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1.  Time related variations in stem cell harvesting of umbilical cord blood 
Scientific Reports  2016;6:21404.
Umbilical cord blood (UCB) contains hematopoietic stem cells and multipotent mesenchymal cells useful for treatment in malignant/nonmalignant hematologic-immunologic diseases and regenerative medicine. Transplantation outcome is correlated with cord blood volume (CBV), number of total nucleated cells (TNC), CD34+ progenitor cells and colony forming units in UCB donations. Several studies have addressed the role of maternal/neonatal factors associated with the hematopoietic reconstruction potential of UCB, including: gestational age, maternal parity, newborn sex and birth weight, placental weight, labor duration and mode of delivery. Few data exist regarding as to how time influences UCB collection and banking patterns. We retrospectively analyzed 17.936 cord blood donations collected from 1999 to 2011 from Tuscany and Apulia Cord Blood Banks. Results from generalized multivariable linear mixed models showed that CBV, TNC and CD34+ cell were associated with known obstetric and neonatal parameters and showed rhythmic patterns in different time domains and frequency ranges. The present findings confirm that volume, total nucleated cells and stem cells of the UCB donations are hallmarked by rhythmic patterns in different time domains and frequency ranges and suggest that temporal rhythms in addition to known obstetric and neonatal parameters influence CBV, TNC and CD34+ cell content in UBC units.
doi:10.1038/srep21404
PMCID: PMC4764902  PMID: 26906327
2.  Evidence of a causal relationship between high serum adiponectin levels and increased cardiovascular mortality rate in patients with type 2 diabetes 
Background
Despite its beneficial role on insulin resistance and atherosclerosis, adiponectin has been repeatedly reported as an independent positive predictor of cardiovascular mortality.
Methods
A Mendelian randomization approach was used, in order to evaluate whether such counterintuitive association recognizes a cause-effect relationship. To this purpose, single nucleotide polymorphism rs822354 in the ADIPOQ locus which has been previously associated with serum adiponectin at genome-wide level, was used as an instrument variable. Our investigation was carried out in the Gargano Heart Study-prospective design, comprising 356 patients with type 2 diabetes, in whom both total and high molecular weight (HMW) adiponectin were measured and cardiovascular mortality was recorded (mean follow-up = 5.4 ± 2.5 years; 58 events/1922 person-year).
Results
The A allele of rs822354 was associated with both total and HMW adiponectin [β (SE) = 0.10 (0.042), p = 0.014 and 0.17 (0.06), p = 0.003; respectively]. In a Poisson model comprising age, sex, smoking habits, BMI, HbA1c, total cholesterol, HDL-cholesterol, triglycerides, insulin therapy and hypertension, both rs822354 (IRR = 1.94, 95 % CI 1.23–3.07; p = 0.005), as well as the genetic equivalent of total adiponectin change (IRR = 1.07, 95 % CI 1.02–1.12; p = 0.003) were significantly associated with cardiovascular mortality. The observed genetic effect was significantly greater than that exerted by the genetic equivalent change of serum adiponectin (p for IRR heterogeneity = 0.012). In the above-mentioned adjusted model, very similar results were obtained when HMW, rather than total, adiponectin was used as the exposure variable of interest.
Conclusions
Our data suggest that the paradoxical association between high serum adiponectin levels and increased cardiovascular mortality rate is based on a cause-effect relationship, thus pointing to an unexpected deleterious role of adiponectin action/metabolism on atherosclerotic processes.
doi:10.1186/s12933-016-0339-z
PMCID: PMC4730617  PMID: 26817832
Adiponectin; ADIPOQ; Mortality; Mendelian randomization; Type 2 diabetes
3.  Competitive allele-specific TaqMan PCR (Cast-PCR) is a sensitive, specific and fast method for BRAF V600 mutation detection in Melanoma patients 
Scientific Reports  2015;5:18592.
BRAF codon 600 mutation testing of melanoma patients is mandatory for the choice of the most appropriate therapy in the clinical setting. Competitive allele specific TaqMan PCR (Cast-PCR) technology allows not only the selective amplification of minor alleles, but it also blocks the amplification of non-mutant allele. We genotyped codon 600 of the BRAF gene in 54 patients’ samples by Cast-PCR and bidirectional direct sequence analysis. All the mutations detected by sequencing were also identified by Cast-PCR. In addition, Cast-PCR assay detected four samples carrying mutations and was able to clearly identify two mutations of uncertain interpretation by Sanger sequencing. The limit of detection of Cast-PCR was evaluated by constructing dilution curves of BRAFV600E and BRAFV600K mutated clinical samples mixed with a not-mutated specimens. Both mutations could be detected until a 1:100 mutated/not mutated ratio. Cloning and sequencing of the clones was used to confirm mutations on representative discrepant cases. Cast PCR performances were not affected by intratumour heterogeneity, and less affected by melanin content. Our results indicate that Cast-PCR is a reliable diagnostic tool for the identification of melanoma patients as eligible to be treated with TKIs and might be implemented in the clinical setting as elective screening method.
doi:10.1038/srep18592
PMCID: PMC4686972  PMID: 26690267
4.  Joint effect of insulin signaling genes on all-cause mortality 
Atherosclerosis  2014;237(2):639-644.
Objective
We have previously reported the combined effect of SNPs perturbing insulin signaling (ENPP1 K121Q, rs1044498; IRS1 G972R, rs1801278; TRIB3 Q84R, rs2295490) on insulin resistance (IR), type 2 diabetes (T2D) and cardiovascular events.
We here investigated whether such a combined effect affects also all-cause mortality in a sample of 1,851 Whites of European ancestry.
