Pontocerebellar hypoplasia type 1: clinical spectrum and relevance of EXOSC3 mutations

Pontocerebellar hypoplasia with spinal muscular atrophy, also known as PCH1, is a group of autosomal recessive disorders characterized by generalized muscle weakness and global developmental delay commonly resulting in early death. Gene defects had been discovered only in single patients until the recent identification of EXOSC3 mutations in several families with relatively mild course of PCH1. We aim to genetically stratify subjects in a large and well-defined cohort to define the clinical spectrum and genotype-phenotype correlation.

Genomic differences between type strain PG1 and field strains of Mycoplasma mycoides subsp. mycoides small-colony type

The recently accomplished complete genomic sequence analysis of the type strain PG1 of Mycoplasma mycoides subsp. mycoides small-colony type revealed four large repeated segments of 24, 13, 12, and 8 kb that are flanked by insertion sequence (IS) elements. Genetic analysis of type strain PG1 and African, European, and Australian field and vaccine strains revealed that the 24-kb genetic locus is repeated only in PG1 and not in other M. mycoides subsp. mycoides SC strains. In contrast, the 13-kb genetic locus was found duplicated in some strains originating from Africa and Australia but not in strains that were isolated from the European outbreaks. The 12- and 8-kb genetic loci were found in two and three copies, respectively, in all 28 strains analyzed. The flanking IS elements are assumed to lead to these tandem duplications, thus contributing to genomic plasticity. This aspect must be considered when designing novel diagnostic approaches and recombinant vaccines.

Snake C-type lectin-like proteins and platelet receptors

Snake venoms are complex mixtures of biologically active proteins and peptides. Many affect haemostasis by activating or inhibiting coagulant factors or platelets, or by disrupting endothelium. Snake venom components are classified into various families, such as serine proteases, metalloproteinases, C-type lectin-like proteins, disintegrins and phospholipases. Snake venom C-type lectin-like proteins have a typical fold resembling that in classic C-type lectins such as the selectins and mannose-binding proteins. Many snake venom C-type lectin-like proteins have now been characterized, as heterodimeric structures with alpha and beta subunits that often form large molecules by multimerization. They activate platelets by binding to VWF or specific receptors such as GPIb, alpha2beta1 and GPVI. Simple heterodimeric GPIb-binding molecules mainly inhibit platelet functions, whereas multimeric ones activate platelets. A series of tetrameric snake venom C-type lectin-like proteins activates platelets by binding to GPVI while another series affects platelet function via integrin alpha2beta1. Some act by inducing VWF to bind to GPIb. Many structures of these proteins, often complexed with their ligands, have been determined. Structure-activity studies show that these proteins are quite complex despite similar backbone folding. Snake C-type lectin-like proteins often interact with more than one platelet receptor and have complex mechanisms of action.

Increased genome instability in Escherichia coli lon mutants: relation to emergence of multiple-antibiotic-resistant (Mar) mutants caused by insertion sequence elements and large tandem genomic amplifications

Thirteen spontaneous multiple-antibiotic-resistant (Mar) mutants of Escherichia coli AG100 were isolated on Luria-Bertani (LB) agar in the presence of tetracycline (4 microg/ml). The phenotype was linked to insertion sequence (IS) insertions in marR or acrR or unstable large tandem genomic amplifications which included acrAB and which were bordered by IS3 or IS5 sequences. Five different lon mutations, not related to the Mar phenotype, were also found in 12 of the 13 mutants. Under specific selective conditions, most drug-resistant mutants appearing late on the selective plates evolved from a subpopulation of AG100 with lon mutations. That the lon locus was involved in the evolution to low levels of multidrug resistance was supported by the following findings: (i) AG100 grown in LB broth had an important spontaneous subpopulation (about 3.7x10(-4)) of lon::IS186 mutants, (ii) new lon mutants appeared during the selection on antibiotic-containing agar plates, (iii) lon mutants could slowly grow in the presence of low amounts (about 2x MIC of the wild type) of chloramphenicol or tetracycline, and (iv) a lon mutation conferred a mutator phenotype which increased IS transposition and genome rearrangements. The association between lon mutations and mutations causing the Mar phenotype was dependent on the medium (LB versus MacConkey medium) and the antibiotic used for the selection. A previously reported unstable amplifiable high-level resistance observed after the prolonged growth of Mar mutants in a low concentration of tetracycline or chloramphenicol can be explained by genomic amplification.

