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1.  Computational Design of Ligand Binding Proteins with High Affinity and Selectivity 
Nature  2013;501(7466):212-216.
The ability to design proteins with high affinity and selectivity for any given small molecule would have numerous applications in biosensing, diagnostics, and therapeutics, and is a rigorous test of our understanding of the physiochemical principles that govern molecular recognition phenomena. Attempts to design ligand binding proteins have met with little success, however, and the computational design of precise molecular recognition between proteins and small molecules remains an “unsolved problem”1. We describe a general method for the computational design of small molecule binding sites with pre-organized hydrogen bonding and hydrophobic interfaces and high overall shape complementary to the ligand, and use it to design protein binding sites for the steroid digoxigenin (DIG). Of 17 designs that were experimentally characterized, two bind DIG; the highest affinity design has the lowest predicted interaction energy and the most pre-organized binding site in the set. A comprehensive binding-fitness landscape of this design generated by library selection and deep sequencing was used to guide optimization of binding affinity to a picomolar level, and two X-ray co-crystal structures of optimized complexes show atomic level agreement with the design models. The designed binder has a high selectivity for DIG over the related steroids digitoxigenin, progesterone, and β-estradiol, which can be reprogrammed through the designed hydrogen-bonding interactions. Taken together, the binding fitness landscape, co-crystal structures, and thermodynamic binding parameters illustrate how increases in binding affinity can result from distal sequence changes that limit the protein ensemble to conformers making the most energetically favorable interactions with the ligand. The computational design method presented here should enable the development of a new generation of biosensors, therapeutics, and diagnostics.
doi:10.1038/nature12443
PMCID: PMC3898436  PMID: 24005320
2.  A Bioartificial Renal Tubule Device Embedding Human Renal Stem/Progenitor Cells 
PLoS ONE  2014;9(1):e87496.
We present a bio-inspired renal microdevice that resembles the in vivo structure of a kidney proximal tubule. For the first time, a population of tubular adult renal stem/progenitor cells (ARPCs) was embedded into a microsystem to create a bioengineered renal tubule. These cells have both multipotent differentiation abilities and an extraordinary capacity for injured renal cell regeneration. Therefore, ARPCs may be considered a promising tool for promoting regenerative processes in the kidney to treat acute and chronic renal injury. Here ARPCs were grown to confluence and exposed to a laminar fluid shear stress into the chip, in order to induce a functional cell polarization. Exposing ARPCs to fluid shear stress in the chip led the aquaporin-2 transporter to localize at their apical region and the Na+K+ATPase pump at their basolateral portion, in contrast to statically cultured ARPCs. A recovery of urea and creatinine of (20±5)% and (13±5)%, respectively, was obtained by the device. The microengineered biochip here-proposed might be an innovative “lab-on-a-chip” platform to investigate in vitro ARPCs behaviour or to test drugs for therapeutic and toxicological responses.
doi:10.1371/journal.pone.0087496
PMCID: PMC3907467  PMID: 24498117
3.  Technological Solutions and Main Indices for the Assessment of Newborns' Nutritive Sucking: A Review 
Sensors (Basel, Switzerland)  2014;14(1):634-658.
Nutritive Sucking (NS) is a highly organized process that is essential for infants' feeding during the first six months of their life. It requires the complex coordination of sucking, swallowing and breathing. The infant's inability to perform a safe and successful oral feeding can be an early detector of immaturity of the Central Nervous System (CNS). Even though the importance of early sucking measures has been confirmed over the years, the need for standardized instrumental assessment tools still exists. Clinicians would benefit from specifically designed devices to assess oral feeding ability in their routine clinical monitoring and decision-making process. This work is a review of the main instrumental solutions developed to assess an infant's NS behavior, with a detailed survey of the main quantities and indices measured and/or estimated to characterize sucking behavior skills and their development. The adopted sensing measuring systems will be described, and their main advantages and weaknesses will be discussed, taking into account their application to clinical practice, or to at-home monitoring as post-discharge assessment tools. Finally, the study will highlight the most suitable sensing solutions and give some prompts for further research.
doi:10.3390/s140100634
PMCID: PMC3926579  PMID: 24451459
nutritive sucking monitoring; oral feeding skills; smart objects; early neonatal assessment; clinical decision-making; at-home monitoring
5.  Downregulation of Nuclear-Encoded Genes of Oxidative Metabolism in Dialyzed Chronic Kidney Disease Patients 
PLoS ONE  2013;8(10):e77847.
Background
Mitochondria, essential eukaryotic cells organelles defined as the “powerhouse of the cell” because of their ability to produce the vast majority of energy necessary for cellular metabolism, may have a primary role in the oxidative stress-related intracellular machinery associated to chronic kidney disease (CKD).