Methods
We investigated a first sample of 721 patients, 232 deaths, 3,389 person-years (py). Replication was assessed in two samples of patients with T2D: the Gargano Mortality Study (GMS) of 714 patients, 127 deaths, 5,426 py and the Joslin Kidney Study (JKS) comprising 416 patients, 214 deaths, 5,325 py.
Results
In the first sample, individuals carrying 1 or ≥ 2 risk alleles had 33% (p=0.06) and 51% (p=0.02) increased risk of mortality, as compared with individuals with no risk alleles. A similar, though not significant, trend was obtained in the two replication samples only for subject carrying ≥ 2 risk alleles. In a pooled analysis, individuals carrying ≥ 2 risk alleles had higher mortality rate as compared to those carrying 0 risk alleles (HR=1.34, 95%CI=1.08–1.67; p=0.008), and as compared to those carrying only one risk allele (HR=1.41, 95%CI=1.13–1.75; p=0.002). This association was independent from several possible confounders including sex, age, BMI, hypertension and diabetes status.
Conclusion
Our data suggest that variants affecting insulin signaling exert a joint effect on all-cause mortality and is consistent with a role of abnormal insulin signaling on mortality risk.
doi:10.1016/j.atherosclerosis.2014.10.005
PMCID: PMC4294190  PMID: 25463099
ENPP1; IRS1; TRIB3; prospective study
5.  Serum Adiponectin and Glomerular Filtration Rate in Patients with Type 2 Diabetes 
PLoS ONE  2015;10(10):e0140631.
High serum adiponectin has been increased in several conditions of kidney disease. Only sparse and conflicting results have been reported in patients with type 2 diabetes (T2D), a subgroup of individuals who are at high risk for renal dysfunction. The aim of this study was to fill up this gap of knowledge by investigating such association in a large sample of Italian diabetic patients. The association between serum adiponectin levels and estimated glomerular filtration rate (eGFR by Chronic Kidney Disease-Epidemiology Collaboration CKD-EPI equation) was investigated in 1,243 patients with T2D from two cross-sectional Italian studies: 878 from San Giovanni Rotondo (SGR) and 365 from Foggia (FG). Serum adiponectin was inversely associated with eGFR in SGR [β (standard error, SE) for 1 standard deviation (SD) of adiponectin = -3.26 (0.64)] and in FG [β(SE)=-5.70(1.28)] sample, as well as in the two studies combined [β(SE)=-3.99(0.59)];(p<0.0001 for all). In this combined analysis, the association was still significant after adjusting for sex, smoking habits, body mass index (BMI), waist circumference, diabetes duration, glycated hemoglobin (HbA1c), albumin creatinine ratio (ACR) and anti-hyperglycemic, anti-hypertensive and anti-dyslipidemic treatments [β (SE)= -2.19 (0.59), p = 0.0001]. A stronger association between each SD adiponectin increment and low eGFR was observed among patients with micro-/macro-albuminuria, as compared to those with normo-albuminuria [adjusted β(SE)=-4.42(1.16) ml/min/1.73m2 vs. -1.50 (0.67) ml/min/1.73m2, respectively; p for adiponectin-by-albuminuric status = 0.022]. For each adiponectin SD increment, the odds of having eGFR < 60 ml/min/1.73m2 increased by 41% (odds ratio, OR = 1.41; 95% confidence interval, CI 1.21–1.64) in SGR sample, 53% (OR = 1.53; 95% CI 1.21–1.94) in FG sample, and 44% (OR = 1.44; 95%CI 1.27–1.64) in the two studies considered together (p<0.0001 for all). In the combined sample, further adjustment for the above mentioned covariates did not change the observed association (OR = 1.36; 95%CI 1.16–1.60; p<0.0001). Our study, so far the largest addressing the relationship between serum adiponectin and GFR in T2D, strongly suggests that the paradoxical inverse association, previously reported in different clinical sets, is also observed in diabetic patients. Further studies are needed to unravel the biology underlying this counterintuitive relationship.
doi:10.1371/journal.pone.0140631
PMCID: PMC4605700  PMID: 26465607
7.  Frontal assessment battery for detecting executive dysfunction in amyotrophic lateral sclerosis without dementia: a retrospective observational study 
BMJ Open  2015;5(9):e007069.
Objective
The frontal assessment battery (FAB) is a quick and reliable method of screening to evaluate frontal lobe dysfunction in amyotrophic lateral sclerosis (ALS). However, previous studies were generally conducted on small samples representing different stages of disease and severity. We assessed the diagnostic accuracy of the FAB in detecting executive functions and its association with demographic and clinical features in ALS without dementia.
Design
Retrospective observational study.
Setting
A multidisciplinary tertiary centre for motor neuron disease.
Participants
We enrolled 95 consecutive patients with ALS diagnosed with El Escorial criteria in the period between January 2006 and December 2010.
Main outcome measures
We screened the patients with ALS using the FAB. An Executive Index (EI) was also calculated by averaging the Z scores of analytic executive tests evaluating information-processing speed (Symbol Digit Modalities Test—Oral version), selective attention (Stroop test) and semantic memory (Verbal Fluency Test).
Results
The FAB detected executive dysfunction in 13.7% of the patients with ALS. Moreover, using the EI standardised cut-off, 37.9% of the patients with ALS showed executive dysfunction. The receiver-operating characteristic curve showed that the optimal cut-off for the FAB in the whole sample was 16, with a sensitivity of 0.889 (95% CIs 0.545 to 1.000), a specificity of 0.593 (95% CI 0.450 to 0.907) and a moderate overall discriminatory power of 0.809. Different levels of respiratory function, duration of disease and depressive symptoms did not affect the FAB validity.
