Newborn screening for propionic, methylmalonic acidemia and vitamin B12 deficiency. Analysis of 588,793 newborns.

OBJECTIVES: We present the results of our experience in the diagnosis and follow up of the positive cases for propionic, methylmalonic acidemias and cobalamin deficiencies (PA/MMA/MMAHC) since the Expanded Newborn Screening was implemented in Madrid Region. METHODS: Dried blood samples were collected 48 h after birth. Amino acids and acylcarnitines were quantitated by MS/MS. Newborns with alterations were referred to the clinical centers for follow-up. Biochemical and molecular genetic studies for confirmation of a disease were performed. RESULTS: In the period 2011-2020, 588,793 children were screened, being 953 of them were referred to clinical units for abnormal result (192 for elevated C3 levels). Among them, 88 were false positive cases, 85 maternal vitamin B12 deficiencies and 19 were confirmed to suffer an IEM (8 PA, 4 MMA, 7 MMAHC). Ten out 19 cases displayed symptoms before the NBS results (6 PA, 1 MMA, 3 MMAHC). C3, C16:1OH+C17 levels and C3/C2 and C3/Met ratios were higher in newborns with PA/MMA/MMAHC. Cases diagnosed with B12 deficiency had mean B12 levels of 187.6 ± 76.9 pg/mL and their mothers 213.7 ± 95.0; 5% of the mothers were vegetarian or had poor eating while 15% were diagnosed of pernicious anemia. Newborns and their mothers received treatment with B12 with different posology, normalizing their levels and the secondary alterations disappeared. CONCLUSIONS: Elevated C3 are a frequent cause for abnormal result in newborn screening with a high rate of false positive cases. Presymptomatic diagnosis of most of PA and some MMA/MMAHC is difficult. Vitamin B12 deficiency secondary to maternal deprivation is frequent with an heterogenous clinical and biochemical spectrum.

Recommendations for the Diagnosis and Therapeutic Management of Hyperammonaemia in Paediatric and Adult Patients.

Hyperammonaemia is a metabolic derangement that may cause severe neurological damage and even death due to cerebral oedema, further complicating the prognosis of its triggering disease. In small children it is a rare condition usually associated to inborn errors of the metabolism. As age rises, and especially in adults, it may be precipitated by heterogeneous causes such as liver disease, drugs, urinary infections, shock, or dehydration. In older patients, it is often overlooked, or its danger minimized. This protocol was drafted to provide an outline of the clinical measures required to normalise ammonia levels in patients of all ages, aiming to assist clinicians with no previous experience in its treatment. It is an updated protocol developed by a panel of experts after a review of recent publications. We point out the importance of frequent monitoring to assess the response to treatment, the nutritional measures that ensure not only protein restriction but adequate caloric intake and the need to avoid delays in the use of specific pharmacological therapies and, especially, extrarenal clearance measures. In this regard, we propose initiating haemodialysis when ammonia levels are >200−350 µmol/L in children up to 18 months of age and >150−200 µmol/L after that age.

Nanobodies Protecting From Lethal SARS-CoV-2 Infection Target Receptor Binding Epitopes Preserved in Virus Variants Other Than Omicron.

The emergence of SARS-CoV-2 variants that escape from immune neutralization are challenging vaccines and antibodies developed to stop the COVID-19 pandemic. Thus, it is important to establish therapeutics directed toward multiple or specific SARS-CoV-2 variants. The envelope spike (S) glycoprotein of SARS-CoV-2 is the key target of neutralizing antibodies (Abs). We selected a panel of nine nanobodies (Nbs) from dromedary camels immunized with the receptor-binding domain (RBD) of the S, and engineered Nb fusions as humanized heavy chain Abs (hcAbs). Nbs and derived hcAbs bound with subnanomolar or picomolar affinities to the S and its RBD, and S-binding cross-competition clustered them in two different groups. Most of the hcAbs hindered RBD binding to its human ACE2 (hACE2) receptor, blocked cell entry of viruses pseudotyped with the S protein and neutralized SARS-CoV-2 infection in cell cultures. Four potent neutralizing hcAbs prevented the progression to lethal SARS-CoV-2 infection in hACE2-transgenic mice, demonstrating their therapeutic potential. Cryo-electron microscopy identified Nb binding epitopes in and out the receptor binding motif (RBM), and showed different ways to prevent virus binding to its cell entry receptor. The Nb binding modes were consistent with its recognition of SARS-CoV-2 RBD variants; mono and bispecific hcAbs efficiently bound all variants of concern except omicron, which emphasized the immune escape capacity of this latest variant.

