell Carcinoma.

OBJECTIVE: Altered microRNAs (miRNAs) expression has been found in many cancer types, including laryngeal squamous cell carcinoma (LSCC). The aim of this study was to determine the role and clinical value of three LSCC-related miRs, such as miR-21-5p, miR-let-7a, and miR-34c-5p in a homogeneous cohort of patients with primary LSCC treated by primary surgery. METHODS: Expression levels of miR-21-5p, miR-let-7a, and miR-34c-5p were detected in 43 pairs of LSCC and adjacent normal tissues by reverse-transcription quantitative PCR. Overall survival and disease-free survival were evaluated using the Kaplan-Meier method, and multivariate analysis was performed using the Cox proportional hazard analysis. RESULTS: miR-21-5p is significantly upregulated, while miR-let-7a is significantly downregulated in LSCC tumor tissues compared with the corresponding adjacent normal tissues. The downregulation of miR-34c-5p expression significantly correlated with a shorter disease-free survival and, in the multivariate analysis, low miR-34c-5p expression was associated with an increased risk of recurrence. CONCLUSIONS: miR-21-5p, miR-let-7a, and miR-34c-5p seem to play a critical role in LSCC carcinogenesis and might have a diagnostic and prognostic clinical value. The miR-let-7a levels could have a predictive role for lymph node metastases and miR-34c-5p might be a promising biomarker of patient outcome.

Endothelial Cell Senescence and Inflammaging: MicroRNAs as Biomarkers and Innovative Therapeutic Tools.

Aging is accompanied by a progressive decline of endothelial function and a progressive drift toward a systemic pro-inflammatory status that has been designated "inflammaging". Both phenomena are accelerated and exacerbated in patients with the most common age-related diseases (ARDs), including cancer. The finding that chronic cell stress activates a pro-inflammatory program leading to acquisition of the senescence-associated secretory phenotype (SASP) and to the propagation of senescence to surrounding cells through the secretome, suggests that cell senescence may have a role in both processes. Here we: i) describe the role of cell senescence in endothelial dysfunction, ii) emphasize the contribution of the endothelial cell SASP to inflammaging, and iii) suggest that selective removal of senescent endothelial cells may not only hinder such harmful processes, but also reduce the risk of developing ARDs and their complications. Although in vivo detection and targeting of senescent endothelial cells are still being investigated, it is likely that therapeutic strategies based on antioxidant and anti-inflammatory compounds would involve generalized anti-aging effects also benefiting endothelial cells. MicroRNA (miRNAs) - single-stranded, non-coding RNAs expressed by all living cells and involved in the epigenetic modulation of all transcriptional programs - may constitute an innovative, valuable tool to detect and target senescent endothelial cells and to devise treatments that can slow down the pro-inflammatory program activated in senescent endothelial cells.

DNA damage response (DDR) and senescence: shuttled inflamma-miRNAs on the stage of inflamm-aging.

A major issue in aging research is how cellular phenomena affect aging at the systemic level. Emerging evidence suggests that DNA damage response (DDR) signaling is a key mechanism linking DNA damage accumulation, cell senescence, and organism aging. DDR activation in senescent cells promotes acquisition of a proinflammatory secretory phenotype (SASP), which in turn elicits DDR and SASP activation in neighboring cells, thereby creating a proinflammatory environment extending at the local and eventually the systemic level. DDR activation is triggered by genomic lesions as well as emerging bacterial and viral metagenomes. Therefore, the buildup of cells with an activated DDR probably fuels inflamm-aging and predisposes to the development of the major age-related diseases (ARDs). Micro (mi)-RNAs - non-coding RNAs involved in gene expression modulation - are released locally and systemically by a variety of shuttles (exosomes, lipoproteins, proteins) that likely affect the efficiency of their biological effects. Here we suggest that some miRNAs, previously found to be associated with inflammation and senescence - miR-146, miR-155, and miR-21 - play a central role in the interplay among DDR, cell senescence and inflamm-aging. The identification of the functions of shuttled senescence-associated miRNAs is expected to shed light on the aging process and on how to delay ARD development.

MicroRNA-34c-5p is related to recurrence in laryngeal squamous cell carcinoma.