Conclusions
In patients with ALS without dementia, a high prevalence of executive dysfunction was present. The FAB showed good validity as a screening instrument to detect executive dysfunction in these patients and may be used when a complete neuropsychological assessment is not possible.
doi:10.1136/bmjopen-2014-007069
PMCID: PMC4563243  PMID: 26338835
EPIDEMIOLOGY
8.  Identification and Clinical Characterization of Adult Patients with Multigenerational Diabetes Mellitus 
PLoS ONE  2015;10(8):e0135855.
Background
Some patients diagnosed as having type 2 diabetes mellitus (T2DM) are, instead, affected by multigenerational diabetes whose clinical characteristics are mostly undefined.
Objective
1. To identify among patients who had been previously defined as affected by T2DM those, in fact, affected by multigenerational diabetes; 2. After excluding patients carrying the most common MODY genes and mitochondrial mutations, we compared clinical features of remaining patients with those of patients with T2DM.
Methods
Among 2,583 consecutive adult patients who had been defined as affected by T2DM, we looked for those with diabetes in ≥3 consecutive generations. All probands were screened for mutations in six MODY genes (HNF4A, GCK, HNF1A, PDX1, HNF1B and NeuroD1) and for the A3243G mitochondrial mutation. After excluding patients with mutations in one of such genes, we compared clinical features of the remaining 67 patients (2.6% of the whole initial sample) affected by multigenerational “familial diabetes of the adulthood” (FDA) and of their diabetic relatives (n = 63) to those with T2DM (n = 1,028) by generalized hierarchical linear models followed by pairwise comparisons.
Results
Age, age at diagnosis, proportion of hypertension (all p<0.001), and waist circumference (p<0.05) were lower in FDA than T2DM. Nonetheless, the two groups had similar age-adjusted incidence rate of all-cause mortality.
Conclusions
Beside younger age at diagnosis, FDA patients show lower waist circumference and reduced proportion of hypertension as compared to those with T2DM; despite such reduced potential cardiovascular risk factors, FDA patients did not show a reduced mortality risk than patients with T2DM.
doi:10.1371/journal.pone.0135855
PMCID: PMC4545999  PMID: 26287533
9.  Statin Treatment and Mortality in Community-Dwelling Frail Older Patients with Diabetes Mellitus: A Retrospective Observational Study 
PLoS ONE  2015;10(6):e0130946.
Background
Older adults are often excluded from clinical trials. Decision making for administration of statins to older patients with diabetes mellitus (DM) is under debate, particularly in frail older patients with comorbidity and high mortality risk. We tested the hypothesis that statin treatment in older patients with DM was differentially effective across strata of mortality risk assessed by the Multidimensional Prognostic Index (MPI), based on information collected with the Standardized Multidimensional Assessment Schedule for Adults and Aged Persons (SVaMA).
Methods
In this retrospective observational study, we estimated the mortality risk in 1712 community-dwelling subjects with DM ≥ 65 years who underwent a SVaMA evaluation to establish accessibility to homecare services/nursing home admission from 2005 to 2013 in the Padova Health District, Italy. Mild (MPI-SVaMA-1), moderate (MPI-SVaMA-2), and high (MPI-SVaMA-3) risk of mortality at baseline and propensity score-adjusted hazard ratios (HR) of three-year mortality were calculated according to statin treatment.
Results
Higher MPI-SVaMA scores were associated with lower rates of statin treatment (MPI-SVaMA-1 = 39% vs MPI-SVaMA-2 = 36% vs MPI-SVaMA-3 = 24.9%. p<0.001) and higher three-year mortality (MPI-SVaMA-1 = 12.9% vs MPI-SVaMA-2 = 24% vs MPI-SVaMA-3 = 34.4%, p<0.001). After adjustment for propensity score quintiles, statin treatment was significantly associated with lower three-year mortality irrespective of MPI-SVaMA group (interaction test p = 0.303). HRs [95% confidence interval (CI)] were 0.19 (0.14–0.27), 0.28 (0.21–0.36), and 0.26 (0.20–0.34) in the MPI-SVaMA-1, MPI-SVaMA-2, and MPI-SVaMA-3 groups, respectively. Subgroup analyses showed that statin treatment was also beneficial irrespective of age. HRs (95% CI) were 0.21 (0.15–0.31), 0.26 (0.20–0.33), and 0.26 (0.20–0.35) among patients aged 65–74, 75–84, and ≥ 85 years, respectively (interaction test p=0.812).
Conclusions
Statin treatment was significantly associated with reduced three-year mortality independently of age and multidimensional impairment in community-dwelling frail older patients with DM.
doi:10.1371/journal.pone.0130946
PMCID: PMC4482517  PMID: 26110884
10.  TRIM8 downregulation in glioma affects cell proliferation and it is associated with patients survival 
BMC Cancer  2015;15:470.
Background
Human gliomas are a heterogeneous group of primary malignant brain tumors whose molecular pathogenesis is not yet solved. In this regard, a major research effort has been directed at identifying novel specific glioma-associated genes. Here, we investigated the effect of TRIM8 gene in glioma.
Methods
TRIM8 transcriptional level was profiled in our own glioma cases collection by qPCR and confirmed in the independent TCGA glioma cohort. The association between TRIM8 expression and Overall Survival and Progression-free Survival in TCGA cohort was determined by using uni-multivariable Cox regression analysis. The effect of TRIM8 on patient glioma cell proliferation was evaluated by performing MTT and clonogenic assays. The mechanisms causing the reduction of TRIM8 expression were explored by using qPCR and in vitro assays.