Diagnosis of inborn errors of metabolism within the expanded newborn screening in the Madrid region.

We present the results of our experience in the diagnosis of inborn errors of metabolism (IEM) since the Expanded Newborn Screening was implemented in our Region. Dried blood samples were collected 48 h after birth. Amino acids and acylcarnitines were quantitated by mass spectrometry (MS)/MS. Newborns with alterations were referred to the clinical centers for follow-up. Biochemical and molecular genetic studies for confirmation of a disease were performed. In the period 2011 to 2019, 592 822 children were screened: 902 of them were referred for abnormal results. An IEM was confirmed in 222 (1/2670): aminoacidopathies: 89 hyperphenylalaninemia (HPA) (51 benign HPA, 32 phenylketonuria, 4 DNAJC12 defect, and 2 primapterinuria), 6 hypermethioninemia, 3 tyrosinemia type 1 (TYR-1), 1 TYR-3, 4 maple syrup urine disease (MSUD), 2 branched-chain amino acid transferase 2 deficiency, 2 homocystinuria, 1 cystinuria, 2 ornithine transcarbamylase (OTC) deficiency, 2 citrullinemia type I (CTLN1); FAO defects: 43 medium-chain acyl-CoA dehydrogenase deficiency (MCADD), 13 very long-chain acyl-CoA dehydrogenase deficiency, 2 long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), 1 multiple acyl-coA dehydrogenation deficiency, 11 systemic primary carnitine deficiency, 2 carnitine palmitoyltransferase type 2 (CPT-II) deficiency, 1 CPT-I deficiency; organic acidurias: 12 glutaric aciduria type 1 (GA-1), 4 methylmalonic acidemia (MMA), 7 MMA including combined cases with homocystinuria (MMAHC), 6 propionic acidemia (PA), 7 3-methylcrotonyl-CoA carboxylase, 1 3-hydroxy-3-methylglutaryl-CoA lyase deficiency lyase deficiency. Only 19 infants (8.5%) were symptomatic at newborn screening result (1 LCHADD, 5 PA, 1 CPT-II deficiency, 1 MMA, 3 MMAHC, 2 MSUD, 2 OTC deficiency, 1 CTLN1, 1 MCADD, 2 TYR-1). No false negative cases were identified. Genetic diagnosis was conclusive in all biochemically confirmed cases, except for two infants with HPA, identifying pathogenic variants in 32 different genes. The conditions with the highest incidence were HPA (1/6661) and MCAD deficiencies (1/13 787).

[Newborn Screening Program in the Community of Madrid: evaluation of positive cases.] / Análisis de casos positivos de cribado neonatal de errores congénitos del metabolismo en la Comunidad de Madrid.

OBJECTIVE: Tandem mass spectrometry (MS/MS) is being used for newborn screening since this laboratory testing technology increases the number of metabolic disorders that can be detected from dried blood-spot specimens. In the Community of Madrid, it was implemented in March 2011 and it includes 13 aminoacidopathies, fatty acid oxidation disorders and organic acidemias. The aim of this study was to describe our experience and evaluate the screening positive cases in a period of 9 years (2011-2019). METHODS: During the period of the study, a total of 592.822 neonates were screened with this expanded program by MS/MS in the Community of Madrid. Amino acids, acylcarnitines, and succinylacetone were quantified in all samples that met the quality criteria. Means, medians, percentiles and standard deviation of the analytes and ratios of interest were calculated. RESULTS: 901 patients (0,15 %) with a positive screening test were referred to clinical evaluation. 230 patients were diagnosed of 30 different inborn errors of metabolism (prevalence 1:2577), 11 of which were not included as a target in the Community of Madrid newborn screening program. The global positive predictive value was 25,6 %. During this period of time, two false negative cases were detected. The most prevalent disorders were phenylketonuria/hyperphenylalaninemia and medium chain acyl-CoA dehydrogenase deficiency (1:6444 and 1:13174 respectively). 93 % of the patients were detected in the presymptomatic stage. CONCLUSIONS: During the last 9 years a large number of cases of IEM have been detected with an acceptable global positive predictive value. These results confirm the utility of inborn errors of metabolism newborn screening as a public health program.