Germline NF1 mutational spectra and loss-of-heterozygosity analyses in patients with pheochromocytoma and neurofibromatosis type 1

BACKGROUND: Neurofibromatosis type 1 (NF1) is a pheochromocytoma-associated syndrome. Because of the low prevalence of pheochromocytoma in NF1, we ascertained subjects by pheochromocytoma that also had NF1 in the hope of describing the germline NF1 mutational spectra of NF1-related pheochromocytoma. MATERIALS AND METHODS: An international registry for NF1-pheochromocytomas was established. Mutation scanning was performed using denaturing HPLC for intragenic variation and quantitative PCR for large deletions. Loss-of-heterozygosity analysis using markers in and around NF1 was performed. RESULTS: There were 37 eligible subjects (ages 14-70 yr). Of 21 patients with corresponding tumor available, 67% showed somatic loss of the nonmutated allele at the NF1 locus vs. 0 of 12 sporadic tumors (P = 0.0002). Overall, 86% of the 37 patients had exonic or splice site mutations, 14% large deletions or duplications; 79% of the mutations are novel. The cysteine-serine rich domain (CSR) was affected in 35% but the RAS GTPase activating protein domain (RGD) in only 13%. There did not appear to be an association between any clinical features, particularly pheochromocytoma presentation and severity, and NF1 mutation genotype. CONCLUSIONS: The germline NF1 mutational spectra comprise intragenic mutations and deletions in individuals with pheochromocytoma and NF1. NF1 mutations tended to cluster in the CSR over the RAS-GAP domain, suggesting that CSR plays a more prominent role in individuals with NF1-pheochromocytoma than in NF1 individuals without this tumor. Loss-of-heterozygosity of NF1 markers in NF1-related pheochromocytoma was significantly more frequent than in sporadic pheochromocytoma, providing further molecular evidence that pheochromocytoma is a true component of NF1.

[Will type I diabetes mellitus be treated with immunosuppressive drugs in the future?]

Even if the pathogenesis of type-I (insulin-dependent) diabetes mellitus is still not clarified in every detail, there is general agreement that this form of diabetes is induced by autoimmune mechanisms leading to beta-cell destruction. Therefore, it should theoretically be feasible to suppress the mechanism leading to type-I diabetes with appropriate and early immunotherapy. The current clinical data clearly document that the rate and duration of remissions in patients with newly diagnosed type-I diabetes can be increased significantly using appropriate immunosuppressive regimens. However, before these therapies can become standard therapy of type-I diabetes, the following important clinical requirements have to be fulfilled: the toxicity (especially to kidneys and beta-cells) has to be reduced, the patients should be diagnosed and treated in 'pre-diabetic' states, more selective immunosuppressive regimens have to be available in order to reduce the occurrence of treatment-associated lymphomas and neoplasias. Since accurate detection of 'pre-diabetic' patients is difficult and presents an immense logistic problem, it may take a long time before large-scale immunosuppressive therapies of type-I diabetes are feasible.

Conversion of CD95 (Fas) Type II into Type I signaling by sub-lethal doses of cycloheximide

CD95 (Fas/Apo-1)-mediated apoptosis was shown to occur through two distinct pathways. One involves a direct activation of caspase-3 by large amounts of caspase-8 generated at the DISC (Type I cells). The other is related to the cleavage of Bid by low concentration of caspase-8, leading to the release of cytochrome c from mitochondria and the activation of caspase-3 by the cytochrome c/APAF-1/caspase-9 apoptosome (Type II cells). It is also known that the protein synthesis inhibitor cycloheximide (CHX) sensitizes Type I cells to CD95-mediated apoptosis, but it remains contradictory whether this effect also occurs in Type II cells. Here, we show that sub-lethal doses of CHX render both Type I and Type II cells sensitive to the apoptogenic effect of anti-CD95 antibodies but not to chemotherapeutic drugs. Moreover, Bcl-2-positive Type II cells become strongly sensitive to CD95-mediated apoptosis by the addition of CHX to the cell culture. This is not the result of a restraint of the anti-apoptotic effect of Bcl-2 at the mitochondrial level since CHX-treated Type II cells still retain their resistance to chemotherapeutic drugs. Therefore, CHX treatment is granting the CD95-mediated pathway the ability to bypass the mitochondria requirement to apoptosis, much alike to what is observed in Type I cells.