Results
We showed that TRIM8 expression correlates with unfavorable clinical outcome in glioma patients. We found that a restored TRIM8 expression induced a significant reduction of clonogenic potential in U87MG and patient’s glioblastoma cells. Finally we provide experimental evidences showing that miR-17 directly targets the 3′ UTR of TRIM8 and post-transcriptionally represses the expression of TRIM8.
Conclusions
Our study provides evidences that TRIM8 may participate in the carcinogenesis and progression of glioma and that the transcriptional repression of TRIM8 might have potential value for predicting poor prognosis in glioma patients.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-015-1449-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s12885-015-1449-9
PMCID: PMC4468980  PMID: 26077989
TRIM8; Glioblastoma; miR-17; Cell proliferation
11.  Vibration Perception Threshold in the Screening of Sensorimotor Distal Symmetric Polyneuropathy 
doi:10.1177/1932296814527818
PMCID: PMC4455454  PMID: 24876629
biothesiometer; diabetes mellitus; diabetic neuropathy; vibration perception threshold
12.  Target Values of Cardiovascular Risk Factors Are Not Associated with All-Cause Mortality in Patients with Type 2 Diabetes Mellitus 
PLoS ONE  2015;10(4):e0124536.
Background
To investigate prospectively the relationship between target values of glycated hemoglobin, blood pressure and LDL-cholesterol, as considered in a combined fashion, and all-cause mortality in patients with type 2 diabetes mellitus.
Methods
Two cohorts of patients with type 2 diabetes mellitus, the Gargano Mortality Study (n=810) and the Foggia Mortality Study (n=929), were investigated. A weighted target risk score was built as a weight linear combination of the recommended targets reached by each patient.
Results
In the Gargano Mortality Study and in the Foggia Mortality Study (mean follow up=7.4 and 5.5 years, respectively), 161 (19.9%) and 220 (23.7%) patients died, with an age and sex adjusted annual incidence rate of 2.1 and 2.8 per 100 person-years, respectively. In both study samples the weighted target risk score tended to be linearly associated with all-cause mortality (HR for one point increment=1.30, 95% CI: 1.11-1.53, p=0.001, and HR=1.08, 95% CI: 0.95-1.24, p=0.243, respectively). When the two cohorts were pooled and analyzed together, a clear association between weighted target risk score and all-cause mortality was observed (HR for one point increment=1.17, 95% CI:1.05-1.30, p=0.004). This counterintuitive association was no longer observable in a model including age, sex, body mass index, smoking habit, estimated glomerular filtration rate, albuminuria and anti-diabetic, anti-hypertensive and anti-dyslipidemic treatment as covariates (HR for one point increment=0.99, 95% CI: 0.87-1.12, p=0.852).
Conclusions
In a real life clinical set of patients with type 2 diabetes mellitus, the combination of recommended target values of established cardiovascular risk factors is not associated with all-cause mortality.
doi:10.1371/journal.pone.0124536
PMCID: PMC4415933  PMID: 25928715
13.  Thrombocytosis after hip and knee surgery in the rehabilitation setting: is it an occasional phenomenon? Relationship with deep venous thrombosis and functional outcome 
Background
Thrombocytosis can follow surgery and has occasionally been observed after major orthopaedic surgery. The aim of the present study was to ascertain the platelet count (PLTC) change in patients admitted to a rehabilitation unit after major joint surgery and whether deep venous thrombosis (DVT) and poor outcomes occurred in those who had thrombocytosis.
Method
PLTC, red blood cells (RBC), haemoglobin (Hb), fibrinogen, and D-dimers were assessed in patients on admission and at discharge after major joint surgery. Functional outcomes were ascertained using the Barthel Scale (BS), the Functional Independence Measure (FIM) and gait evaluation. Thrombocytosis was considered to have occurred when PLTC was greater than or equal to 500 × 1009/L. All subjects with thrombocytosis had ultrasonography to assess DVT occurrence. The patients were divided into “young” and “old” groups according to an age cut-off of 75 years to investigate potential age-related differences.
Results
Two hundred and seventy-five patients were identified and 142 (36 M and 106 F, mean age 77.2 ± 10.7) were enrolled. Seventy-six (53.5%) underwent total hip arthroplasty (THA), 40 (51.1%) underwent hip internal fixation and 26 (18.3%) subjects underwent total knee arthroplasty (TKA). The young and old groups included 60 and 82 patients, respectively. Fifty-nine (42.4%) patients had PLTC above 400 × 1009/L. Of these, 28 (20.1%) had thrombocytosis with PLTC above 500 × 1009/L, and 15 of them (10.7%) had very high values above 600 × 1009/L. Increased levels of fibrinogen and D-dimers were also detected. No subject with thrombocytosis had DVT. Outcome was not affected by PLTC. At discharge, significant improvement in all functional assessments was observed in young compared to old people; gait: 2.9 ± 0.2 vs. 2.2 ± 0.8; BS: 97 ± 6.9 vs. 70.5 ± 25.6; and FIM: 116.4 ± 10.9 vs. 83.6 ± 31.2 (p < 0.004), respectively. BS and FIM mean scores were positively correlated with Hb level.
Conclusion
Elevated PLTC and thrombocytosis were not uncommon in patients after major joint surgery, but no subject developed DVT. Platelet count change did not affect the outcome. Higher age and lower haemoglobin level correlated with poorer functional recovery.
doi:10.1186/s12891-015-0550-1
PMCID: PMC4403830  PMID: 25884173
Platelets; Thrombocytosis; Orthopaedic surgery; Functional outcome; Deep venous thrombosis
14.  Serum Resistin and Glomerular Filtration Rate in Patients with Type 2 Diabetes 
PLoS ONE  2015;10(3):e0119529.