Methods
To better assess this research assumption, we decided to study the key factors regulating mitochondrial oxidative metabolism in CKD patients in peritoneal dialysis (PD, n = 15) using several bio-molecular methodologies.
Results
RT-PCR experiments demonstrate that the expression level of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) and nuclear respiratory factor-1 (NRF-1), two genes primarily involved in mitochondrial biogenesis and functions, were significantly hypo-expressed in peripheral blood mononuclear cells of PD patients compared to healthy subjects (HS, n = 15). Additionally, mRNA levels of several PGC1-α downstream target genes (TFAM, COX6C,COX7C, UQCRH and MCAD) were profoundly down-regulated in PD cells. TFAM protein analysis confirmed gene-expression results. High plasmatic concentration of Malondialdehyde found in PD patients, confirmed the contribution of the oxidative stress to these biological effects. Finally, Nuclear factor erythroid-derived 2-like 2 (NRF2 or NFE2L2), a transcription factor for numerous antioxidant/detoxifying enzymes and one of its target genes, superoxide dismutase-2 mitochondrial (SOD2) were up-regulated in PD compared to HS.
Conclusions
Our results revealed, for the first time, that CKD-PD patients’ PBMC, through a complex intracellular biochemical machinery, are able to modulate their mitochondrial functions probably in the attempt to reduce oxidative metabolic damage and to turn on a valuable defense cellular strategy against oxidative stress.
doi:10.1371/journal.pone.0077847
PMCID: PMC3810143  PMID: 24204994
6.  The role of exercise capacity in the health and longevity of centenarians 
Maturitas  2012;73(2):115-120.
Ageing is a continuum of biological processes characterized by progressive adaptations which can be influenced by both genetic and physiological factors. In terms of human maturation, physically and cognitively functional centenarians certainly represent an impressive example of successful healthy ageing. However, even in these unique individuals, with the passage of time, declining lung function and sarcopenia lead to a progressive fall in maximal strength, maximal oxygen uptake, and therefore reduced exercise capacity. The subsequent mobility limitation can initiate a viscous downward spiral of reduced physical function and health. Emerging literature has shed some light on this multi-factorial decline in function associated with aging and the positive role that exercise and physical capacity can play in the elderly. Recognizing the multiple factors that influence ageing, the aim of this review is to highlight the recently elucidated limitations to physical function of the extremely old and therefore evaluate the role of exercise capacity in the health and longevity of centenarians.
doi:10.1016/j.maturitas.2012.07.009
PMCID: PMC3618983  PMID: 22883374
Mechanical efficiency; VO2max; Independence; Lifestyle; Mobility
7.  Optical Fiber-Based MR-Compatible Sensors for Medical Applications: An Overview 
Sensors (Basel, Switzerland)  2013;13(10):14105-14120.
During last decades, Magnetic Resonance (MR)—compatible sensors based on different techniques have been developed due to growing demand for application in medicine. There are several technological solutions to design MR-compatible sensors, among them, the one based on optical fibers presents several attractive features. The high elasticity and small size allow designing miniaturized fiber optic sensors (FOS) with metrological characteristics (e.g., accuracy, sensitivity, zero drift, and frequency response) adequate for most common medical applications; the immunity from electromagnetic interference and the absence of electrical connection to the patient make FOS suitable to be used in high electromagnetic field and intrinsically safer than conventional technologies. These two features further heightened the potential role of FOS in medicine making them especially attractive for application in MRI. This paper provides an overview of MR-compatible FOS, focusing on the sensors employed for measuring physical parameters in medicine (i.e., temperature, force, torque, strain, and position). The working principles of the most promising FOS are reviewed in terms of their relevant advantages and disadvantages, together with their applications in medicine.
doi:10.3390/s131014105
PMCID: PMC3859111  PMID: 24145918
fiber optic sensors; MR-compatibility; interferometry; fiber Bragg grating; hyperthermia; respiratory monitoring; MR-compatible robotic assistive device
8.  miR-1915 and miR-1225-5p Regulate the Expression of CD133, PAX2 and TLR2 in Adult Renal Progenitor Cells 
PLoS ONE  2013;8(7):e68296.
