High resolution melting analysis for the identification of novel mutations in DKC1 and TERT genes in patients with dyskeratosis congenita.

Dyskeratosis congenita (DC) is a rare inherited bone-marrow failure syndrome with high clinical heterogeneity. Cells derived from DC patients present short telomeres at early ages, as a result of mutations in genes encoding components of the telomerase complex (DKC1, TERC, TERT, NHP2 and NOP10), or the shelterin complex (TINF2). However, mutations have been identified only in around 50% of the cases, indicating that other genes could be involved in the development of this disease. Indeed, mutations in TCBA1 or chromosome segment C16orf57 have been described recently. We have used HRM technology to perform genetic analysis in the above mentioned genes, in Spanish patients showing both, some clinical features of DC and short telomeres. The mutations have been identified by PCR amplification of DC genes followed by high resolution melting (HRM) and direct DNA sequencing analysis. We have identified seven new families with DC, three with X-linked DC and four with autosomal dominant DC, in which we have found two novel mutations in DKC1 (p.His68Arg and p.Lys390del) and four novel mutations in TERT gene (p.Pro530Leu, p.Arg698Trp, p.Arg971His and p.Arg698Gln). The results show that the use of HRM analysis enables a rapid and inexpensive identification of mutations in dyskeratosis congenita associated genes.

ECLIM-SEHOP, a new platform to set up and develop international academic clinical trials for childhood cancer and blood disorders in Spain.

INTRODUCTION: Cancer and blood disorders in children are rare. The progressive improvement in survival over the last decades largely relies on the development of international academic clinical trials that gather the sufficient number of patients globally to elaborate solid conclusions and drive changes in clinical practice. The participation of Spain into large international academic trials has traditionally lagged behind of other European countries, mainly due to the burden of administrative tasks to open new studies, lack of financial support and limited research infrastructure in our hospitals. METHODS: The objective of ECLIM-SEHOP platform (Ensayos Clínicos Internacionales Multicéntricos-SEHOP) is to overcome these difficulties and position Spain among the European countries leading the advances in cancer and blood disorders, facilitate the access of our patients to novel diagnostic and therapeutic approaches and, most importantly, continue to improve survival and reducing long-term sequelae. ECLIM-SEHOP provides to the Spanish clinical investigators with the necessary infrastructural support to open and implement academic clinical trials and registries. RESULTS: In less than 3 years from its inception, the platform has provided support to 20 clinical trials and 8 observational studies, including 8 trials and 4 observational studies where the platform performs all trial-related tasks (integral support: trial setup, monitoring, etc.) with more than 150 patients recruited since 2017 to these studies. In this manuscript, we provide baseline metrics for academic clinical trial performance that permit future comparisons. CONCLUSIONS: ECLIM-SEHOP facilitates Spanish children and adolescents diagnosed with cancer and blood disorders to access state-of-the-art diagnostic and therapeutic strategies.

Osmotic gradient ektacytometry: A valuable screening test for hereditary spherocytosis and other red blood cell membrane disorders.

INTRODUCTION: New generation osmotic gradient ektacytometry has become a powerful procedure for measuring red blood cell deformability and therefore for the diagnosis of red blood cell membrane disorders. In this study, we aim to provide further support to the usefulness of osmotic gradient ektacytometry for the differential diagnosis of hereditary spherocytosis by measuring the optimal cutoff values of the parameters provided by this technique. METHODS: A total of 65 cases of hereditary spherocytosis, 7 hereditary elliptocytosis, 3 hereditary xerocytosis, and 171 normal controls were analyzed with osmotic gradient ektacytometry in addition to the routine red blood cell laboratory techniques. The most robust osmoscan parameters for hereditary spherocytosis diagnosis were determined using receiver operating characteristic curve analysis. RESULTS: The best diagnostic criteria for hereditary spherocytosis were the combination of decreased minimal elongation index up to 3% and increased minimal osmolality point up to 5.2% when compared to the mean of controls. Using this established criterion, osmotic gradient ektacytometry reported a sensitivity of 93.85% and a specificity of 98.38% for the diagnosis of hereditary spherocytosis. CONCLUSION: Osmotic gradient ektacytometry is an effective diagnostic test for hereditary spherocytosis and enables its differential diagnosis with other red blood cell membrane diseases based on specific pathology profiles.