Background
High serum levels of the pro-inflammatory adipokine resistin have been associated with decreased renal function in the general population. The goal of this study was to investigate whether such association is also present among diabetic subjects, who are at increased risk of renal function loss.
Methods
The cross-sectional association between serum resistin levels and estimated glomerular filtration rate (eGFR) was investigated in 1,560 type 2 diabetic (T2D) patients of European ancestry comprised in two different cohorts: 762 patients from San Giovanni Rotondo (SGR; Italy) and 798 patients from Boston (US).
Results
Serum resistin was inversely associated with eGFR in SGR [β (SE) for one SD of resistin increment = -1.01 (0.70) ml/min/1.73m2, p = 0.019] and in Boston [β (SE) = -5.31 (0.74) ml/min/1.73m2, p < 0.001] samples, as well as in the two studies combined [β (SE) = -3.42 (0.52) ml/min/1.73m2, p < 0.001]. The association was unaffected by adjustment for smoking habits, BMI, waist circumference, diabetes duration, HbA1c, insulin treatment, hypertension and lipid-lowering therapy: β (SE) for one SD of resistin increment = -1.07 (0.70), p = 0.02; -5.50 (0.88), p < 0.001; and -2.81 (0.55) ml/min/1.73m2, p < .001, in SGR, Boston and the two studies combined, respectively. The association was significantly stronger in men than in women (p for resistin-by-gender interaction = 0.003). For each resistin SD increment, the odds of having eGFR < 0 ml/min/1.73m2 increased by 22% (OR = 1.22; 95% CI 1.02–1.44; p = 0.025) in SGR sample, 69% (OR = 1.69; 95% CI 1.38–2.07; p < 0.001) in Boston sample, and 47% (OR = 1.47; 95% CI 1.29–1.68; p < 0.001) in the two studies considered together. Similar associations were observed in the adjusted model: OR 95% CI for each SD resistin increment being 1.23 (1.03–1.46), p = 0.021; 1.52 (1.20–1.92), p < 0.001; 1.33 (1.16–1.53), p < 0.001, in SGR, Boston and the two studies combined, respectively.
Conclusions
This is the first report of an association between high serum resistin and low eGFR in patients with T2D of European ancestry.
doi:10.1371/journal.pone.0119529
PMCID: PMC4374786  PMID: 25811174
15.  Association between Resistin Levels and All-Cause and Cardiovascular Mortality: A New Study and a Systematic Review and Meta-Analysis 
PLoS ONE  2015;10(3):e0120419.
Context
Studies concerning the association between circulating resistin and mortality risk have reported, so far, conflicting results.
Objective
To investigate the association between resistin and both all-cause and cardiovascular (CV) mortality risk by 1) analyzing data from the Gargano Heart Study (GHS) prospective design (n=359 patients; 81 and 58 all-cause and CV deaths, respectively); 2) performing meta-analyses of all published studies addressing the above mentioned associations.
Data Source and Study Selection
MEDLINE and Web of Science search of studies reporting hazard ratios (HR) of circulating resistin for all-cause or CV mortality.
Data Extraction
Performed independently by two investigators, using a standardized data extraction sheet.
Data Synthesis
In GHS, adjusted HRs per one standard deviation (SD) increment in resistin concentration were 1.28 (95% CI: 1.07-1.54) and 1.32 (95% CI: 1.06-1.64) for all-cause and CV mortality, respectively. The meta-analyses included 7 studies (n=4016; 961 events) for all-cause mortality and 6 studies (n=4,187: 412 events) for CV mortality. Pooled HRs per one SD increment in resistin levels were 1.21 (95% CI: 1.03-1.42, Q-test p for heterogeneity<0.001) and 1.05 (95% CI: 1.01-1.10, Q-test p for heterogeneity=0.199) for all-cause and CV mortality, respectively. At meta-regression analyses, study mean age explained 9.9% of all-cause mortality studies heterogeneity. After adjusting for age, HR for all-cause mortality was 1.24 (95% CI: 1.06-1.45).
Conclusions
Our results provide evidence for an association between circulating resistin and mortality risk among high-risk patients as are those with diabetes and coronary artery disease.
doi:10.1371/journal.pone.0120419
PMCID: PMC4368155  PMID: 25793385
16.  A MiRNA Signature for Defining Aggressive Phenotype and Prognosis in Gliomas 
PLoS ONE  2014;9(10):e108950.
Gliomas represent a disparate group of tumours for which there are to date no cure. Thus, there is a recognized need for new diagnostic and therapeutic approaches based on increased understanding of their molecular nature. We performed the comparison of the microRNA (miRNA) profile of 8 WHO grade II gliomas and 24 higher grade tumours (2 WHO grade III and 22 glioblastomas) by using the Affymetrix GeneChip miRNA Array v. 1.0. A relative quantification method (RT-qPCR) with standard curve was used to confirm the 22 miRNA signature resulted by array analysis. The prognostic performances of the confirmed miRNAs were estimated on the Tumor Cancer Genome Atlas (TCGA) datasets. We identified 22 miRNAs distinguishing grade II gliomas from higher grade tumours. RT-qPCR confirmed the differential expression in the two patients' groups for 13 out of the 22 miRNAs. The analysis of the Glioblastoma Multiforme (GBM) and Lower Grade Glioma (LGG) datasets from TCGA demonstrated the association with prognosis for 6 of those miRNAs. Moreover, in the GBM dataset miR-21 and miR-210 were predictors of worse prognosis in both univariable and multivariable Cox regression analyses (HR 1.19, p = 0.04, and HR 1.18, p = 0.029 respectively). Our results support a direct contribution of miRNAs to glioma cancerogenesis and suggest that miR-21 and miR-210 may play a role in the aggressive clinical behaviour of glioblastomas.
doi:10.1371/journal.pone.0108950
PMCID: PMC4184816  PMID: 25279461
17.  Circulating adiponectin and cardiovascular mortality in patients with type 2 diabetes mellitus: evidence of sexual dimorphism 
Background
The pathogenesis of cardiovascular (CV) mortality, whose rate is increased in type 2 diabetes, is poorly understood.