Anticipating antiport in P-type ATPases

Cation-transporting P-type ATPases show a high degree of structural and functional homology. Nevertheless, for many members of this large family, the molecular mechanism of transport is unclear; namely, whether transport is electrogenic or not and if countertransport is involved remains to be established. In a few well-studied cases such as the Na(+)-K(+)-ATPase, plasma membrane Ca(2+) ATPase (PMCA) and sarcoplasmic reticulum Ca(2+) ATPase (SERCA) countertransport has been clearly demonstrated. New data based on the crystal structure of SERCA now strongly indicate that countertransport could be mandatory for all P-type ATPases. This concept should be verified for other known and for all newly characterized P-type ATPases.

Establishing glycaemic control with continuous subcutaneous insulin infusion in children and adolescents with type 1 diabetes: experience of the PedPump Study in 17 countries

AIMS/HYPOTHESIS: To assess the use of paediatric continuous subcutaneous infusion (CSII) under real-life conditions by analysing data recorded for up to 90 days and relating them to outcome. METHODS: Pump programming data from patients aged 0-18 years treated with CSII in 30 centres from 16 European countries and Israel were recorded during routine clinical visits. HbA(1c) was measured centrally. RESULTS: A total of 1,041 patients (age: 11.8 +/- 4.2 years; diabetes duration: 6.0 +/- 3.6 years; average CSII duration: 2.0 +/- 1.3 years; HbA(1c): 8.0 +/- 1.3% [means +/- SD]) participated. Glycaemic control was better in preschool (n = 142; 7.5 +/- 0.9%) and pre-adolescent (6-11 years, n = 321; 7.7 +/- 1.0%) children than in adolescent patients (12-18 years, n = 578; 8.3 +/- 1.4%). There was a significant negative correlation between HbA(1c) and daily bolus number, but not between HbA(1c) and total daily insulin dose. The use of <6.7 daily boluses was a significant predictor of an HbA(1c) level >7.5%. The incidence of severe hypoglycaemia and ketoacidosis was 6.63 and 6.26 events per 100 patient-years, respectively. CONCLUSIONS/INTERPRETATION: This large paediatric survey of CSII shows that glycaemic targets can be frequently achieved, particularly in young children, and the incidence of acute complications is low. Adequate substitution of basal and prandial insulin is associated with a better HbA(1c).

Inhibition of L-type amino acid transport with non-physiological amino acids in the Pahenu2 mouse model of phenylketonuria

Phenylketonuria, an autosomal recessive Mendelian disorder, is one of the most common inborn errors of metabolism. Although currently treated by diet, many suboptimal outcomes occur for patients. Neuropathological outcomes include cognitive loss, white matter abnormalities, and hypo- or demyelination, resulting from high concentrations and/or fluctuating levels of phenylalanine. High phenylalanine can also result in competitive exclusion of other large neutral amino acids from the brain, including tyrosine and tryptophan (essential precursors of dopamine and serotonin). This competition occurs at the blood brain barrier, where the L-type amino acid transporter, LAT1, selectively facilitates entry of large neutral amino acids. The hypothesis of these studies is that certain non-physiological amino acids (NPAA; DL-norleucine (NL), 2-aminonorbornane (NB; 2-aminobicyclo-(2,1,1)-heptane-2-carboxylic acid), α-aminoisobutyrate (AIB), and α-methyl-aminoisobutyrate (MAIB)) would competitively inhibit LAT1 transport of phenylalanine (Phe) at the blood-brain barrier interface. To test this hypothesis, Pah-/- mice (n=5, mixed gender; Pah+/-(n=5) as controls) were fed either 5% NL, 0.5% NB, 5% AIB or 3% MAIB (w/w 18% protein mouse chow) for 3 weeks. Outcome measurements included food intake, body weight, brain LNAAs, and brain monoamines measured via LCMS/MS or HPLC. Brain Phe values at sacrifice were significantly reduced for NL, NB, and MAIB, verifying the hypothesis that these NPAAs could inhibit Phe trafficking into the brain. However, concomitant reductions in tyrosine and methionine occurred at the concentrations employed. Blood Phe levels were not altered indicating no effect of NPAA competitors in the gut. Brain NL and NB levels, measured with HPLC, verified both uptake and transport of NPAAs. Although believed predominantly unmetabolized, NL feeding significantly increased blood urea nitrogen. Pah-/-disturbances of monoamine metabolism were exacerbated by NPAA intervention, primarily with NB (the prototypical LAT inhibitor). To achieve the overarching goal of using NPAAs to stabilize Phe transport levels into the brain, a specific Phe-reducing combination and concentration of NPAAs must be found. Our studies represent the first in vivo use of NL, NB and MAIB in Pah-/- mice, and provide proof-of-principle for further characterization of these LAT inhibitors. Our data is the first to document an effect of MAIB, a specific system A transport inhibitor, on large neutral amino acid transport.

