Design of a rapid, multiplex, one-pot miRNA assay optimized by label-free analysis.

MicroRNAs are widely studied as circulating biomarkers for early stage diagnosis of several diseases. Detection and quantification of miRNAs is currently performed through complex and time consuming procedures. Herein we demonstrate a rapid, multiplex, one-pot detection method based on two-step amplification of the signal measured by Reflective Phantom Interface (RPI) label-free optical biosensor. We achieved sub-pM quantification of different miRNAs in about 1.5 h, through specific capture with surface DNA probes combined to a 35-fold mass amplification by an antibody targeting DNA-RNA hybrids and polyclonal secondary antibody, all performed without washing steps. The assay is the result of a modelling and optimization of the multi-step process that has been made possible by the RPI characterization of each individual interaction involved.

Kappa free light chains is a valid tool in the diagnostics of MS: A large multicenter study.

OBJECTIVE: To validate kappa free light chain (KFLC) and lambda free light chain (LFLC) indices as a diagnostic biomarker in multiple sclerosis (MS). METHODS: We performed a multicenter study including 745 patients from 18 centers (219 controls and 526 clinically isolated syndrome (CIS)/MS patients) with a known oligoclonal IgG band (OCB) status. KFLC and LFLC were measured in paired cerebrospinal fluid (CSF) and serum samples. Gaussian mixture modeling was used to define a cut-off for KFLC and LFLC indexes. RESULTS: The cut-off for the KFLC index was 6.6 (95% confidence interval (CI) = 5.2-138.1). The cut-off for the LFLC index was 6.9 (95% CI = 4.5-22.2). For CIS/MS patients, sensitivity of the KFLC index (0.88; 95% CI = 0.85-0.90) was higher than OCB (0.82; 95%CI = 0.79-0.85; p < 0.001), but specificity (0.83; 95% CI = 0.78-0.88) was lower (OCB = 0.92; 95% CI = 0.89-0.96; p < 0.001). Both sensitivity and specificity for the LFLC index were lower than OCB. CONCLUSION: Compared with OCB, the KFLC index is more sensitive but less specific for diagnosing CIS/MS. Lacking an elevated KFLC index is more powerful for excluding MS compared with OCB but the latter is more important for ruling in a diagnosis of CIS/MS.

Congenital chylothorax: Current evidence-based prenatal and post-natal diagnosis and management.

Congenital chylothorax is an uncommon condition but represents the main cause of congenital pleural effusion during the neonatal period. It usually appears before birth, both as an isolated disorder or in association with hydrops fetalis, negatively affecting the subsequent neonatal outcome. Prenatal treatment is usually considered to ensure a satisfactory lung development in case of moderate to severe pleural effusion or in the presence of hydrops, although consensus on treatment timing and modalities has not been reached to date. Both medical and surgical therapeutic strategies are available to treat this condition and novel treatment options have been recently attempted with acceptable results in both prenatal and post-natal setting. The heterogeneous clinical presentation of congenital chylothorax together with its rarity, its numerous etiologies and the absence of a highly effective treatment renders the diagnostic and therapeutic approach difficult to standardize. In addition, adequate visualization of the lymphatic system is complex, especially in small neonates, although new promising techniques have been developed lately and may contribute to improved management of this serious but infrequent condition. This review focuses on the current evidence base for the diagnosis and treatment options for congenital chylothorax, suggesting a rational diagnostic and therapeutic approach both in the prenatal and in the neonatal period.

Early fetal hydropic changes are associated with moderate dilatation of the brain ventricular system: A clue to a possible link between cervical lymphatic engorgement and ventricular dilatation?