Adult renal progenitor cells (ARPCs) were recently identified in the cortex of the renal parenchyma and it was demonstrated that they were positive for PAX2, CD133, CD24 and exhibited multipotent differentiation ability. Recent studies on stem cells indicated that microRNAs (miRNAs), a class of noncoding small RNAs that participate in the regulation of gene expression, may play a key role in stem cell self-renewal and differentiation. Distinct sets of miRNAs are specifically expressed in pluripotent stem cells but not in adult tissues, suggesting a role for miRNAs in stem cell self-renewal. We compared miRNA expression profiles of ARPCs with that of mesenchymal stem cells (MSCs) and renal proximal tubular cells (RPTECs) finding distinct sets of miRNAs that were specifically expressed in ARPCs. In particular, miR-1915 and miR-1225-5p regulated the expression of important markers of renal progenitors, such as CD133 and PAX2, and important genes involved in the repair mechanisms of ARPCs, such as TLR2. We demonstrated that the expression of both the renal stem cell markers CD133 and PAX2 depends on lower miR-1915 levels and that the increase of miR-1915 levels improved capacity of ARPCs to differentiate into adipocyte-like and epithelial-like cells. Finally, we found that the low levels of miR-1225-5p were responsible for high TLR2 expression in ARPCs. Therefore, together, miR-1915 and miR-1225-5p seem to regulate important traits of renal progenitors: the stemness and the repair capacity.
doi:10.1371/journal.pone.0068296
PMCID: PMC3704645  PMID: 23861881
9.  Alterations of Neuromuscular Function after the World's Most Challenging Mountain Ultra-Marathon 
PLoS ONE  2013;8(6):e65596.
We investigated the physiological consequences of the most challenging mountain ultra-marathon (MUM) in the world: a 330-km trail run with 24000 m of positive and negative elevation change. Neuromuscular fatigue (NMF) was assessed before (Pre-), during (Mid-) and after (Post-) the MUM in experienced ultra-marathon runners (n = 15; finish time  = 122.43 hours ±17.21 hours) and in Pre- and Post- in a control group with a similar level of sleep deprivation (n = 8). Blood markers of muscle inflammation and damage were analyzed at Pre- and Post-. Mean ± SD maximal voluntary contraction force declined significantly at Mid- (−13±17% and −10±16%, P<0.05 for knee extensor, KE, and plantar flexor muscles, PF, respectively), and further decreased at Post- (−24±13% and −26±19%, P<0.01) with alteration of the central activation ratio (−24±24% and −28±34% between Pre- and Post-, P<0.05) in runners whereas these parameters did not change in the control group. Peripheral NMF markers such as 100 Hz doublet (KE: −18±18% and PF: −20±15%, P<0.01) and peak twitch (KE: −33±12%, P<0.001 and PF: −19±14%, P<0.01) were also altered in runners but not in controls. Post-MUM blood concentrations of creatine kinase (3719±3045 Ul·1), lactate dehydrogenase (1145±511 UI·L−1), C-Reactive Protein (13.1±7.5 mg·L−1) and myoglobin (449.3±338.2 µg·L−1) were higher (P<0.001) than at Pre- in runners but not in controls. Our findings revealed less neuromuscular fatigue, muscle damage and inflammation than in shorter MUMs. In conclusion, paradoxically, such extreme exercise seems to induce a relative muscle preservation process due likely to a protective anticipatory pacing strategy during the first half of MUM and sleep deprivation in the second half.
doi:10.1371/journal.pone.0065596
PMCID: PMC3694082  PMID: 23840345
10.  Heart Rate and Arterial Pressure Changes during Whole-Body Deep Hypothermia 
ISRN Pediatrics  2013;2013:140213.
Whole-body deep hypothermia (DH) could be a new therapeutic strategy for asphyxiated newborn. This retrospective study describes how DH modified the heart rate and arterial blood pressure if compared to mild hypothermia (MH). Fourteen in DH and 17 in MH were cooled within the first six hours of life and for the following 72 hours. Hypothermia criteria were gestational age ≥36 weeks; birth weight ≥1800 g; clinical signs of moderate/severe hypoxic-ischemic encephalopathy. Rewarming was obtained in the following 6–12 hours (0.5°C/h) after cooling. Heart rates were the same between the two groups; there was statistically significant difference at the beginning of hypothermia and during rewarming. Three babies in the DH group and 2 in the MH group showed HR < 80 bpm and QTc > 520 ms. Infant submitted to deep hypothermia had not bradycardia or Qtc elongation before cooling and after rewarming. Blood pressure was significantly lower in DH compared to MH during the cooling, and peculiar was the hypotension during rewarming in DH group. Conclusion. The deeper hypothermia is a safe and feasible, only if it is performed by a well-trained team. DH should only be associated with a clinical trial and prospective randomized trials to validate its use.
doi:10.1155/2013/140213
PMCID: PMC3649319  PMID: 23691350
11.  Metabolic Effects of Aerobic Training and Resistance Training in Type 2 Diabetic Subjects 
Diabetes Care  2012;35(4):676-682.
OBJECTIVE
To assess differences between the effects of aerobic and resistance training on HbA1c (primary outcome) and several metabolic risk factors in subjects with type 2 diabetes, and to identify predictors of exercise-induced metabolic improvement.