OBJETIVO: La tecnología de espectrometría de masas en tándem (MS/MS) en los programas de cribado neonatal ha permitido la detección de gran número de errores congénitos del metabolismo (ECM). En la comunidad de Madrid se implementó en marzo de 2011 incluyendo 13 aminoacidopatías, defectos de la ß-oxidación de ácidos grasos y acidemias orgánicas. El objetivo de este estudio fue describir nuestra experiencia y analizar los casos positivos de cribado en un periodo de 9 años (2011-2019). METODOS: Durante el periodo de estudio se realizó el cribado mediante MS/MS a 592822 recién nacidos en la Comunidad de Madrid. Se cuantificaron aminoácidos, acilcarnitinas y succinilacetona en todas las muestras que cumplieron los criterios de calidad. Se calcularon medias, medianas, percentiles y desviación típica de los analitos y ratios de interés. RESULTADOS: Se derivaron a las unidades clínicas de seguimiento por sospecha de una ECM un total de 901 (0,15 %) casos. Se confirmaron 230 casos de 30 ECM diferentes (prevalencia 1:2577), 11 de los cuales no eran inicialmente objetivo de detección del programa. El valor predictivo positivo global fue de 25,6 %. Durante este periodo se detectaron dos falsos negativos. Las enfermedades con mayor prevalencia fueron fenilcetonuria/hiperfenilalaninemia y deficiencia de acil-CoA deshidrogenasa de cadena media (1:6444 y 1: 13174 respectivamente). 93 % de los casos fueron detectados en fase presintomática. CONCLUSIONES: En estos 9 años de experiencia se han detectado numerosos casos de ECM con un valor predictivo positivo global aceptable. Estos resultados confirman la utilidad del cribado neonatal de ECM como programa de salud pública.

Beneficial Effect of N-Carbamylglutamate in a Neonatal Form of Multiple Acyl-CoA Dehydrogenase Deficiency.

Background. Multiple acyl-CoA dehydrogenase deficiency is an autosomal recessive disorder of the amino acid metabolism and fatty acid oxidation due to the deficiency of the electron transfer protein or electron transfer protein ubiquinone oxidoreductase. The clinical picture ranges from a severe neonatal lethal presentation to late myopathic forms responsive to riboflavin. Up to now, there is no effective treatment for the neonatal form, which exhibits severe metabolic acidosis, hyperammonemia, hypoketotic hypoglycemia, and rhabdomyolysis. We present the case of a child who has had a good long-term outcome after a typical neonatal onset, with a dramatic drop in ammonia levels during the initial metabolic decompensation crisis and adequate control even during intercurrent diseases thereafter with N-carbamylglutamate treatment.

Análisis de casos positivos de cribado neonatal de errores congénitos del metabolismo en la Comunidad de Madrid / Newborn Screening Program in the Community of Madrid: evaluation of positive cases

OBJETIVO: La tecnología de espectrometría de masas en tándem (MS/MS) en los programas de cribado neonatal ha permitido la detección de gran número de errores congénitos del metabolismo (ECM). En la comunidad de Madrid se implementó en marzo de 2011 incluyendo 13 aminoacidopatías, defectos de la beta-oxidación de ácidos grasos y acidemias orgánicas. El objetivo de este estudio fue describir nuestra experiencia y analizar los casos positivos de cribado en un periodo de 9 años (2011-2019). MÉTODOS: Durante el periodo de estudio se realizó el cribado mediante MS/MS a 592822 recién nacidos en la Comunidad de Madrid. Se cuantificaron aminoácidos, acilcarnitinas y succinilacetona en todas las muestras que cumplieron los criterios de calidad. Se calcularon medias, medianas, percentiles y desviación típica de los analitos y ratios de interés. RESULTADOS: Se derivaron a las unidades clínicas de seguimiento por sospecha de una ECM un total de 901 (0,15 %) casos. Se confirmaron 230 casos de 30 ECM diferentes (prevalencia 1:2577), 11 de los cuales no eran inicialmente objetivo de detección del programa. El valor predictivo positivo global fue de 25,6 %. Durante este periodo se detectaron dos falsos negativos. Las enfermedades con mayor prevalencia fueron fenilcetonuria/hiperfenilalaninemia y deficiencia de acil-CoA deshidrogenasa de cadena media (1:6444 y 1: 13174 respectivamente). 93 % de los casos fueron detectados en fase presintomática. CONCLUSIONES: En estos 9 años de experiencia se han detectado numerosos casos de ECM con un valor predictivo positivo global aceptable. Estos resultados confirman la utilidad del cribado neonatal de ECM como programa de salud pública