While high serum adiponectin is associated with increased CV mortality in the general population, no data are available in type 2 diabetes.
We here investigated whether this counterintuitive association was observable also in diabetic patients and whether it was sex-specific.
Methods
Three prospective cohorts were analyzed: 1) Gargano Heart Study (GHS; 359 patients, 58 events/1,934 person-years; py); 2) Health Professional Follow-up Study (HPFS; 833 men, 146 events/10,024 py); 3) Nurses’ Health Study (NHS; 902 women, 144 events/15,074 py).
Results
In GHS serum adiponectin predicted CV mortality in men (hazard ratio, HR, and 95% CI per standard deviation, SD, increment = 1.54, 1.19-2.01), but not women (HR = 0.98, 0.48-2.01).
Circulating adiponectin predicted CV mortality in men from HPFS (HR = 1.44, 1.21-1.72), but not in women from NHS (HR = 1.08, 0.86-1.35), used as replication samples. In a pooled analysis, HRs were 1.47 (1.27-1.70) in 1,075 men and 1.07 (0.86-1.33) in 1,019 women (p for HRs heterogeneity across sexes = 0.018).
Conclusions
This is the first report showing that high circulating adiponectin predicts increased CV mortality in men, but not in women with type 2 diabetes. Further studies are necessary to unravel the mechanisms through which adiponectin influences CV mortality in a sex-specific manner.
Electronic supplementary material
The online version of this article (doi:10.1186/s12933-014-0130-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s12933-014-0130-y
PMCID: PMC4172916  PMID: 25200659
Adipokines; Prospective studies; Paradoxical effect; Sex-linked genes
18.  Modeling interactions between Human Equilibrative Nucleoside Transporter-1 and other factors involved in the response to gemcitabine treatment to predict clinical outcomes in pancreatic ductal adenocarcinoma patients 
Background
Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive malignancy, characterized by largely unsatisfactory responses to the currently available therapeutic strategies. In this study we evaluated the expression of genes involved in gemcitabine uptake in a selected cohort of patients with PDAC, with well-defined clinical-pathological features.
Methods
mRNA levels of hENT1, CHOP, MRP1 and DCK were evaluated by means of qRT-PCR in matched pairs of tumor and adjacent normal tissue samples collected from PDAC patients treated with gemcitabine after surgical tumor resection. To detect possible interaction between gene expression levels and to identify subgroups of patients at different mortality/progression risk, the RECursive Partitioning and Amalgamation (RECPAM) method was used.
Results
RECPAM analysis showed that DCK and CHOP were most relevant variables for the identification of patients with different mortality risk, while hENT1 and CHOP were able to identify subgroups of patients with different disease progression risk. Conclusion: hENT1, CHOP, MRP1 and DCK appear correlated to PDAC, and this interaction might influence disease behavior.
doi:10.1186/s12967-014-0248-4
PMCID: PMC4172900  PMID: 25199538
Pancreatic ductal adenocarcinoma; hENT1; CHOP; MRP1; DCK; RECPAM
19.  Development and Validation of a Predicting Model of All-Cause Mortality in Patients With Type 2 Diabetes 
Diabetes Care  2013;36(9):2830-2835.
OBJECTIVE
To develop and validate a parsimonious model for predicting short-term all-cause mortality in patients with type 2 diabetes mellitus (T2DM).
RESEARCH DESIGN AND METHODS
Two cohorts of patients with T2DM were investigated. The Gargano Mortality Study (GMS, n = 679 patients) was the training set and the Foggia Mortality Study (FMS, n = 936 patients) represented the validation sample. GMS and FMS cohorts were prospectively followed up for 7.40 ±2.15 and 4.51 ±1.69 years, respectively, and all-cause mortality was registered. A new forward variable selection within a multivariate Cox regression was implemented. Starting from the empty model, each step selected the predictor that, once included into the multivariate Cox model, yielded the maximum continuous net reclassification improvement (cNRI). The selection procedure stopped when no further statistically significant cNRI increase was detected.
RESULTS
Nine variables (age, BMI, diastolic blood pressure, LDL cholesterol, triglycerides, HDL cholesterol, urine albumin-to-creatinine ratio, and antihypertensive and insulin therapy) were included in the final predictive model with a C statistic of 0.88 (95% CI 0.82–0.94) in the GMS and 0.82 (0.76–0.87) in the FMS. Finally, we used a recursive partition and amalgamation algorithm to identify patients at intermediate and high mortality risk (hazard ratio 7.0 and 24.4, respectively, as compared with those at low risk). A web-based risk calculator was also developed.
CONCLUSIONS
We developed and validated a parsimonious all-cause mortality equation in T2DM, providing also a user-friendly web-based risk calculator. Our model may help prioritize the use of available resources for targeting aggressive preventive and treatment strategies in a subset of very high-risk individuals.
doi:10.2337/dc12-1906
PMCID: PMC3747924  PMID: 23637348
20.  Cognitive Impairment in Myotonic Dystrophy Type 1 Is Associated with White Matter Damage 
PLoS ONE  2014;9(8):e104697.