Mechanics of translation in the fosbury-flop.

The mechanics of the translations involved in the Fosbury-flop technique of high jumping were examined in the light of appropriate experimental data, generated using a 3-dimensional cinematographical method. The curved run-up was found to cause the athletes to lean toward the center of the curve at the start of the take-off phage (TD). The center of gravity (c.g.) of all the jumpers had a negative vertical velocity at TI). The lateral deviation of the path of the c.g. during the take-off phage was small, implying small centripetal forces. The initial trajectory of the parabolic path of the c.g. after the take-off made an angle of between 40 degrees and 48 degrees with the horizontal plane. During the take-off phase the take-off leg was found to flex at least as much as previouslyreported for athletes using the straddle style. One of the subjects in the study was able to clear the bar although the peak height of the parabolic path followed by his c.g. was at the same level as the bar.

Mechanics of rotation in the Fosbury-flop.

The mechanics of the rotations involved in the Fosbury-flop technique of high jumping were examined in the light of appropriate experimental data generated by use of a 3-dimensional cinematographical method. At the start of the take-off phage, the six subjects who used the Fosbury-flop had little angular momentum. Most of the angular momentum necessary for a proper bar clerance was not produced during the run-up, but during the take-off phase. A curved approach run appeared to favor the production of "somersaulting" angular momentum (the component of angular momentum which mainly contributes to lower the shoulders and lift the knees during the flight phase), as well as a greater vertical range of motion of the c.g. during the take-off phase. These conclusions give little support to the hypothesis that the main purpose of the curved run-up is to produce angular momentum during the run-up. On the other hand, they favor the hypothesis that the main purpose of the curved approach run is to make the athletes lean away from the bar. One athlete with particularly small "somersaluting" angular momentum made effective use of a hitch-kick motion to accomplish an effcicient bar clearance.

Use of separate hand locations to calculate ground reaction force exerted on a vaulting pole.

A model was developed to permit calculation of the force exerted by the ground on a vaulting pole given the flexibility characteristics of the pole, the grip height of the upper hand, and the coordinates of each of the two hands relative to the base of the pole. The flexural rigidity of the pole was assumed constant throughout the length of the pole and not subject to hysteresis or dynamic loading effects. The model was based on the following rationale: knowing the initial angle of the base of the pole (beta 0) and the force vector (F1) exerted by the ground on the pole, it was possible to estimate the shape of the pole and the coordinates of the two hands following an iterative procedure. Conversely, it was possible to find a combination of beta 0 and F1 that made the pole fit two specified hand locations.

Comparison of the kinematics of the full-instep and pass kicks in soccer.

PURPOSE: The goal of this study was to gain a better understanding of the mechanics of the inside-of-the-foot passing shot used in soccer ("pass kick"). METHODS: The motions of the pass kick were compared with those of the full-instep kick ("full kick"). The study followed an inverse dynamics approach, using three-dimensional cinematographic techniques. RESULTS: At impact, the pelvis and the thigh-shank plane pointed more toward the right in the pass kick; the shank-foot plane also pointed further outward relative to the thigh-shank plane. Knee extension accounted for most of the speed of the foot in both kicks (86% in the full kick; 67% in the pass kick). In the pass kick, pelvis tilt toward the right and hip adduction contributed to a medial component of foot velocity (8.4 m.s-1) normal to the thigh-shank plane, which made the resultant foot velocity vector more oblique to the plane than in the full kick. This facilitated ball impact with the medial aspect of the foot. The slower ball speed in the pass kick was because of a slower foot speed (18.3 m.s-1 vs 21.6 m.s-1). Limitations in the maximum medial velocity that can be generated may force players to restrain the within-plane (and therefore also the resultant) velocity of the foot to be able to impact the ball squarely with the medial aspect of the foot. CONCLUSIONS: To impact the ball with the medial aspect of the foot in the pass kick, the player orients the pelvis, the right leg, and the foot more toward the right and introduces a medial component of foot velocity. However, most of the speed of the foot is still generated through knee extension.

Linear kinematics of the men's 110-m and women's 100-m hurdles races.