The aim of this study is to assess whether early cervical lymphatic obstruction is associated with a sonographically detectable dilatation of the ventricular system in the 1st trimester of pregnancy. In particular, the objective is to assess whether fetuses with non-immune hydrops fetalis (NIHF), cystic hygroma, or enlarged nuchal translucency (NT) have a greater atrial width/biparietal diameter (AW/BPD) ratio than normal at time of the combined first trimester screening scan. This retrospective study included 96 first trimester fetuses (33 normal and 63 with various degree of cervical lymphatic engorgement). Inclusion criteria were CRL in the 45-84 mm range and availability of one or more three-dimensional volume datasets of the fetal head, acquired from the BPD plane. Each three-dimensional volume dataset was opened and multiplanar correlation employed to align the three orthogonal planes. The ratio between the atrial width and the BPD (AW/BPD ratio) was used to evaluate the possible presence of increased amount of cerebrospinal fluid. Abnormal cases were placed into 4 categories: 1) enlarged non-septated NT 2.5-3.9 mm, no hydrops; 2) grossly enlarged non-septated NT / edema >3.9 mm; 3) cystic hygroma and/ or NIHF; 4) major anomalies with NT <2.5 mm. Presence of dilatation of the laterocervical jugular lymphatic sacs, karyotype and presence of congenital anomalies were also recorded. The One-way ANOVA test was used to compare means. Intra- and inter-observer variability were also assessed. The AW/BPD ratio was found to be significantly higher in fetuses with grossly enlarged NT/nuchal edema and NIHF/septated cystic hygroma than in normal (p <0.05 and p <0.01, respectively). Also, the AW/BPD ratio was significantly higher in NIHF/septated cystic hygroma than in enlarged NT 2.5-3.9 mm (p <0.05). In case of enlarged NT (2.5-3.9 mm), the AW/BPD ratio is significantly higher in presence of JLS (p <0.01). At the end of the first trimester, presence of cervical lymphatic engorgement, in terms of grossly enlarged NT, nuchal edema, septated cystic hygroma, and NIHF, is statistically associated with a moderate dilatation of the ventricular system. Of note, among fetuses with moderately enlarged NT, those with evidence of dilatation of the JLS show a statistically significant increase in the AW/BPD ratio.

Brain lymphatic drainage system in fetus and newborn: Birth of a new era of exploration.

A peculiar brain lymphatic drainage system has been recently fully recognized in animals and humans. It comprises different draining pathways, including the lymphatic system, the perivascular drainage pathway, and the cerebrospinal fluid (CSF) drainage routes. Although scant data are available about its function during the neonatal period, it may play a role in neonatal brain diseases. In this review, we focus on the actual knowledge of brain lymphatic drainage system, and we hypothesize potential implications of its impairment and dysfunction in major neonatal neurological diseases.

Small-angle neutron scattering and molecular dynamics structural study of gelling DNA nanostars.

DNA oligomers with properly designed sequences self-assemble into well defined constructs. Here, we exploit this methodology to produce bulk quantities of tetravalent DNA nanostars (each one composed of 196 nucleotides) and to explore the structural signatures of their aggregation process. We report small-angle neutron scattering experiments focused on the evaluation of both the form factor and the temperature evolution of the scattered intensity at a nanostar concentration where the system forms a tetravalent equilibrium gel. We also perform molecular dynamics simulations of one isolated tetramer to evaluate the form factor numerically, without resorting to any approximate shape. The numerical form factor is found to be in very good agreement with the experimental one. Simulations predict an essentially temperature-independent form factor, offering the possibility to extract the effective structure factor and its evolution during the equilibrium gelation.

Label-free detection of DNA single-base mismatches using a simple reflectance-based optical technique.

Rapid and quantitative detection of the binding of nucleic acids to surface-immobilized probes remains a challenge in many biomedical applications. We investigated the hybridization of a set of fully complementary and defected 12-base long DNA oligomers by using the Reflective Phantom Interface (RPI), a recently developed multiplexed label-free detection technique. Based on the simple measurement of reflected light intensity, this technology enables to quantify the hybridization directly as it occurs on the surface with a sensitivity of 10 pg mm(-2). We found a strong effect of single-base mismatches and of their location on hybridization kinetics and equilibrium binding. In line with previous studies, we found that DNA-DNA binding is weaker on a surface than in the bulk. Our data indicate that this effect is a consequence of weak nonspecific binding of the probes to the surface.