Objective
To investigate grey (GM) and white matter (WM) abnormalities and their effects on cognitive and behavioral deficits in a large, phenotypically and genotypically well-characterized cohort of classic adult (aDM1, age at onset ≥20 years) or juvenile (jDM1, age at onset <20 years) patients with myotonic dystrophy type 1 (DM1).
Methods
A case-control study including 51 DM1 patients (17 jDM1 and 34 aDM1) and 34 controls was conducted at an academic medical center. Clinical, cognitive and structural MRI evaluations were obtained. Quantitative assessments of regional GM volumes, WM hyperintensities (WMHs), and microstructural WM tract damage were performed. The association between structural brain damage and clinical and cognitive findings was assessed.
Results
DM1 patients showed a high prevalence of WMHs, severe regional GM atrophy including the key nodes of the sensorimotor and main cognitive brain networks, and WM microstructural damage of the interhemispheric, corticospinal, limbic and associative pathways. WM tract damage extends well beyond the focal WMHs. While aDM1 patients had severe patterns of GM atrophy and WM tract damage, in jDM1 patients WM abnormalities exceeded GM involvement. In DM1, WMHs and microstructural damage, but not GM atrophy, correlated with cognitive deficits.
Conclusions
WM damage, through a disconnection between GM structures, is likely to be the major contributor to cognitive impairment in DM1. Our MRI findings in aDM1 and jDM1 patients support the hypothesis of a degenerative (premature aging) origin of the GM abnormalities and of developmental changes as the principal substrates of microstructural WM alterations in DM1.
doi:10.1371/journal.pone.0104697
PMCID: PMC4130603  PMID: 25115999
21.  Evaluation of Genome-Wide Expression Profiles of Blood and Sputum Neutrophils in Cystic Fibrosis Patients Before and After Antibiotic Therapy 
PLoS ONE  2014;9(8):e104080.
In seeking more specific biomarkers of the cystic fibrosis (CF) lung inflammatory disease that would be sensitive to antibiotic therapy, we sought to evaluate the gene expression profiles of neutrophils in CF patients before treatment in comparison with non-CF healthy individuals and after antibiotic treatment. Genes involved in neutrophil-mediated inflammation, i.e. chemotaxis, respiratory burst, apoptosis, and granule exocytosis, were the targets of this study. Microarray analysis was carried out in blood and airway neutrophils from CF patients and in control subjects. A fold change (log) threshold of 1.4 and a cut-off of p<0.05 were utilized to identify significant genes. Community networks and principal component analysis were used to distinguish the groups of controls, pre- and post-therapy patients. Control subjects and CF patients before therapy were readily separated, whereas a clear distinction between patients before and after antibiotic therapy was not possible. Blood neutrophils before therapy presented 269 genes down-regulated and 56 up-regulated as compared with control subjects. Comparison between the same patients before and after therapy showed instead 44 genes down-regulated and 72 up-regulated. Three genes appeared to be sensitive to therapy and returned to “healthy” condition: phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), hydrogen voltage-gated channel 1 (HVCN1), and β-arrestin 1 (ARRB1). The up-regulation of these genes after therapy were confirmed by real time PCR. In airway neutrophils, 1029 genes were differentially expressed post- vs pre-therapy. Of these, 30 genes were up-regulated and 75 down-regulated following antibiotic treatment. However, biological plausibility determined that only down-regulated genes belonged to the gene classes studied for blood neutrophils. Finally, it was observed that commonly expressed genes showed a greater variability in airway neutrophils than that found in blood neutrophils, both before and after therapy. These results indicate more specific targets for future interventions in CF patients involving respiratory burst, apoptosis, and granule exocytosis.
doi:10.1371/journal.pone.0104080
PMCID: PMC4118979  PMID: 25084273
22.  MRI Predicts Efficacy of Constraint-Induced Movement Therapy in Children With Brain Injury 
Neurotherapeutics  2013;10(3):511-519.
Using resting state (RS) functional magnetic resonance imaging (fMRI) and diffusion tensor imaging (DTI), we identified the predictors of clinical improvement following constraint-induced movement therapy (CIMT) in pediatric patients with chronic hemiplegia.From 14 children with congenital or acquired brain injury and 10 sex- and age-matched healthy controls, brain dual-echo, DTI and RS fMRI sequences were acquired before CIMT. The Quality of Upper Extremities Skills Test and the Gross Motor Function Measure (GMFM) were administered at baseline, at the end of CIMT (10 weeks), and after 6 months. Mean diffusivity and fractional anisotropy (FA) were measured in the lesion responsible for the clinical symptomatology, the affected and unaffected corticospinal tract (CST), motor transcallosal fibers, and uncinate fasciculus (as an internal control). Independent component analysis was used to identify the sensorimotor RS network. The ability of baseline MRI variables to predict clinical changes over time was assessed using multivariate linear models. At baseline, patients had increased mean diffusivity in the symptomatic lesion and decreased FA in the symptomatic lesion, affected corticospinal tract, and motor transcallosal fibers. A reduced RS functional connectivity was found in the bilateral cerebellum, left precentral gyrus, and right secondary sensorimotor cortex. At follow up, Quality of Upper Extremities Skills Test and GMFM scales improved significantly. Baseline average lesion FA predicted clinical improvement at week 10, and baseline functional connectivity of the right secondary sensorimotor cortex and cerebellum predicted GMFM improvement at month 6. DTI and RS fMRI offer promising and objective markers to predict clinical outcomes following CIMT in pediatric patients with congenital or acquired hemiplegia.