RESEARCH DESIGN AND METHODS
Type 2 diabetic patients (n = 40) were randomly assigned to aerobic training or resistance training. Before and after 4 months of intervention, metabolic phenotypes (including HbA1c, glucose clamp–measured insulin sensitivity, and oral glucose tolerance test–assessed β-cell function), body composition by dual-energy X-ray absorptiometry, visceral (VAT) and subcutaneous (SAT) adipose tissue by magnetic resonance imaging, cardiorespiratory fitness, and muscular strength were measured.
RESULTS
After training, increase in peak oxygen consumption (VO2peak) was greater in the aerobic group (time-by-group interaction P = 0.045), whereas increase in strength was greater in the resistance group (time-by-group interaction P < 0.0001). HbA1c was similarly reduced in both groups (−0.40% [95% CI −0.61 to −0.18] vs. −0.35% [−0.59 to −0.10], respectively). Total and truncal fat, VAT, and SAT were also similarly reduced in both groups, whereas insulin sensitivity and lean limb mass were similarly increased. β-Cell function showed no significant changes. In multivariate analyses, improvement in HbA1c after training was independently predicted by baseline HbA1c and by changes in VO2peak and truncal fat.
CONCLUSIONS
Resistance training, similarly to aerobic training, improves metabolic features and insulin sensitivity and reduces abdominal fat in type 2 diabetic patients. Changes after training in VO2peak and truncal fat may be primary determinants of exercise-induced metabolic improvement.
doi:10.2337/dc11-1655
PMCID: PMC3308269  PMID: 22344613
12.  The Cox-Maze Procedure for Lone Atrial Fibrillation: A Single Center Experience over Two Decades 
Background
The Cox-Maze procedure (CMP) has achieved high success rates in the therapy of atrial fibrillation (AF) while becoming progressively less invasive. This report evaluates our experience with the CMP in the treatment of lone AF over two decades and compares the original cut-and-sew CMP-III to the ablation-assisted CMP-IV, which uses bipolar radiofrequency and cryoenergy to create the original lesion pattern.
Methods and Results
Data were collected prospectively on 212 consecutive patients (mean age: 53.5±10.4, 78% males), who underwent a stand-alone CMP from 1992 through 2010. Median duration of preoperative AF was 6 (IQR 2.9–11.5) years, with 48% paroxysmal and 52% persistent or longstanding persistent AF. Univariate analysis with preoperative and perioperative variables used as covariates for the CMP-III (n=112) and the CMP-IV (n=100) was performed. Overall, 30-day mortality was 1.4% with no intraoperative deaths. Freedom from AF was 93% and freedom from AF off antiarrhythmics was 82% at a mean follow-up time of 3.6 ± 3.1 years. Freedom from symptomatic AF at 10 years was 85%. Only one late stroke occurred with 80% of patients being off anticoagulation. The less invasive CMP-IV had significantly shorter cross-clamp times (41±13 vs. 92±26 minutes, p<0.001) while achieving high success rates with 90% freedom from AF and 84% freedom from AF off antiarrhythmics at 2 years.
Conclusions
The CMP, while simplified and shortened by alternative energy sources, has excellent results even with improved follow-up and stricter definition of failure.
doi:10.1161/CIRCEP.111.963819
PMCID: PMC3288520  PMID: 22095640
ablation; arrhythmia (Heart Rhythm Disorders); atrial fibrillation; surgery; tachyarrhythmias
13.  Foot-strike haemolysis after a 60-km ultramarathon 
Blood Transfusion  2012;10(3):377-383.
Background.
The various contributors to sport-related anaemia include increased plasma volume, exercise-induced oxidative stress, increased body temperature, acidosis, gastrointestinal bleeding, acute and chronic inflammation as well as compression and damage of red blood cells (RBC) in the capillaries within the contracting muscles. The effective contribution of foot-strike haemolysis is unclear.
Materials and methods.
We studied 18 Caucasian male athletes (mean age, 42 years; range, 34–52 years) before and immediately after a 60-km ultramarathon. Laboratory investigations included the haematological profile along with haptoglobin, potassium, aspartate aminotransferase (AST), creatine kinase (CK), lactate dehydrogenase (LDH) and albumin concentrations and a haemolysis index (HI).
Results.
No significant variations were found in post-exercise values of haemoglobin, RBC count and haematocrit. Mean corpuscular volume and haptoglobin were significantly decreased, whereas RBC distribution width was increased. The concentration of haptoglobin was reduced by approximately 50%, whereas enzyme concentrations were all remarkably increased. The HI remained below 0.5 g/L. After adjusting for plasma volume change, the increases were 1.7% for potassium (P=0.17), 30% for AST (P<0.01), 49% for LDH (P<0.01) and 2.39-fold for CK (P<0.01). A statistically significant association was found between haemoconcentration-adjusted variations of CK and those of AST (r=0.803; P<0.01) and LDH (r=0.551; P=0.02).
Discussion.