OBJECTIVES/HYPOTHESIS: Altered microRNA expression has been found in many cancer types, including laryngeal squamous cell carcinoma (LSCC). We investigated the association of LSCC-related miR-34c-5p with disease-free survival and overall survival. STUDY DESIGN: Retrospective cohort study. METHODS: Expression levels of miR-34c-5p were detected in 90 LSCC formalin-fixed paraffin-embedded tissues by reverse-transcription quantitative polymerase chain reaction. Overall survival and disease-free survival were evaluated using the Kaplan-Meier method, and multivariate analysis was performed using Cox proportional hazard analysis. RESULTS: A downregulation of miR-34c-5p expression significantly correlated with worse disease-free and overall survival. In the multivariate analysis, low miR-34c-5p expression was associated with an increased risk of recurrence. CONCLUSIONS: A downregulation of miR-34c-5p in LSCC is independently associated with unfavorable disease-free survival, suggesting that miR-34c-5p might be a promising marker for evaluating the risk of recurrences. LEVEL OF EVIDENCE: NA.

Epigenetic mechanisms of endothelial dysfunction in type 2 diabetes.

The development of type-2 diabetes mellitus (T2DM) and its complications is largely due to the complex interaction between genetic factors and environmental influences, mainly dietary habits and lifestyle, which can either accelerate or slow down disease progression. Recent findings suggest the potential involvement of epigenetic mechanisms as a crucial interface between the effects of genetic predisposition and environmental factors. The common denominator of environmental factors promoting T2DM development and progression is that they trigger an inflammatory response, promoting inflammation-mediated insulin resistance and endothelial dysfunction. Proinflammatory stimuli, including hyperglycemia, oxidative stress, and other inflammatory mediators, can affect epigenetic mechanisms, altering the expression of specific genes in target cells without changes in underlying DNA sequences. DNA methylation and post-translational histone modifications (PTHMs) are the most extensively investigated epigenetic mechanisms. Over the past few years, non-coding RNA, including microRNAs (miRNAs), have also emerged as key players in gene expression modulation. MiRNAs can be actively released or shed by cells in the bloodstream and taken up in active form by receiving cells, acting as efficient systemic communication tools. The miRNAs involved in modulation of inflammatory pathways (inflammamiRs), such as miR-146a, and those highly expressed in endothelial lineages and hematopoietic progenitor cells (angiomiRs), such as miR-126, are the most extensively studied circulating miRNAs in T2DM. However, data on circulating miRNA signatures associated with specific diabetic complications are still lacking. Since immune cells and endothelial cells are primarily involved in the vascular complications of T2DM, their relative contribution to circulating miRNA signatures needs to be elucidated. An integrated approach encompassing different epigenetic mechanisms would have the potential to provide new mechanistic insights into the genesis of diabetes and its severe vascular complications and identify a panel of epigenetic markers with diagnostic/prognostic and therapeutic relevance.

Aged-related increase of high sensitive Troponin T and its implication in acute myocardial infarction diagnosis of elderly patients.

High sensitive cardiac Troponin T (hs-cTnT) represents an important tool in acute myocardial infarction (AMI) diagnosis. Even though the hs-cTnT evaluation is relevant for AMI diagnosis in elderly patients characterized by clinical and instrumental atypical presentation, the overall reliability in elderly patients is unknown. We aimed at: (1) defining the hs-cTnT 99th percentile value in an aged healthy reference population and (2) testing hs-cTnT diagnostic accuracy in elderly patients with a suspected AMI. 294 healthy subjects (50-105 years old) and 299 elderly patients (75-96 years old) with suspected AMI at presentation, were enrolled. Conventional cTnT, hs-cTnT, NT-proBNP and creatinine levels were determined in all participants. Our main results are: (1) a significant hs-cTnT age-related increase was observed in an healthy reference population ranging 50-105 years old; (2) hs-cTnT levels showed an age-related multimodal distribution in the healthy reference population: 16 ng/L corresponds to the 99th percentile in subjects ranging 50-75 years old, whereas 70.6 ng/L corresponds to the 99th percentile in subjects ≥75 years old; (3) 86.8 ng/L resulted the hs-cTnT cut-off value with the highest efficiency in AMI diagnosis of geriatric patients. Our data suggest that the hs-cTnT cut-off value must be age-tailored to improve the AMI diagnostic accuracy.