Twenty-three male and nine female hurdlers were filmed using three-dimensional methods during competition at the 1988 United States Olympic Trials. An entire four-step cycle was analyzed, including the clearances of the men's fifth hurdle and the women's fourth hurdle. The results showed an increase in vertical velocity and a decrease in forward horizontal velocity during the takeoff of the hurdle step. The forward velocity was recovered mainly in the second support phase after the hurdle. The downward motion of the center of mass (c.m.) was not stopped until the second support phase after the hurdle clearance. The peak of the c.m. parabola was almost directly over the hurdle in the men, and 0.30 m before the hurdle in the women. It was shown that the women used a parabola with a larger margin over the top of the hurdle than the men: A lower parabola would shorten the hurdle step, and would require the lengthening of the three interhurdle steps. It would also make the duration of the airborne phase too short, which would not give the legs enough time to prepare for landing after the execution of their motions over the hurdle. Therefore, women should not be coached to imitate the men's hurdle clearance technique.

Angular momentum in the men's 110-m and women's 100-m hurdles races.

Twenty-three male and nine female hurdlers were filmed using three-dimensional methods at the 1988 United States Olympic Trials. With respect to the athletes, the X, Y, and Z axes pointed to the right, forward, and upward, respectively. During hurdle clearance, the X component of angular momentum was negative (clockwise rotation in a view from the right). Early in the airborne phase, it was associated with the motion of the trail leg. The downward motion of the lead leg was produced mainly by transfer of angular momentum from the trail leg, rather than by the lifting of the head-trunk. The Y component of angular momentum was negative (counterclockwise in a back view of a hurdler taking off from the right foot). It was necessary for the abduction of the trail leg. When this abduction slowed down, the angular momentum was taken up by the lowering (and slight adduction) of the left leg and elevation of the right elbow. The hurdle clearance required a positive Z component of angular momentum (counterclockwise in an overhead view): The clockwise angular momentum of the right arm as it swept backward was not enough to compensate for the larger counterclockwise angular momentum required for the forward motion of the trail leg. Our improved understanding of the rotations involved in hurdling will be useful for the correction of technique defects in individual athletes.

A three-dimensional analysis of angular momentum in the hammer throw.

Eight hammer throwers were studied using three-dimensional cinematography. The local term of angular momentum of each thrower (HTL) followed a wide conical path, while the remote term (HTR) and the hammer angular momentum (HH) followed much narrower conical paths. HT, the sum of HTL and HTR, followed a conical path similar to that of HTL, although with smaller amplitude. HH was half of a cycle out of phase with HT. It was also larger but followed a narrower conical path. As a result, the conical paths of HH and HT counteracted each other, and HS, the total angular momentum vector of the thrower-hammer system, had little conical motion. The paths of the angular momentum vectors, the trunk tilt, and the height of the hammer plane relative to the system center of mass were interrelated. Some throwers kept the hammer plane high and the trunk tilting back in all the turns; other throwers kept the hammer plane low and the trunk tilting forward in the early turns, but the hammer plane rose in their late turns and the trunk tilted back. Two theories were proposed to explain why the athletes who had forward trunk tilt in the early turns tilted backward in the final part of the throw.

Vertical and radial motions of the body during the take-off phase of high jumping.

By placing the muscles of the take-off leg in faster eccentric or slower concentric conditions, a high jumper can increase the ground reaction force and the height of the jump. Film analysis of seven high jumpers showed that the radial velocity of the center of mass with respect to the supporting foot was more negative or less positive than the vertical velocity throughout the take-off phase. This favored faster eccentric or slower concentric conditions of the leg muscles. The radial distance from the hip of the take-off leg to the center of mass (RG/H) first decreased by 0.030 m, due to negative radial motions of the arms and swinging leg. This contributed to a smaller negative radial velocity of the hip (VRH), and thus to slower eccentric conditions of the muscles of the take-off leg. Therefore, it may have helped to cushion the initial impact with the ground. Subsequently, RG/H increased by 0.120 m, due to positive radial velocities of the arms, the swinging leg, and the head and trunk. This contributed first to larger negative (and later to smaller positive) VRH values, and thus to faster eccentric and slower concentric conditions of the muscles of the take-off leg.

The pattern of hammer speed during a hammer throw and influence of gravity on its fluctuations.