Triggering of volcanic activity by large earthquakes

Statistical analyses of temporal relationships between large earthquakes and volcanic eruptions suggest seismic waves may trigger eruptions even over great (>1000 km) distances, although the causative mechanism is not well constrained. In this study the relationship between large earthquakes and subtle changes in volcanic activity was investigated in order to gain greater insight into the relationship between dynamic stresses propagated by surface waves and volcanic response. Daily measurements from the Ozone Monitoring Instrument (OMI), onboard the Aura satellite, provide constraints on volcanic sulfur-dioxide (SO2) emission rates as a measure of subtle changes in activity. Time series of SO2 emission rates were produced from OMI data for thirteen persistently active volcanoes from 1 October 2004 to 30 September 2010. In order to quantify the affect of earthquakes at teleseismic distances, we modeled surface-wave amplitudes from the source mechanisms of moment magnitude (Mw) ≥7 earthquakes, and calculated the Peak Dynamic Stress (PDS). We assessed the influence of earthquakes on volcanic activity in two ways: 1) by identifying increases in the SO2 time series data and looking for causative earthquakes and 2) by examining the average emission rate before and after each earthquake. In the first, the SO2 time series for each volcano was used to calculate a baseline threshold for comparison with post-earthquake emission. Next, we generated a catalog of responses based on sustained SO2 emission increases above this baseline. Delay times between each SO2 response and each prior earthquake were analyzed using both the actual earthquake catalog, and a randomly generated catalog of earthquakes. This process was repeated for each volcano. Despite varying multiple parameters, this analysis did not demonstrate a clear relationship between earthquake-generated PDS and SO2 emission. However, the second analysis, which was based on the occurrence of large earthquakes indicated a response at most volcanoes. Using the PDS calculations as a filtering criterion for the earthquake catalog, the SO2 mass for each volcano was analyzed in 28-day windows centered on the earthquake origin time. If the average SO2 mass after the earthquake was greater than an arbitrary percentage of pre-earthquake mass, we identified the volcano as having a response to the event. This window analysis provided insight on what type of volcanic activity is more susceptible to triggering by dynamic stress. The volcanoes with very open systems included in this study, Ambrym, Gaua, Villarrica, Erta Ale and, Turrialba, showed a clear response to dynamic stress while the volcanoes with more closed systems, Merapi, Semeru, Fuego, Pacaya, and Bagana, showed no response.

Expanding the spectrum of mutations in GH1 and GHRHR: genetic screening in a large cohort of patients with congenital isolated growth hormone deficiency

CONTEXT: It is estimated that 3-30% of cases with isolated GH deficiency (IGHD) have a genetic etiology, with a number of mutations being reported in GH1 and GHRHR. The aim of our study was to genetically characterize a cohort of patients with congenital IGHD and analyze their characteristics. PATIENTS AND METHODS: A total of 224 patients (190 pedigrees) with IGHD and a eutopic posterior pituitary were screened for mutations in GH1 and GHRHR. To explore the possibility of an association of GH1 abnormalities with multiple pituitary hormone deficiencies, we have screened 62 patients with either multiple pituitary hormone deficiencies (42 pedigrees), or IGHD with an ectopic posterior pituitary (21 pedigrees). RESULTS: Mutations in GH1 and GHRHR were identified in 41 patients from 21 pedigrees (11.1%), with a higher prevalence in familial cases (38.6%). These included previously described and novel mutations in GH1 (C182X, G120V, R178H, IVS3+4nt, a>t) and GHRHR (W273S, R94L, R162W). Autosomal dominant, type II IGHD was the commonest form (52.4%), followed by type IB (42.8%) and type IA (4.8%). Patients with type II IGHD had highly variable phenotypes. There was no difference in the endocrinology or magnetic resonance imaging appearance between patients with and without mutations, although those with mutations presented with more significant growth failure (height, -4.7 +/- 1.6 SDS vs. -3.4 +/- 1.7 SDS) (P = 0.001). There was no apparent difference between patients with mutations in GH1 and GHRHR. CONCLUSIONS: IGHD patients with severe growth failure and a positive family history should be screened for genetic mutations; the evolving endocrinopathy observed in some of these patients suggests the need for long-term follow-up.