This is the first study demonstrating that long-distance running does not induce clinically significant changes in haemoglobin, haematocrit, RBC count or potassium concentration. The significant post-exercise decrease of haptoglobin reflects a certain degree of haemolysis, but the concentration of cell-free haemoglobin remaining below 0.5 g/L and the non-significant variation in RBC count both indicate that the foot-strike haemolysis is very modest or even clinically negligible.
doi:10.2450/2012.0167-11
PMCID: PMC3417738  PMID: 22682343
sport; physical exercise; marathon; haemolysis
14.  Altered chromatin organization and SUN2 localization in mandibuloacral dysplasia are rescued by drug treatment 
Histochemistry and Cell Biology  2012;138(4):643-651.
Mandibuloacral dysplasia type A (MADA) is a rare laminopathy characterized by growth retardation, craniofacial anomalies, bone resorption at specific sites including clavicles, phalanges and mandibula, mottled cutaneous pigmentation, skin rigidity, partial lipodystrophy, and insulin resistance. The disorder is caused by recessive mutations of the LMNA gene encoding for A-type lamins. The molecular feature of MADA consists in the accumulation of the unprocessed lamin A precursor, which is detected at the nuclear rim and in intranuclear aggregates. Here, we report the characterization of prelamin A post-translational modifications in MADA cells that induce alterations in the chromatin arrangement and dislocation of nuclear envelope-associated proteins involved in correct nucleo-cytoskeleton relationships. We show that protein post-translational modifications change depending on the passage number, suggesting the onset of a feedback mechanism. Moreover, we show that treatment of MADA cells with the farnesyltransferase inhibitors is effective in the recovery of the chromatin phenotype, altered in MADA, provided that the cells are at low passage number, while at high passage number, the treatment results ineffective. Moreover, the distribution of the lamin A interaction partner SUN2, a constituent of the nuclear envelope, is altered by MADA mutations, as argued by the formation of a highly disorganized lattice. Treatment with statins partially rescues proper SUN2 organization, indicating that its alteration is caused by farnesylated prelamin A accumulation. Given the major role of SUN1 and SUN2 in the nucleo-cytoskeleton interactions and in regulation of nuclear positioning in differentiating cells, we hypothesise that mechanisms regulating nuclear membrane–centrosome interplay and nuclear movement may be affected in MADA fibroblasts.
Electronic supplementary material
The online version of this article (doi:10.1007/s00418-012-0977-5) contains supplementary material, which is available to authorized users.
doi:10.1007/s00418-012-0977-5
PMCID: PMC3432780  PMID: 22706480
Mandibuloacral dysplasia type A (MADA); Prelamin A forms; SUN2; Heterochromatin defects; Statins; Trichostatin A
15.  1H Nuclear Magnetic Resonance Study of Olive Oils Commercially Available as Italian Products in the United States of America 
Nutrients  2012;4(5):343-355.
Multivariate analysis of 1H NMR data has been used for the characterization of 12 blended olive oils commercially available in the U.S. as Italian products. Chemometric methods such as unsupervised Principal Component Analysis (PCA) allowed good discrimination and gave some affinity indications for the U.S. market olive oils compared to other single cultivars of extra virgin olive oil such as Coratina and Ogliarola from Apulia, one of Italy’s leading olive oil producers, Picual (Spain), Kalamata (Greece) and Sfax (Tunisia). The olive oils commercially available as Italian products in the U.S. market clustered into 3 groups. Among them only the first (7 samples) and the second group (2 samples) showed PCA ranges similar to European references. Two oils of the third group (3 samples) were more similar to Tunisian references. In conclusion, our study revealed that most EVOO (extra virgin olive oils) tested were closer to Greek (in particular) and Spanish olive oils than Apulia EVOO. The PCA loadings disclose the components responsible for the discrimination as unsaturated (oleic, linoleic, linolenic) and saturated fatty acids. All are of great importance because of their nutritional value and differential effects on the oxidative stability of oils. It is evident that this approach has the potential to reveal the origin of EVOO, although the results support the need for a larger database, including EVOO from other Italian regions.
doi:10.3390/nu4050343
PMCID: PMC3367261  PMID: 22690321
NMR spectroscopy; extra virgin olive oil; U.S. market oils; food origin characterization
16.  Homeostatic expansion of autoreactive immunoglobulin-secreting cells in the Rag2 mouse model of Omenn syndrome 
The Journal of Experimental Medicine  2010;207(7):1525-1540.