OBJECTIVE: Tandem mass spectrometry (MS/MS) is being used for newborn screening since this laboratory testing technology increases the number of metabolic disorders that can be detected from dried blood-spot specimens. In the Community of Madrid, it was implemented in March 2011 and it includes 13 aminoacidopathies, fatty acid oxidation disorders and organic acidemias. The aim of this study was to describe our experience and evaluate the screening positive cases in a period of 9 years (2011-2019). METHODS: During the period of the study, a total of 592.822 neonates were screened with this expanded program by MS/MS in the Community of Madrid. Amino acids, acylcarnitines, and succinylacetone were quantified in all samples that met the quality criteria. Means, medians, percentiles and standard deviation of the analytes and ratios of interest were calculated. RESULTS: 901 patients (0,15 %) with a positive screening test were referred to clinical evaluation. 230 patients were diagnosed of 30 different inborn errors of metabolism (prevalence 1:2577), 11 of which were not included as a target in the Community of Madrid newborn screening program. The global positive predictive value was 25,6 %. During this period of time, two false negative cases were detected. The most prevalent disorders were phenylketonuria/hyperphenylalaninemia and medium chain acyl-CoA dehydrogenase deficiency (1:6444 and 1:13174 respectively). 93 % of the patients were detected in the presymptomatic stage. CONCLUSIONS: During the last 9 years a large number of cases of IEM have been detected with an acceptable global positive predictive value. These results confirm the utility of inborn errors of metabolism newborn screening as a public health program

Betaine anhydrous in homocystinuria: results from the RoCH registry.

BACKGROUND: The Registry of Adult and Paediatric Patients Treated with Cystadane® - Homocystinuria (RoCH) is a non-interventional, observational, multi-centre, post-authorization safety study that aimed to identify safety of betaine anhydrous (Cystadane®) in the treatment of patients with inborn errors of homocysteine metabolism (homocystinuria) in order to minimise the treatment associated risks and establish better knowledge on its clinical use. The registry included patients of all ages with homocystinuria who were treated with betaine anhydrous in conjunction with other therapies. Clinical data were collected retrospectively from 2007 to 2013, then prospectively up to February 2014. All adverse events (AEs) reported during the study were recorded. The clinical and biological status of patients was monitored at least once a year. RESULTS: A total of 125 patients with homocystinuria (adults [> 18 years]: 50; paediatric [≤18 years]: 75) were enrolled at 29 centres in France and Spain. Patients were treated with betaine anhydrous for a mean duration of 7.4 ± 4.3 years. The median total daily dose of betaine anhydrous at the first and last study visits was 6 g/day for cystathionine ß-synthase (CBS)-deficient vitamin B6 responders and 9 g/day for methylenetetrahydrofolate reductase-deficient patients, while the median daily dose increased in CBS-deficient B6 non-responders (from 6 to 9 g/day) and cobalamin metabolism-defective patients (from 3 to 6 g/day) between the first and last visits. Treatment caused a mean overall reduction of 29% in plasma homocysteine levels in the study population. A total of 277 AEs were reported during the study, of which two non-serious AEs (bad taste and headache) and one serious AE (interstitial lung disease) were considered to be drug related. Overall, betaine anhydrous was well tolerated with no major safety concerns. CONCLUSIONS: Data from the RoCH registry provided real-world evidence on the clinical safety and efficacy of betaine anhydrous in the management of homocystinuria in paediatric and adult patients.

Publisher Correction: Z-ring membrane anchors associate with cell wall synthases to initiate bacterial cell division.

The original version of this Article contained errors in Figures 1 and 3. In Fig. 1b, the label 'IP-PBP3' above the second of the three blots incorrectly read 'IP-PBP1B'. In Fig. 3b, the label 'PBP1B' under the first bar of each chart incorrectly read 'PBP1A'. These errors have been corrected in both the PDF and HTML versions of the Article.