Electronic supplementary material
The online version of this article (doi:10.1007/s13311-013-0189-2) contains supplementary material, which is available to authorized users.
doi:10.1007/s13311-013-0189-2
PMCID: PMC3701764  PMID: 23605556
MRI; DTI; Tractography; Resting-state functional connectivity; Brain injury; Constraint-induced movement therapy
23.  Evaluation of microRNA-10b prognostic significance in a prospective cohort of breast cancer patients 
Molecular Cancer  2014;13:142.
Background
MicroRNA-10b (miR-10b) has a prominent role in regulating tumor invasion and metastasis by targeting the HOXD10 transcriptional repressor and has been found up-regulated in several tumor types.
Methods
We evaluated the expression of miR-10b in paired tumor and normal specimens obtained from a prospective cohort of breast cancer patients with at least 36 months follow-up enrolled according to the REMARK guidelines (n = 150). RNA quality was measured and only samples with RNA Integrity Number (RIN) ≥7.0 were analyzed.
Results
The relative expression of miR-10b in tumor as compared to its normal counterpart (RER) was determined by RT-qPCR. miR-10b RERs were higher in the subgroup of patients with synchronous metastases (n = 11, Median 0.25; IQR 0.11-1.02) as compared with patients without metastases (n = 90, Median 0.09; IQR 0.04-0.29) (p = 0.028). In the subgroup of patients without synchronous metastases (n = 90), higher miR-10b RERs were associated with increased risk of disease progression and death in both univariable (HR 1.16, p = 0.021 and HR 1.20, p = 0.015 respectively for 0.10 unitary increase of miR-10b RERs levels) and multivariable (HR1.30, p < 0.001, and HR 1.31, p = 0.003 respectively for 0.10 unitary increase of miR-10b RERs levels) Cox regression models. The addition of miR-10b RERs to the Nottingham Prognostic Index (NPI) provided an improvement in discrimination power and risk reclassification abilities for the clinical outcomes at 36 months. Survival C-indices significantly increased from 0.849 to 0.889 (p = 0.009) for OS and from 0.735 to 0.767 (p = 0.050) for DFS.
Conclusions
Our results provide evidences that the addition of miR-10b RERs to the prognostic factors used in clinical routine could improve the prediction abilities for both overall mortality and disease progression in breast cancer patients.
doi:10.1186/1476-4598-13-142
PMCID: PMC4055397  PMID: 24897960
Breast cancer; microRNA; Metastasis; RT-qPCR
24.  The EphA2 Receptor Drives Self-Renewal and Tumorigenicity in Stem-Like Tumor-Propagating Cells from Human Glioblastomas 
Cancer cell  2012;22(6):765-780.
SUMMARY
In human glioblastomas (hGBMs), tumor-propagating cells with stem-like characteristics (TPCs) represent a key therapeutic target. We found that the EphA2 receptor tyrosine kinase is overexpressed in hGBM TPCs. Cytofluorimetric sorting into EphA2High and EphA2Low populations demonstrated that EphA2 expression correlates with the size and tumor-propagating ability of the TPC pool in hGBMs. Both, ephrinA1-Fc, which caused EphA2 downregulation in TPCs, and siRNA-mediated knockdown of EPHA2 expression suppressed TPCs self-renewal ex vivo and intracranial tumorigenicity, pointing to EphA2 downregulation as a causal event in the loss of TPCs tumorigenicity. Infusion of ephrinA1-Fc into intracranial xenografts elicited strong tumor-suppressing effects, suggestive of therapeutic applications.
doi:10.1016/j.ccr.2012.11.005
PMCID: PMC3922047  PMID: 23238013
25.  Joint effect of insulin signalling genes on cardiovascular events and on whole body and endothelial insulin resistance 
Atherosclerosis  2012;226(1):140-145.
Objective
Insulin resistance (IR) and cardiovascular disease (CVD) share a common soil. We investigated the combined role of single nucleotide polymorphisms (SNPs) affecting insulin signaling (ENPP1 K121Q, rs1044498; IRS1 G972R, rs1801278; TRIB3 Q84R, rs2295490) on CVD, age at myocardial infarction (MI), in vivo insulin sensitivity and in vitro insulin-stimulated nitric oxide synthase (NOS) activity.
Design and Setting
1. We first studied, incident cardiovascular events (a composite endpoint comprising myocardial infarction -MI-, stroke and cardiovascular death) in 733 patients (2,186 person-years, 175 events). 2. In a replication attempt, age at MI was tested in 331 individuals. 3. OGTT-derived insulin sensitivity index (ISI) was assessed in 829 individuals with fasting glucose < 126 mg/dl. 4. NOS activity was measured in 40 strains of human vein endothelial cells (HUVECs).
Results
1. Risk variants jointly predicted cardiovascular events (HR=1.181; p=0.0009) and, when added to clinical risk factors, significantly improved survival C-statistics; they also allowed a significantly correct reclassification (by net reclassification index) in the whole sample (135/733 individuals) and, even more, in obese patients (116/204 individuals). 2. Risk variants were jointly associated with age at MI (p=0.006). 3. A significant association was also observed with ISI (p=0.02). 4. Finally, risk variants were jointly associated with insulin-stimulated NOS activity in HUVECs (p=0.009).
Conclusions
Insulin signaling genes variants jointly affect cardiovascular disease, very likely by promoting whole body and endothelium-specific insulin resistance. Further studies are needed to address whether their genotyping help identify very high-risk patients who need specific and/or more aggressive preventive strategies.
doi:10.1016/j.atherosclerosis.2012.10.035
PMCID: PMC3529747  PMID: 23107043
genetic susceptibility; non synonymous polymorphism; insulin sensitivity; insulin dependent endothelial function

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