Hammer speed at release is one of the most important factors contributing to the distance of a hammer throw. Hammer speed follows a generally increasing trend during the throw, with one fluctuation per turn. The purpose of the present paper was to quantify the influence of gravity on the speed fluctuations. Eight experienced hammer throwers were studied with three-dimensional filming methods. Instantaneous values of hammer velocity and speed were calculated from the film data. The rate of change of hammer speed due to the tangential component of gravity was computed, and integrated to calculate the accumulated contribution of gravity to hammer speed at all instants of the throw. These values were subtracted from the corresponding values of hammer speed. The amplitude of the fluctuations was reduced in the corrected speed functions, indicating a contribution of gravity to the original fluctuations. However, the fluctuations were still clearly present in the corrected speed functions, indicating the existence of other causal factors.

A kinematic study of center of mass motions in the hammer throw.

Eight highly-skilled hammer throwers were studied using film analysis procedures. The location and velocity of the center of mass (c.m.) of each thrower, hammer and thrower-hammer system were calculated. The vertical component of motion of all three c.m.s followed cyclic patterns with one fluctuation per turn. The fluctuation of the c.m. of the thrower was ahead of that of the hammer by approximately a third of a cycle, and this made the periods of upward vertical acceleration of the system c.m. coincide approximately with the double-support phases. In the horizontal direction, the c.m.s of the thrower and of the hammer followed roughly trochoid patterns as a result of the combination of rotation with forward displacement across the throwing circle. Their rotations were out of synchrony by approximate synchrony with the hammer, or an essentially straight trajectory. The results of this study suggest that the investigation of the hammer throw might be facilitated by the use of a quasi-inertial non-rotating reference frame that follows the general motion of the system c.m. while ignoring its fluctuations within each turn.

Influence of the direction of the cable force and of the radius of the hammer path on speed fluctuations during hammer throwing.

Hammer speed increases gradually during a throw, but this general increasing trend has one fluctuation superimposed in each turn. In some throwers, gravity and the forward translation of the system produce most of the fluctuation; in others, a marked fluctuation remains after the effects of gravity and of the forward translation of the system have been subtracted out. The remaining fluctuation could be produced through two mechanisms: (a) pulling on the hammer cable in a direction alternately ahead and behind the position of the centroid of the hammer path and (b) alternately shortening and lengthening the distance between the hammer head and the centroid of its path. Three-dimensional film analysis of eight highly-skilled throwers showed that the portion of the hammer speed fluctuation not due to gravity nor to the forward motion is produced mainly by pulling alternately ahead and behind the position of the centroid of the hammer path.

Three-dimensional cinematography with control object of unknown shape.

A technique for reconstruction of three-dimensional (3D) motion which involves a simple filming procedure but allows the deduction of coordinates in large object volumes was developed. Internal camera parameters are calculated from measurements of the film images of two calibrated crosses while external camera parameters are calculated from the film images of points in a control object of unknown shape but at least one known length. The control object, which includes the volume in which the activity is to take place, is formed by a series of poles placed at unknown locations, each carrying two targets. From the internal and external camera parameters, and from locations of the images of point in the films of the two cameras, 3D coordinates of the point can be calculated. Root mean square errors of the three coordinates of points in a large object volume (5m x 5m x 1.5m) were 15 mm, 13 mm, 13 mm and 6 mm, and relative errors in lengths averaged 0.5%, 0.7% and 0.5%, respectively.

[Results of hematopoietic stem cell transplantation in hemoglobinopathies: thalassemia major and sickle cell disease]. / Resultados del trasplante de progenitores hematopoyéticos en hemoglobinopatías: talasemia maior y enfermedad drepanocítica.