Z-ring membrane anchors associate with cell wall synthases to initiate bacterial cell division.

During the transition from elongation to septation, Escherichia coli establishes a ring-like peptidoglycan growth zone at the future division site. This preseptal peptidoglycan synthesis does not require the cell division-specific peptidoglycan transpeptidase PBP3 or most of the other cell division proteins, but it does require FtsZ, its membrane-anchor ZipA and at least one of the bi-functional transglycosylase-transpeptidases, PBP1A or PBP1B. Here we show that PBP1A and PBP1B interact with ZipA and localise to preseptal sites in cells with inhibited PBP3. ZipA stimulates the glycosyltransferase activity of PBP1A. The membrane-anchored cell division protein FtsN localises at preseptal sites and stimulates both activities of PBP1B. Genes zipA and ftsN can be individually deleted in ftsA* mutant cells, but the simultaneous depletion of both proteins is lethal and cells do not establish preseptal sites. Our data support a model according to which ZipA and FtsN-FtsA have semi-redundant roles in connecting the cytosolic FtsZ ring with the membrane-anchored peptidoglycan synthases during the preseptal phase of envelope growth.

The hypermorph FtsA* protein has an in vivo role in relieving the Escherichia coli proto-ring block caused by excess ZapC.

Assembly of the proto-ring, formed by the essential FtsZ, FtsA and ZipA proteins, and its progression into a divisome, are essential events for Escherichia coli division. ZapC is a cytoplasmic protein that belongs to a group of non-essential components that assist FtsZ during proto-ring assembly. Any overproduction of these proteins leads to faulty FtsZ-rings, resulting in a cell division block. We show that ZapC overproduction can be counteracted by an excess of the ZipA-independent hypermorph FtsA* mutant, but not by similar amounts of wild type FtsA+. An excess of FtsA+ allowed regular spacing of the ZapC-blocked FtsZ-rings, but failed to promote recruitment of the late-assembling proteins FtsQ, FtsK and FtsN and therefore, to activate constriction. In contrast, overproduction of FtsA*, besides allowing correct FtsZ-ring localization at midcell, restored the ability of FtsQ, FtsK and FtsN to be incorporated into active divisomes.

Life without Division: Physiology of Escherichia coli FtsZ-Deprived Filaments.

When deprived of FtsZ, Escherichia coli cells (VIP205) grown in liquid form long nonseptated filaments due to their inability to assemble an FtsZ ring and their failure to recruit subsequent divisome components. These filaments fail to produce colonies on solid medium, in which synthesis of FtsZ is induced, upon being diluted by a factor greater than 4. However, once the initial FtsZ levels are recovered in liquid culture, they resume division, and their plating efficiency returns to normal. The potential septation sites generated in the FtsZ-deprived filaments are not annihilated, and once sufficient FtsZ is accumulated, they all become active and divide to produce cells of normal length. FtsZ-deprived cells accumulate defects in their physiology, including an abnormally high number of unsegregated nucleoids that may result from the misplacement of FtsK. Their membrane integrity becomes compromised and the amount of membrane proteins, such as FtsK and ZipA, increases. FtsZ-deprived cells also show an altered expression pattern, namely, transcription of several genes responding to DNA damage increases, whereas transcription of some ribosomal or global transcriptional regulators decreases. We propose that the changes caused by the depletion of FtsZ, besides stopping division, weaken the cell, diminishing its resiliency to minor challenges, such as dilution stress. IMPORTANCE: Our results suggest a role for FtsZ, in addition to its already known effect in the constriction of E. coli, in protecting the nondividing cells against minor stress. This protection can even be exerted when an inactive FtsZ is produced, but it is lost when the protein is altogether absent. These results have implications in fields like synthetic biology or antimicrobial discovery. The construction of synthetic divisomes in the test tube may need to preserve unsuspected roles, such as this newly found FtsZ property, to guarantee the stability of artificial containers. Whereas the effects on viability caused by inhibiting the activity of FtsZ may be partly overcome by filamentation, the absence of FtsZ is not tolerated by E. coli, an observation that may help in the design of effective antimicrobial compounds.

Carglumic acid enhances rapid ammonia detoxification in classical organic acidurias with a favourable risk-benefit profile: a retrospective observational study.