Hypomorphic RAG mutations, leading to limited V(D)J rearrangements, cause Omenn syndrome (OS), a peculiar severe combined immunodeficiency associated with autoimmune-like manifestations. Whether B cells play a role in OS pathogenesis is so far unexplored. Here we report the detection of plasma cells in lymphoid organs of OS patients, in which circulating B cells are undetectable. Hypomorphic Rag2R229Q knock-in mice, which recapitulate OS, revealed, beyond severe B cell developmental arrest, a normal or even enlarged compartment of immunoglobulin-secreting cells (ISC). The size of this ISC compartment correlated with increased expression of Blimp1 and Xbp1, and these ISC were sustained by elevated levels of T cell derived homeostatic and effector cytokines. The detection of high affinity pathogenic autoantibodies toward target organs indicated defaults in B cell selection and tolerance induction. We hypothesize that impaired B cell receptor (BCR) editing and a serum B cell activating factor (BAFF) abundance might contribute toward the development of a pathogenic B cell repertoire in hypomorphic Rag2R229Q knock-in mice. BAFF-R blockade reduced serum levels of nucleic acid-specific autoantibodies and significantly ameliorated inflammatory tissue damage. These findings highlight a role for B cells in OS pathogenesis.
doi:10.1084/jem.20091928
PMCID: PMC2901059  PMID: 20547828
17.  Prelamin A processing and functional effects in restrictive dermopathy 
Cell Cycle  2010;9(23):4766-4768.
doi:10.4161/cc.9.23.14210
PMCID: PMC3048041  PMID: 21127399
restrictive dermopathy; prelamin A; chromatin; nuclear envelope; ZMPSTE24; laminopathies
18.  α- and β-Adducin polymorphisms affect podocyte proteins and proteinuria in rodents and decline of renal function in human IgA nephropathy 
Adducins are cytoskeletal actin-binding proteins (α, β, γ) that function as heterodimers and heterotetramers and are encoded by distinct genes. Experimental and clinical evidence implicates α- and β-adducin variants in hypertension and renal dysfunction. Here, we have addressed the role of α- and β-adducin on glomerular function and disease using β-adducin null mice, congenic substrains for α- and β-adducin from the Milan hypertensive (MHS) and Milan normotensive (MNS) rats and patients with IgA nephropathy. Targeted deletion of β-adducin in mice reduced urinary protein excretion, preceded by an increase of podocyte protein expression (phospho-nephrin, synaptopodin, α-actinin, ZO-1, Fyn). The introgression of polymorphic MHS β-adducin locus into MNS (Add2, 529R) rats was associated with an early reduction of podocyte protein expression (nephrin, synaptopodin, α-actinin, ZO-1, podocin, Fyn), followed by severe glomerular and interstitial lesions and increased urinary protein excretion. These alterations were markedly attenuated when the polymorphic MHS α-adducin locus was also present (Add1, 316Y). In patients with IgA nephropathy, the rate of decline of renal function over time was associated to polymorphic β-adducin (ADD2, 1797T, rs4984) with a significant interaction with α-adducin (ADD1, 460W, rs4961). These findings suggest that adducin genetic variants participate in the development of glomerular lesions by modulating the expression of specific podocyte proteins.
Electronic supplementary material
The online version of this article (doi:10.1007/s00109-009-0549-x) contains supplementary material, which is available to authorized users.
doi:10.1007/s00109-009-0549-x
PMCID: PMC2832889  PMID: 19838659
Adducin; Genetic renal disease; Glomerular disease; IgA nephropathy; Podocytes; Proteinuria
19.  Mitochondrial dysregulation and oxidative stress in patients with chronic kidney disease 
BMC Genomics  2009;10:388.
Background
Chronic renal disease (CKD) is characterized by complex changes in cell metabolism leading to an increased production of oxygen radicals, that, in turn has been suggested to play a key role in numerous clinical complications of this pathological condition. Several reports have focused on the identification of biological elements involved in the development of systemic biochemical alterations in CKD, but this abundant literature results fragmented and not exhaustive.
Results
To better define the cellular machinery associated to this condition, we employed a high-throughput genomic approach based on a whole transcriptomic analysis associated with classical molecular methodologies. The genomic screening of peripheral blood mononuclear cells revealed that 44 genes were up-regulated in both CKD patients in conservative treatment (CKD, n = 9) and hemodialysis (HD, n = 17) compared to healthy subjects (HS, n = 8) (p < 0.001, FDR = 1%). Functional analysis demonstrated that 11/44 genes were involved in the oxidative phosphorylation system. Western blotting for COXI and COXIV, key constituents of the complex IV of oxidative phosphorylation system, performed on an independent testing-group (12 healthy subjects, 10 CKD and 14 HD) confirmed an higher synthesis of these subunits in CKD/HD patients compared to the control group. Only for COXI, the comparison between CKD and healthy subjects reached the statistical significance. However, complex IV activity was significantly reduced in CKD/HD patients compared to healthy subjects (p < 0.01). Finally, CKD/HD patients presented higher reactive oxygen species and 8-hydroxydeoxyguanosine levels compared to controls.