An insulin infusion advisory system for type 1 diabetes patients based on non-linear model predictive control methods

In this paper, an Insulin Infusion Advisory System (IIAS) for Type 1 diabetes patients, which use insulin pumps for the Continuous Subcutaneous Insulin Infusion (CSII) is presented. The purpose of the system is to estimate the appropriate insulin infusion rates. The system is based on a Non-Linear Model Predictive Controller (NMPC) which uses a hybrid model. The model comprises a Compartmental Model (CM), which simulates the absorption of the glucose to the blood due to meal intakes, and a Neural Network (NN), which simulates the glucose-insulin kinetics. The NN is a Recurrent NN (RNN) trained with the Real Time Recurrent Learning (RTRL) algorithm. The output of the model consists of short term glucose predictions and provides input to the NMPC, in order for the latter to estimate the optimum insulin infusion rates. For the development and the evaluation of the IIAS, data generated from a Mathematical Model (MM) of a Type 1 diabetes patient have been used. The proposed control strategy is evaluated at multiple meal disturbances, various noise levels and additional time delays. The results indicate that the implemented IIAS is capable of handling multiple meals, which correspond to realistic meal profiles, large noise levels and time delays.

[Occurrence of Clostridium perfringens type A and type C in piglets of the Swiss swine population]

Necrotizing enteritis (NE) of newborn piglets still represents an economical problem in Swiss pig breeding and production. The aim of our study was to identify risk factors for NE and evaluate the prevalence of C. perfringens with the toxingenes cpb and cpb2 in Swiss pig breeding farms. The prevalence of theses C. perfringens was investigated using fecal swabs followed by bacteriological culturing and genotyping. Close proximity to other breeding farms and large herd sizes were shown to predispose to NE. C. perfringens type C, carrying the genes cpa, cpb and cpb2 were frequently identified in herds with acute outbreaks of NE. Farms not affected by NE or those using prophylactic vaccination against NE were predominantly positive for C. perfringens type A strains with cpb2 and showed much lower prevalence of C. perfringens type C, compared to acutely affected herds. Our results demonstrate that C. perfringens type A strains with cpb2 are not associated with NE. Besides typical necropsy finding, only the identification of cpb can be used for the diagnosis of NE in affected herds.

A mutation in the human ortholog of the Saccharomyces cerevisiae ALG6 gene causes carbohydrate-deficient glycoprotein syndrome type-Ic

Carbohydrate-deficient glycoprotein syndrome (CDGS) represents a class of genetic diseases characterized by abnormal N-linked glycosylation. CDGS patients show a large number of glycoprotein abnormalities resulting in dysmorphy, encephalopathy, and other organ disorders. The majority of CDGSs described to date are related to an impaired biosynthesis of dolichyl pyrophosphate-linked Glc3Man9GlcNAc2 in the endoplasmic reticulum. Recently, we identified in four related patients a novel type of CDGS characterized by an accumulation of dolichyl pyrophosphate-linked Man9GlcNAc2. Elaborating on the analogy of this finding with the phenotype of alg5 and alg6 Saccharomyces cerevisiae strains, we have cloned and analyzed the human orthologs to the ALG5 dolichyl phosphate glucosyltransferase and ALG6 dolichyl pyrophosphate Man9GlcNAc2 alpha1,3-glucosyltransferase in four novel CDGS patients. Although ALG5 was not altered in the patients, a C-->T transition was detected in ALG6 cDNA of all four CDGS patients. The mutation cosegregated with the disease in a Mendelian recessive manner. Expression of the human ALG5 and ALG6 cDNA could partially complement the respective S. cerevisiae alg5 and alg6 deficiency. By contrast, the mutant ALG6 cDNA of CDGS patients failed to revert the hypoglycosylation observed in alg6 yeasts, thereby proving a functional relationship between the alanine to valine substitution introduced by the C-->T transition and the CDGS phenotype. The mutation in the ALG6 alpha1,3-glucosyltransferase gene defines an additional type of CDGS, which we propose to refer to as CDGS type-Ic.