BACKGROUND: Isovaleric aciduria (IVA), propionic aciduria (PA) and methylmalonic aciduria (MMA) are inherited organic acidurias (OAs) in which impaired organic acid metabolism induces hyperammonaemia arising partly from secondary deficiency of N-acetylglutamate (NAG) synthase. Rapid reduction in plasma ammonia is required to prevent neurological complications. This retrospective, multicentre, open-label, uncontrolled, phase IIIb study evaluated the efficacy and safety of carglumic acid, a synthetic structural analogue of NAG, for treating hyperammonaemia during OA decompensation. METHODS: Eligible patients had confirmed OA and hyperammonaemia (plasma NH3 > 60 µmol/L) in ≥1 decompensation episode treated with carglumic acid (dose discretionary, mean (SD) first dose 96.3 (73.8) mg/kg). The primary outcome was change in plasma ammonia from baseline to endpoint (last available ammonia measurement at ≤18 hours after the last carglumic acid administration, or on Day 15) for each episode. Secondary outcomes included clinical response and safety. RESULTS: The efficacy population (received ≥1 dose of study drug and had post-baseline measurements) comprised 41 patients (MMA: 21, PA: 16, IVA: 4) with 48 decompensation episodes (MMA: 25, PA: 19, IVA: 4). Mean baseline plasma ammonia concentration was 468.3 (±365.3) µmol/L in neonates (29 episodes) and 171.3 (±75.7) µmol/L in non-neonates (19 episodes). At endpoint the mean plasma NH3 concentration was 60.7 (±36.5) µmol/L in neonates and 55.2 (±21.8) µmol/L in non-neonates. Median time to normalise ammonaemia was 38.4 hours in neonates vs 28.3 hours in non-neonates and was similar between OA subgroups (MMA: 37.5 hours, PA: 36.0 hours, IVA: 40.5 hours). Median time to ammonia normalisation was 1.5 and 1.6 days in patients receiving and not receiving concomitant scavenger therapy, respectively. Although patients receiving carglumic acid with scavengers had a greater reduction in plasma ammonia, the endpoint ammonia levels were similar with or without scavenger therapy. Clinical symptoms improved with therapy. Twenty-five of 57 patients in the safety population (67 episodes) experienced AEs, most of which were not drug-related. Overall, carglumic acid seems to have a good safety profile for treating hyperammonaemia during OA decompensation. CONCLUSION: Carglumic acid when used with or without ammonia scavengers, is an effective treatment for restoration of normal plasma ammonia concentrations in hyperammonaemic episodes in OA patients.

Metabolic Syndrome in Children and Adolescents Living with HIV.

BACKGROUND: Metabolic syndrome (MetS) is considered an independent risk factor for developing cardiovascular disease. It is well known that the prevalence of metabolic disorders have increased in pediatric HIV-infected children. The objective of this study is to assess the prevalence and characteristics of MetS in HIV-infected children and adolescents in Spain. METHODS: A cross-sectional multicenter study in 152 patients from the pediatric cohort of the Spanish AIDS Research Network (CoRISpe) was performed. MetS was defined according to the new International Diabetes Federation (IDF) diagnostic criteria and the modified National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP III) criteria. Measurements included anthropometry, waist circumference, blood pressure, fasting lipids, glucose and insulin and lipodystrophy assessment. Demographic, clinical, immunological, virological and antiretroviral therapy data were obtained from the Network database. RESULTS: An abnormally low high-density lipoprotein-cholesterol level was the most prevalent disturbance (21.05%) found. Three patients met IDF criteria for MetS (1.97%), and MetS was significantly associated with lipohypertrophy (P=0.029) in the analysis. When the modified NCEP-ATP III criteria were used, the prevalence of MetS was 5.92% (9 patients), and MetS was significantly associated with Tanner stage ≥2 (P=0.041), lipohypertrophy (P=0.001) and higher Z scores for weight and body mass index (P=0.002 and P<0.001). Insulin resistance was observed in 17 patients (11.18%) and was associated with MetS (as per the modified NCEP-ATP III criteria) (P=0.03) and lower high-density lipoprotein-cholesterol values (P=0.036). CONCLUSIONS: The prevalence of MetS in our cohort was 1.97% or 5.92%, depending on the diagnostic criteria used. MetS should be actively assessed, particularly in children who show lipohypertrophy.