Conclusion
Taken together these results suggest, for the first time, that CKD/HD patients may have an impaired mitochondrial respiratory system and this condition may be both the consequence and the cause of an enhanced oxidative stress.
doi:10.1186/1471-2164-10-388
PMCID: PMC2737002  PMID: 19698090
20.  Different prelamin A forms accumulate in human fibroblasts: a study in experimental models and progeria 
Lamin A is a component of the nuclear lamina mutated in a group of human inherited disorders known as laminopathies. Among laminopathies, progeroid syndromes and lipodystrophies feature accumulation of prelamin A, the precursor protein which, in normal cells, undergoes a multi-step processing to yield mature lamin A. It is of utmost importance to characterize the prelamin A form accumulated in each laminopathy, since existing evidence shows that drugs acting on protein processing can improve some pathological aspects. We report that two antibodies raised against differently modified prelamin A peptides show a clear specificity to full-length prelamin A or carboxymethylated farnesylated prelamin A, respectively. Using these antibodies, we demonstrated that inhibition of the prelamin A endoprotease ZMPSTE24 mostly elicits accumulation of full-length prelamin A in its farnesylated form, while loss of the prelamin A cleavage site causes accumulation of carboxymethylated prelamin A in progeria cells. These results suggest a major role of ZMPSTE24 in the first prelamin A cleavage step.
doi:10.4081/ejh.2009.e6
PMCID: PMC3167279
prelamin A; FTI-277; AFCMe; HIV-protease inhibitors; anti-prelamin A antibodies; laminopathies.
21.  Use of genome sequence data in the design and testing of SSR markers for Phytophthora species 
BMC Genomics  2008;9:620.
Background
Microsatellites or single sequence repeats (SSRs) are a powerful choice of marker in the study of Phytophthora population biology, epidemiology, ecology, genetics and evolution. A strategy was tested in which the publicly available unigene datasets extracted from genome sequences of P. infestans, P. sojae and P. ramorum were mined for candidate SSR markers that could be applied to a wide range of Phytophthora species.
Results
A first approach, aimed at the identification of polymorphic SSR loci common to many Phytophthora species, yielded 171 reliable sequences containing 211 SSRs. Microsatellites were identified from 16 target species representing the breadth of diversity across the genus. Repeat number ranged from 3 to 16 with most having seven repeats or less and four being the most commonly found. Trinucleotide repeats such as (AAG)n, (AGG)n and (AGC)n were the most common followed by pentanucleotide, tetranucleotide and dinucleotide repeats. A second approach was aimed at the identification of useful loci common to a restricted number of species more closely related to P. sojae (P. alni, P. cambivora, P. europaea and P. fragariae). This analysis yielded 10 trinucleotide and 2 tetranucleotide SSRs which were repeated 4, 5 or 6 times.
Conclusion
Key studies on inter- and intra-specific variation of selected microsatellites remain. Despite the screening of conserved gene coding regions, the sequence diversity between species was high and the identification of useful SSR loci applicable to anything other than the most closely related pairs of Phytophthora species was challenging. That said, many novel SSR loci for species other than the three 'source species' (P. infestans, P. sojae and P. ramorum) are reported, offering great potential for the investigation of Phytophthora populations. In addition to the presence of microsatellites, many of the amplified regions may represent useful molecular marker regions for other studies as they are highly variable and easily amplifiable from different Phytophthora species.
doi:10.1186/1471-2164-9-620
PMCID: PMC2647557  PMID: 19099584
22.  Health benefits of physical activity 
doi:10.1503/cmaj.1060094
PMCID: PMC1569919  PMID: 17001065
23.  The IgA nephropathy Biobank. An important starting point for the genetic dissection of a complex trait 
BMC Nephrology  2005;6:14.
Background
IgA nephropathy (IgAN) or Berger's disease, is the most common glomerulonephritis in the world diagnosed in renal biopsied patients. The involvement of genetic factors in the pathogenesis of the IgAN is evidenced by ethnic and geographic variations in prevalence, familial clustering in isolated populations, familial aggregation and by the identification of a genetic linkage to locus IGAN1 mapped on 6q22–23. This study seems to imply a single major locus, but the hypothesis of multiple interacting loci or genetic heterogeneity cannot be ruled out. The organization of a multi-centre Biobank for the collection of biological samples and clinical data from IgAN patients and relatives is an important starting point for the identification of the disease susceptibility genes.
Description
The IgAN Consortium organized a Biobank, recruiting IgAN patients and relatives following a common protocol. A website was constructed to allow scientific information to be shared between partners and to divulge obtained data (URL: ). The electronic database, the core of the website includes data concerning the subjects enrolled. A search page gives open access to the database and allows groups of patients to be selected according to their clinical characteristics. DNA samples of IgAN patients and relatives belonging to 72 multiplex extended pedigrees were collected. Moreover, 159 trios (sons/daughters affected and healthy parents), 1068 patients with biopsy-proven IgAN and 1040 healthy subjects were included in the IgAN Consortium Biobank. Some valuable and statistically productive genetic studies have been launched within the 5th Framework Programme 1998–2002 of the European project No. QLG1-2000-00464 and preliminary data have been published in "Technology Marketplace" website: .