Congenital Chylothorax of the Newborn: Diagnosis and Treatment in Three Pictures.

There is general agreement regarding the evident need for an international, multicenter trial including long-term follow-up to establish the correct criteria for diagnosing and managing congenital chylothorax. In an attempt to identify these criteria, which could then be used to draft a prospective multicenter trial, we propose three flow-charts showing three algorithms that could be used to: 1) obtain a definitive diagnosis of pleural chylous effusion; 2) specifically focus on chyle leakage evolution and etiology of chylothorax; and 3) focus on the management of congenital chylothorax. The aim of the algorithms we propose is to build the basis on which a strongly needed multicenter trial might be structured.

Transfusion of stored red blood cells in critically ill trauma patients: a retrospective study.

OBJECTIVE: The many published studies on the effects of the transfusion of stored red blood cells on clinical outcomes yielded discordant results. Therefore, we chose to study patients with severe trauma. The clinical outcomes considered included in-hospital mortality, the occurrence of sepsis, length of stay in intensive care unit and in hospital, and days of mechanical ventilation. PATIENTS AND METHODS: We selected all patients with traumatic injury, who received at least 2 red cell units in the first day of admission. Patients were divided into two groups: those who had received fresh red cells only (fresh group) and those who had received at least one "old" red cell unit (old group). The red cells were considered fresh if they had been stored <14 days. RESULTS: The fresh and old groups included 376 and 321 patients, respectively. Baseline demographic and clinical characteristics were comparable between the groups. However, old group received more red cell and plasma units during whole hospital stay (red cells: 11 ± 7 vs 6 ± 4, p < 0.001; plasma: 7 [0-9] vs 3 [0-6]). Among outcomes, only length of stay in intensive care unit (old vs fresh: 18 ± 9 vs 12 ± 8 days, p < 0.001) and in hospital (77 ± 35 vs 45 ± 30 days, p < 0.001) differed significantly between groups. The association remained statistically significant in a multivariate analysis including known confounding factors. CONCLUSIONS: Patients with major trauma transfused with old (≥14 days) red cells had a longer length of stay in intensive care unit and in hospital, without any difference in mortality, occurence of sepsis or days of mechanical ventilation.

LYMPHODYSPLASIA AND KRAS MUTATION: A CASE REPORT AND LITERATURE REVIEW.

Cardio-facio-cutaneous (CFC) syndrome is a very rare and sporadic disease whose characteristics include dysmorphic facial appearance, ectodermal abnormalities, cardiac abnormalities, growth retardation and neurodevelopmental delay. This syndrome is classified as one of the RAS syndromes which are caused by altered signal transduction of the RAS/MAPK (mitogen activated protein kinase) pathway due to the mutation of genes including BRAF, MEK1/2, HRAS and KRAS. Other RAS syndromes, such as Costello syndrome and Noonan syndrome, share clinical features with CFC. Moreover, patients with the same clinical phenotype may have different molecular diagnoses. Clinical diagnosis is the starting pointfor correct classification. We describe the clinical data of one case of CFC syndrome, genetically determined by KRAS mutation, that involved chylothorax, lymphedema, sinus pericranii, craniosynostosis, and seizures. This is the second case report of the literature.

Lymphoscintigraphy patterns in newborns and children with congenital lymphatic dysplasia.

We performed lymphoscintigraphy on 31 patients (newborns and children) affected by congenital lymphatic dysplasia according to our previously published protocol. Congenital lymphatic dysplasia may present with various degrees of clinical severity, ranging from nonimmune hydrops fetalis with visceral effusions to lymphedema alone. We recommend that lymphoscintigraphy should be strongly considered in all patients with signs of lymphatic dysplasia, including those with minimal and initial signs of lymphatic impairment, in order to obtain a very early diagnosis and to start treatment. Lymphoscintigraphy is safe and useful in the diagnosis of lymphatic dysplasia in the newborn and children. Moreover, it is well tolerated by patients and well accepted by their parents.