BACKGROUND: The prevalence of hemoglobinopathies in Spain is increasing as a result of immigration. Thalassemia major presents with chronic hemolytic anemia that requires regular red blood cell transfusions within the first year of life. Patients with sickle cell disease suffer from chronic anemia, vasculopathy and progressive damage in almost any organ. There is decreased life expectancy in both conditions. Allogeneic hematopoietic stem cell transplantation represents the only potentially curative option. PATIENTS: Seventeen patients (fourteen thalassemia major, and three sickle cell disease) underwent allogeneic hematopoietic stem cell transplantations. RESULTS: In the thalassemia group, nine donors were HLA-geno-identical siblings, two were partially matched related donors (one HLA allele mismatch), and three unrelated donors. All three patients with sickle cell disease were transplanted from HLA-geno-identical siblings. The source of stem cells was bone marrow in sixteen cases. Median patient age at transplant was six years (range: 1-16) in the thalassemia group, and twelve years (range: 8-15) in the sickle cell disease group. The graft was successful in all patients. Secondary graft rejection was observed in two thalassemia patients rendering them dependent on blood transfusions. Complete chimerism was observed in thirteen patients and, although mixed chimerism occurred in two, with all of them showing normal hemoglobin levels after transplantation and not requiring further transfusion support. Patients affected by sickle cell disease did not present with new vaso-occlusive crises, and stabilization of pulmonary and neurological function was observed. Chronic graft-versus-host disease was detected in three patients affected by thalassemia, and hypogonadotrophic hypogonadism in five patients. CONCLUSIONS: We conclude that for thalassemia major and sickle cell disease, allogenic hematopoietic stem cell transplantation from HLA-geno-identical siblings offers a high probability of complication-free survival. Despite good results, morbidity and mortality associated with transplantation from unrelated donors is a risk that might be considered, in contrast to a non-curative medical treatment that offers a long term survival. For thalassemia major groups it could be an option, but not for sickle cell disease, which is still in the investigational phase.

[Severe congenital neutropenia: analysis of clinical features, diagnostic methods, treatment and long-term outcome]. / Neutropenia congénita grave: análisis de las características clínicas, estudios diagnósticos, tratamiento y seguimiento a largo plazo.

INTRODUCTION: Severe congenital neutropenia (SCN), a heterogeneous condition with onset at early ages, is characterised by primary myelopoiesis failure with an absolute neutrophil count (ANC) < 0.5 x10(9)/L, severe infections and risk of leukaemic transformation. OBJECTIVE: The aim of the study was to ascertain the long term outcome of patients with SCN. MATERIAL AND METHODS: The clinical features, diagnostic methods, treatment and outcome of 11 patients with SCN were analysed. RESULTS: The median age at diagnosis was 4 months (range: 3 days-12 years). The primary clinical manifestation was severe infection. Median ANC at diagnosis: 0.2 x 10(9)/L (range: 0-0.37). Bone marrow aspirate showed maturation arrest at promyelocyte stage in all cases. Genetic studies revealed 3 mutations, two in ELA-2 gene and 1 in G6PC3 gene, showing a correlation between genotype and phenotype. Granulocyte Colony Stimulating Factor (G-CSF) was the first-line treatment in 9 patients; six of whom showed a good response at doses between 5 and 15 µg/kg/day for 3-7 days/week. The remaining 3 patients failed to respond to G-CSF and allogeneic stem cell transplantation (SCT) was indicated. Furthermore, SCT was the treatment of choice in two cases. Median follow-up of the cohort was 5 years (range: 1-10 years) with 100% survival and no cases of leukaemic transformation. CONCLUSIONS: We conclude that genetic study is useful for establishing a correlation between genotype and phenotype. The treatment of choice for SCN is G-CSF to which 2/3 of patients should respond; while SCT is reserved for cases of poor response or those evolving to myelodysplastic syndrome (MDS) or leukaemia; thus close follow-up of this condition is essential.

[Spanish Paediatric Infectious Diseases Society consensus document on the treatment of fungal infections based on the immune response]. / Documento de consenso de la Sociedad de Infectología Pediátrica sobre el tratamiento de la infección fúngica basado en la respuesta inmunológica.

Despite the emergence of new diagnostic and therapeutic methods, invasive fungal infections are still a major cause of morbidity and mortality in immunocompromised and critical patients. Therefore, adjuvant treatments to the standard antifungal therapy are being investigated, with immunity-based therapy being one of the most important. Both immunomodulatory (dendritic and T cells transfusions, colony stimulating factors, interferón-gamma, interleukin 12, fungal vaccines, transfer factors and certain drugs such as chloroquine) and immunotherapeutic modalities (granulocyte transfusions, monoclonal antibodies and intravenous immunoglobulin) have been described. This document aims to summarise currently available data on immunity-based therapy of fungal infections and to provide basic knowledge on the immune response to fungal infections. This helps to understand how, in selected cases, immunity-based therapy may improve the response to standard antifungal treatment. The potential indications of immunity-based therapy in the paediatric patient are reviewed, although there is still a lack of scientific evidence for its use in children.