Biopsia selectiva del ganglio centinela en cáncer de mama: sin recurrencias axilares tras un seguimiento medio de 4, 5 años / Selective biopsy of the sentinel lymph node in breast cancer: Without axillary recurrences after a mean follow-up of 4.5 years

Objetivo. Analizar la tasa de recurrencias axilares (RA) en pacientes con cáncer de mama en estadios iniciales y biopsia selectiva del ganglio centinela negativa (BSGC) sin linfadenectomía (LDN) posterior. Material y métodos. Se han incluido un total de 173 pacientes a quienes se aplicó la BSGC tras diagnóstico de cáncer de mama en estadios iniciales. En 32 pacientes la BSGC fue positiva y estas pacientes fueron sometidas a LDN. Se han seguido 141 pacientes con BSGC negativa sin LDN, por un periodo medio de 55 meses (rango 74-36). Resultados. La tasa de detección del GC fue del 99,42%. Después de un periodo de seguimiento medio de 4,5 años no se han detectado RA. Dos pacientes desarrollaron recidiva local, otras 2 desarrollaron metástasis a distancia y 4 desarrollaron un cáncer intercurrente. Se produjeron 4 muertes, ninguna de ellas asociadas al cáncer de mama. Conclusiones. Los resultados obtenidos apoyan la BSGC como una técnica exacta en la estadificación axilar de los pacientes con cáncer de mama en estadios iniciales, ofreciendo en los casos de BSGC negativa un control axilar seguro después de 4,5 años de seguimiento medio (AU)

Objective. To analyze the rate of axillary recurrences (AR) in patients with early breast cancer who had not undergone an axillary node dissection (ALND) because of a negative sentinel lymph node biopsy (SLNB). Material and methods. The study includes 173 patients operated on for breast cancer and selective node biopsy. In 32 patients the SLNB was positive and undergone subsequent ALND. We followed up 141 patients with negative SLNB without LDN, with a median follow up of 55 months (range 74-36). Results. The detection rate of SLN was of 99.42%. After a median follow-up of 4.5 years, there were no axillary recurrences. Two patients developed local recurrence, other two patients developed distant metastases and four patients developed a metachronous tumor. Four patients died, none of them because of breast cancer. Conclusions. The results obtained support the SLNB as an accurate technique in the axillary stratification of patients with breast cancer, offering in the cases of negative SLNB a safe axillary control after 4.5 year follow-up (AU)

FtsZ placement in nucleoid-free bacteria.

We describe the placement of the cytoplasmic FtsZ protein, an essential component of the division septum, in nucleoid-free Escherichia coli maxicells. The absence of the nucleoid is accompanied in maxicells by degradation of the SlmA protein. This protein, together with the nucleoid, prevents the placement of the septum in the regions occupied by the chromosome by a mechanism called nucleoid occlusion (NO). A second septum placement mechanism, the MinCDE system (Min) involving a pole-to-pole oscillation of three proteins, nonetheless remains active in maxicells. Both Min and NO act on the polymerization of FtsZ, preventing its assembly into an FtsZ-ring except at midcell. Our results show that even in the total absence of NO, Min oscillations can direct placement of FtsZ in maxicells. Deletion of the FtsZ carboxyl terminal domain (FtsZ*), a central hub that receives signals from a variety of proteins including MinC, FtsA and ZipA, produces a Min-insensitive form of FtsZ unable to interact with the membrane-anchoring FtsA and ZipA proteins. This protein produces a totally disorganized pattern of FtsZ localization inside the maxicell cytoplasm. In contrast, FtsZ*-VM, an artificially cytoplasmic membrane-anchored variant of FtsZ*, forms helical or repetitive ring structures distributed along the entire length of maxicells even in the absence of NO. These results show that membrane anchoring is needed to organize FtsZ into rings and underscore the role of the C-terminal hub of FtsZ for their correct placement.

[Selective biopsy of the sentinel lymph node in breast cancer: without axillary recurrences after a mean follow-up of 4.5 years]. / Biopsia selectiva del ganglio centinela en cáncer de mama: sin recurrencias axilares tras un seguimiento medio de 4,5 años.