Dynamics of pleural fluid effusion and chylothorax in the fetus and newborn: role of the lymphatic system.

Pleural fluid effusion particularly chylothorax is a relatively rare occurrence in the newborn, but when it occurs it is often life-threatening. In this article, we describe and illustrate the morphologic features of the visceral and parietal pleura including pleural lymphatics and the physiology and pathophysiology of pleural fluid balance. The role and function of the lymphatic system in controlling the volume and composition of pleural liquid are detailed and a conceptual scheme presented. Finally, the crucial role of inadequate lymphatic drainage (either functional overload from an imbalance in Starling forces or mechanical insufficiency from lymphatic dysplasia) is emphasized.

Are there lymphatic vessels in the placenta?

The role of lymphatics in placentation has been scantily studied and the true existence of placental lymphatics is under debate. Numerous blood and lymphatic-lineage molecule markers are now available and they are expressed in human placental tissue. D2-40 expression at the placental stromal level seems to indicate that network-forming, podoplanin-expressing cells may act as a reticular-lymphatic-like conductive network. This exciting area at the intersection of perinatology and lymphology needs further investigation.

Congenital fetal and neonatal visceral chylous effusions: neonatal chylothorax and chylous ascites revisited. A multicenter retrospective study.

This retrospective study was carried out at eight Neonatal Intensive Care Units (NICU) Centers worldwide on 33 newborns presenting at birth with pleural, pericardial, or abdominal chylous effusions. Diagnosis of chylous effusion is based on findings of fluid with a milk-like appearance, a concentration of triglycerides in pleural effusion >1.1 mmol/l, and a total cell count >1,000 cells/ml with a predominance of >80% lymphocytes. Thirty-three newborns met the inclusion criteria and were studied. Six subjects who presented at birth with fetal effusion were treated by in-utero pleuro-amniotic shunt. Five of these patients are alive at follow-up. At birth, pleural drainage was performed in 29/33 patients and abdominal drainage was carried out in 3/33. Total parenteral nutrition (TPN) was given to 32/33 patients; 19/23 patients were fed a medium-chain triglycerides (MCT). No adverse effects were observed. Eight patients were treated with Octreotide at dosages ranging from 1 to 7 mcg/kg/hour for 8 to 35 days. All patients showed decreased chylous production. Two patients were treated by pleurodesis. Twenty-two babies are alive after at least 6 months follow-up, 9/33 are deceased, and 2 were lost to follow-up. Clinical conditions of survivors are basically good except for lung involvement [chronic lung disease (CLD) or lung lymphangiectasia] and lymphedema. All patients were using a MCT diet at follow-up with good control of chylous effusion. Visceral chylous effusions of the fetus and neonate are rare disorders, and there currently is only partial agreement on decision-making strategies. We suggest the need for an international prospective trial in an effort to establish the efficacy and effectiveness of diagnostic and therapeutic options described in this article.

Perinatal deaths and lymphatic system involvement: a diagnostic flow-chart applying immunohistochemical methods.

A diagnostic flow chart is presented for use in case of perinatal death or still birth with non-immune hydrops fetalis, visceral effusions, or increased nuchal translucency. Immunohistochemical staining with CD-31, CD-34, D2-40, and smooth muscle actin is recommended.

Congenital lymphatic dysplasia in Kabuki syndrome: first report of an unusual association.

Kabuki syndrome was first described in Japan in 1981 as a rare disorder of unknown cause. Its main features include characteristic facies, postnatal growth retardation, and mental delay. To date, there is no molecular marker for Kabuki syndrome, which is considered genetically heterogeneous and still is a clinically-based diagnosis. Here we describe the first case of a patient affected by Kabuki syndrome associated with lymphatic dysplasia. We suggest accurate evaluation of all Kabuki patients as early as possible in order to diagnose lymphedema or other clinical manifestations of lymphatic system involvement. Early identification of lymphatic system maldevelopment provides the best chance for reducing the risk of developing progressive lymphedema with associated tissue changes (fibrosis, sclerosis, and fat deposition).