Conclusion
The first world IgAN Biobank with a readily accessible database has been constituted. The knowledge gained from the study of Mendelian diseases has shown that the genetic dissection of a complex trait is more powerful when combined linkage-based, association-based, and sequence-based approaches are performed. This Biobank continuously expanded contains a sample size of adequately matched IgAN patients and healthy subjects, extended multiplex pedigrees, parent-child trios, thus permitting the combined genetic approaches with collaborative studies.
doi:10.1186/1471-2369-6-14
PMCID: PMC1318455  PMID: 16329758
24.  Effects of angiotensin converting enzyme inhibitors and angiotensin II receptor antagonists on mortality and renal outcomes in diabetic nephropathy: systematic review 
BMJ : British Medical Journal  2004;329(7470):828.
Objective To evaluate the effects of angiotensin converting enzyme (ACE) inhibitors and angiotensin II receptor antagonists (AIIRAs) on renal outcomes and all cause mortality in patients with diabetic nephropathy.
Data sources Medline, Embase, the Cochrane controlled trials register, conference proceedings, and contact with investigators.
Study selection Trials comparing ACE inhibitors or AIIRAs with placebo or with each other in patients with diabetic nephropathy.
Data extraction Mortality, renal outcomes (end stage renal disease, doubling of serum creatinine concentration, prevention of progression of microalbuminuria to macroalbuminuria, remission of microalbuminuria), and quality of trials.
Data synthesis 36 of 43 identified trials compared ACE inhibitors with placebo (4008 patients), four compared AIIRAs with placebo (3331 patients), and three compared ACE inhibitors with AIIRAs (206 patients). We obtained unpublished data for 11 trials. ACE inhibitors significantly reduced all cause mortality (relative risk 0.79, 95% confidence interval 0.63 to 0.99) compared with placebo but AIIRAs did not (0.99, 0.85 to 1.17), although baseline mortality was similar in the trials. Both agents had similar effects on renal outcomes. Reliable estimates of the unconfounded relative effects of ACE inhibitors compared with AIIRAs could not be obtained owing to small sample sizes.
Conclusion Although the survival benefits of ACE inhibitors for patients with diabetic nephropathy are known, the relative effects of ACE inhibitors and AIIRAs on survival are unknown owing to the lack of adequate head to head trials.
doi:10.1136/bmj.38237.585000.7C
PMCID: PMC521570  PMID: 15459003
25.  Disease-associated Bias in T Helper Type 1 (Th1)/Th2 CD4+ T Cell Responses Against MAGE-6 in HLA-DRB1*0401+ Patients With Renal Cell Carcinoma or Melanoma 
T helper type 1 (Th1)-type CD4+ antitumor T cell help appears critical to the induction and maintenance of antitumor cytotoxic T lymphocyte (CTL) responses in vivo. In contrast, Th2- or Th3/Tr-type CD4+ T cell responses may subvert Th1-type cell-mediated immunity, providing a microenvironment conducive to disease progression. We have recently identified helper T cell epitopes derived from the MAGE-6 gene product; a tumor-associated antigen expressed by most melanomas and renal cell carcinomas. In this study, we have assessed whether peripheral blood CD4+ T cells from human histocompatibility leukocyte antigens (HLA)-DRβ1*0401+ patients are Th1- or Th2-biased to MAGE-6 epitopes using interferon (IFN)-γ and interleukin (IL)-5 enzyme-linked immunospot assays, respectively. Strikingly, the vast majority of patients with active disease were highly-skewed toward Th2-type responses against MAGE-6–derived epitopes, regardless of their stage (stage I versus IV) of disease, but retained Th1-type responses against Epstein-Barr virus– or influenza-derived epitopes. In marked contrast, normal donors and cancer patients with no current evidence of disease tended to exhibit either mixed Th1/Th2 or strongly Th1-polarized responses to MAGE-6 peptides, respectively. CD4+ T cell secretion of IL-10 and transforming growth factor (TGF)-β1 against MAGE-6 peptides was not observed, suggesting that specific Th3/Tr-type CD4+ subsets were not common events in these patients. Our data suggest that immunotherapeutic approaches will likely have to overcome or complement systemic Th2-dominated, tumor-reactive CD4+ T cell responses to provide optimal clinical benefit.
doi:10.1084/jem.20012142
PMCID: PMC2193999  PMID: 12208877
melanoma; renal cell carcinoma; helper T lymphocyte; MAGE-6; epitope

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