OBJECTIVE: To analyze the rate of axillary recurrences (AR) in patients with early breast cancer who had not undergone an axillary node dissection (ALND) because of a negative sentinel lymph node biopsy (SLNB). MATERIAL AND METHODS: The study includes 173 patients operated on for breast cancer and selective node biopsy. In 32 patients the SLNB was positive and undergone subsequent ALND. We followed up 141 patients with negative SLNB without LDN, with a median follow up of 55 months (range 74-36). RESULTS: The detection rate of SLN was of 99.42%. After a median follow-up of 4.5 years, there were no axillary recurrences. Two patients developed local recurrence, other two patients developed distant metastases and four patients developed a metachronous tumor. Four patients died, none of them because of breast cancer. CONCLUSIONS: The results obtained support the SLNB as an accurate technique in the axillary stratification of patients with breast cancer, offering in the cases of negative SLNB a safe axillary control after 4.5 year follow-up.

Bacterial division proteins FtsZ and ZipA induce vesicle shrinkage and cell membrane invagination.

Permeable vesicles containing the proto-ring anchoring ZipA protein shrink when FtsZ, the main cell division protein, polymerizes in the presence of GTP. Shrinkage, resembling the constriction of the cytoplasmic membrane, occurs at ZipA densities higher than those found in the cell and is modulated by the dynamics of the FtsZ polymer. In vivo, an excess of ZipA generates multilayered membrane inclusions within the cytoplasm and causes the loss of the membrane function as a permeability barrier. Overproduction of ZipA at levels that block septation is accompanied by the displacement of FtsZ and two additional division proteins, FtsA and FtsN, from potential septation sites to clusters that colocalize with ZipA near the membrane. The results show that elementary constriction events mediated by defined elements involved in cell division can be evidenced both in bacteria and in vesicles.

Role of Escherichia coli FtsN protein in the assembly and stability of the cell division ring.

Deprivation of FtsN, the last protein in the hierarchy of divisome assembly, causes the disassembly of other elements from the division ring, even extending to already assembled proto-ring proteins. Therefore the stability and function of the divisome to produce rings active in septation is not guaranteed until FtsN is recruited. Disassembly follows an inverse sequential pathway relative to assembly. In the absence of FtsN, the frequencies of FtsN and FtsQ rings are affected similarly. Among the proto-ring components, ZipA are more sensitive than FtsZ or FtsA rings. In contrast, removal of FtsZ leads to an almost simultaneous disappearance of the other elements from rings. Although restoration of FtsN allows for a quick reincorporation of ZipA into proto-rings, the de novo joint assembly of the three components when FtsZ levels are restored to FtsZ-deprived filaments is even faster. This suggests that the recruitment of ZipA into FtsZ-FtsA incomplete proto-rings may require first a period for the reversal of these partial assemblies.

Efficacy of sapropterin dihydrochloride in increasing phenylalanine tolerance in children with phenylketonuria: a phase III, randomized, double-blind, placebo-controlled study.

OBJECTIVE: To evaluate the ability of sapropterin dihydrochloride (pharmaceutical preparation of tetrahydrobiopterin) to increase phenylalanine (Phe) tolerance while maintaining adequate blood Phe control in 4- to 12-year-old children with phenylketonuria (PKU). STUDY DESIGN: This international, double-blind, randomized, placebo-controlled study screened for sapropterin response among 90 enrolled subjects in Part 1. In Part 2, 46 responsive subjects with PKU were randomized (3:1) to sapropterin, 20 mg/kg/d, or placebo for 10 weeks while continuing on a Phe-restricted diet. After 3 weeks, a dietary Phe supplement was added every 2 weeks if Phe control was adequate. RESULTS: The mean (+/-SD) Phe supplement tolerated by the sapropterin group had increased significantly from the pretreatment amount (0 mg/kg/d) to 20.9 (+/-15.4) mg/kg/d (P < .001) at the last visit at which subjects had adequate blood Phe control (<360 micromol/L), up to week 10. Over the 10-week period, the placebo group tolerated only an additional 2.9 (+/-4.0) mg/kg/d Phe supplement; the mean difference from the sapropterin group (+/-SE) was 17.7 +/- 4.5 mg/kg/d (P < .001). No severe or serious related adverse events were observed. CONCLUSIONS: Sapropterin is effective in increasing Phe tolerance while maintaining blood Phe control and has an acceptable safety profile in this population of children with PKU.