Selective D2-40 lymphatic endothelium immunoreactivity in developing human fetal skin appendages.

D2-40 is a novel monoclonal antibody that recognizes a 40,000 Da O-linked sialoglycoprotein podoplanin. Although its use is becoming more common, little work has been done with human foetuses. We initiated an evaluation of D2-40 antibody immunoreactivity in developing cutaneous adnexa of human fetuses at various gestational ages. Starting from a retrospective cohort of 1,098 human fetal autopsies we identified and selected a total of 48 fetuses ranging from the 12th week gestational age to term appropriate for this study. We demonstrated that the gems of the hair follicles were D2-40 negative in fetuses from the 12th to 15th week gestation, positive in fetuses between the 16th and 20th week of gestation, negative in fetuses from the 21st week gestation to term. Normal adult controls were also negative. This is the first report to demonstrate intense D2-40 immunoreactivity in the gems of hair follicles of developing human skin.

Field-induced clearing in sphere-sphere and rod-sphere binary mixtures of charged colloidal particles.

Binary mixtures of larger and smaller colloids having charges of equal sign display unusual response to low-frequency electric fields. We show here that the previously reported negative torque acting on rodlike particles when in presence of a sea of smaller particles is accompanied by a field-induced clearing of the suspension. Measurements of transmitted intensity performed on mixtures of large (dilute) and small (semidilute) spherical particles indicate that such a clearing effect is universal and due to an anisotropic E2 -dependent redistribution of the small spheres around the large ones. We interpret this behavior as resulting from O(E2) electro-osmotic flows whose magnitude is greatly enhanced by the presence of the small particles.

Potential relevance of cerebrospinal fluid and serum levels and intrathecal synthesis of active matrix metalloproteinase-2 (MMP-2) as markers of disease remission in patients with multiple sclerosis.

BACKGROUND: Little is known about the involvement of matrix metalloproteinase-2 (MMP-2) and its tissue inhibitor TIMP-2 in multiple sclerosis (MS). OBJECTIVE: To elucidate the actual implication of MMP-2 and TIMP-2 in MS. METHODS: Cerebrospinal fluid (CSF) and serum levels of active MMP-2 and TIMP-2 were measured by activity assay system and ELISA, respectively, in 67 patients with relapsing-remitting MS (RRMS), categorized according clinical and magnetic resonance imaging (MRI), and in 129 controls. RESULTS: Cerebrospinal fluid and serum active MMP-2/TIMP-2 ratio mean values and an intrathecal active MMP-2 production were more increased in RRMS than in non-inflammatory conditions (P < 0.001, P < 0.05, and P < 0.0001, respectively) and in MRI inactive than in MRI active RRMS (P < 0.02, P < 0.01 and P < 0.001, respectively). An intrathecal synthesis of active MMP-2 was more frequent in RRMS than in inflammatory disorders (P < 0.01). Serum active MMP-2/TIMP-2 ratio and MS disease duration were positively correlated (P < 0.02). CONCLUSION: These findings suggest a potential role for MMP-2 activity in the termination of MS neuroinflammation related to remission of the disease and seem to indicate that serum MMP-2/TIMP-2 ratio may represent a useful biomarker for monitoring MS recovery phase.

Liquid crystal alignment on a chiral surface: interfacial interaction with sheared DNA films.

We explore the alignment of various achiral liquid crystals on films of aligned double-stranded helical DNA. In all cases and both for the nematic and smectic A phases, we find a distinctly chiral interfacial structure, with the mean orientation of the liquid crystal in contact with the DNA-treated surfaces chirally rotated through a substantial angle with respect to the mean DNA orientation. This rotation originates in the chirality of double-stranded DNA and depends on the liquid crystal molecular structure. We discuss the role of dipolar and hydrophobic coupling in determining the observed orientation.