A G613A missense in the Hutchinson’s progeria lamin A/C gene causes a lone,autosomal dominant atrioventricular block

Background

LMNA/C mutations have been linked to the premature aging syndrome Hutchinson’s progeria, dilated cardiomyopathy 1A, skeletal myopathies (such as the autosomal dominant variant of Emery-Dreifuss muscular dystrophy and limb-girdle muscular dystrophy), Charcot-Marie-Tooth disorder type 2B1, mandibuloacral dysplasia, autosomal dominant partial lipodystrophy, and axonal neuropathy. Atrioventricular block (AVB) can be associated with several cardiac disorders and it can also be a highly heritable, primitive disease.One of the most common pathologies associated with AVB is dilated cardiomyopathy (DCM), which is characterized by cardiac dilatation and reduced systolic function. In this case, onset has been correlated with several mutations in genes essential for the proper maturation of cardiomyocytes, such as the gene for lamin A/C. However, no clear genotype–phenotype relationship has been reported to date between LMNA/C mutations and cardiomyopathies.

Results

DNA and medical histories were collected from (n?=?11) members of different generations of one family, the proband of which was implanted with a pacemaker for lone, type II AVB. Exome sequencing analysis was performed on three relatives with AVB, and the mutations therein identified validated in a further three AVB-affected family members.In the initial three AVB family members, we identified 10 shared nonsynonymous single-nucleotide variations with a rare or unreported allele frequency in the 1000 Genomes Project database. Follow-up genetic screening in the additional three affected relatives disclosed a correlation between the lone AVB phenotype and the single-nucleotide polymorphism rs56816490, which generates an E317K change in lamin A/C. Although this mutation has already been described by others in a DCM-affected proband with familiarity for AVB and sudden death, the absence of DCM in our large, AVB-affected family is indicative of genotype–phenotype correlation between rs56816490 and a familial, autosomal dominant form of lone AVB.

Conclusions

Screening for G613A in LMNA/C in patients with lone AVB and their relatives might prevent sudden death in families affected by AVB but without familiarity for DCM. Lone AVB is an age-related disease caused by mutations in LMN A/C gene rather than a complication of DCM.

     

Proteomic analysis of formalin-fixed, paraffin-embedded lung neuroendocrine tumor samples from hospital archives

Hospital tissue repositories host an invaluable supply of diseased samples with matched retrospective clinical information. In this work, a recently optimized method for extracting full-length proteins from formalin-fixed, paraffin-embedded (FFPE) tissues was evaluated on lung neuroendocrine tumor (LNET) samples collected from hospital repositories. LNETs comprise a heterogeneous spectrum of diseases, for which subtype-specific diagnostic markers are lacking. Six archival samples diagnosed as typical carcinoid (TC) or small cell lung carcinoma (SCLC) were subjected to a full-length protein extraction followed by a GeLC-MS/MS analysis, enabling the identification of over 300 distinct proteins per tumor subtype. All identified proteins were categorized through DAVID software, revealing a differential distribution of functional classes, such as those involved in RNA processing, response to oxidative stress and ion homeostasis. Moreover, using spectral counting for protein abundance estimation and beta-binomial test as statistical filter, a list of 28 differentially expressed proteins was generated and submitted to pathway analysis by means of Ingenuity Pathway Analysis software. Differential expression of chromogranin-A (more expressed in TCs) and stathmin (more expressed in SCLCs) was consistently confirmed by immunohistochemistry. Therefore, FFPE hospital archival samples can be successfully subjected to proteomic investigations aimed to biomarker discovery following a GeLC-MS/MS label-free approach.     

Direct and Inverted Repeats Elicit Genetic Instability by Both Exploiting and Eluding DNA Double-Strand Break Repair Systems in Mycobacteria

Repetitive DNA sequences with the potential to form alternative DNA conformations, such as slipped structures and cruciforms, can induce genetic instability by promoting replication errors and by serving as a substrate for DNA repair proteins, which may lead to DNA double-strand breaks (DSBs). However, the contribution of each of the DSB repair pathways, homologous recombination (HR), non-homologous end-joining (NHEJ) and single-strand annealing (SSA), to this sort of genetic instability is not fully understood. Herein, we assessed the genome-wide distribution of repetitive DNA sequences in the Mycobacterium smegmatis, Mycobacterium tuberculosis and Escherichia coli genomes, and determined the types and frequencies of genetic instability induced by direct and inverted repeats, both in the presence and in the absence of HR, NHEJ, and SSA. All three genomes are strongly enriched in direct repeats and modestly enriched in inverted repeats. When using chromosomally integrated constructs in M. smegmatis, direct repeats induced the perfect deletion of their intervening sequences ∼1,000-fold above background. Absence of HR further enhanced these perfect deletions, whereas absence of NHEJ or SSA had no influence, suggesting compromised replication fidelity. In contrast, inverted repeats induced perfect deletions only in the absence of SSA. Both direct and inverted repeats stimulated excision of the constructs from the attB integration sites independently of HR, NHEJ, or SSA. With episomal constructs, direct and inverted repeats triggered DNA instability by activating nucleolytic activity, and absence of the DSB repair pathways (in the order NHEJ>HR>SSA) exacerbated this instability. Thus, direct and inverted repeats may elicit genetic instability in mycobacteria by 1) directly interfering with replication fidelity, 2) stimulating the three main DSB repair pathways, and 3) enticing L5 site-specific recombination.     

Properties of a putative cambialistic superoxide dismutase from the aerotolerant bacterium Streptococcus thermophilus strain LMG 18311

The aerotolerance of the lactic - fermentative bacterium Streptococcus thermophilus is mainly based on the key antioxidant function of superoxide dismutase (StSOD). In this work, the comparison of recombinant StSOD (rStSOD) forms obtained from two different initiation triplets indicated that the enzyme from S. thermophilus strain LMG 18311 spans 201 residues. rStSOD is organised as a homodimer, even though protein aggregates are formed in concentrated solutions. The capability of binding and exchanging Fe or Mn in the active site classifies rStSOD as a putative cambialistic enzyme; the moderate preference for iron is counteracted by a 1.5-fold higher activity measured for the Mn-containing form. The enzyme is thermostable, being its half-inactivation time 10 min at 73.5°C; the energetic parameters of the heat inactivation process are regulated by the level of Mn cofactor. The effect of Mn content on the rStSOD sensitivity towards inhibitors and inactivators was also evaluated. Sodium azide acts as a weak inhibitor of rStSOD and its Mn content does not greatly affect this sensitivity. Concerning the physiological inactivator hydrogen peroxide, the Mn-enriched rStSOD displays a great resistance; a moderate sensitivity is instead observed in the presence of a low Mn content. Contrary to hydrogen peroxide, sodium peroxynitrite is a powerful inactivator, a behaviour enhanced in the Mn-enriched enzyme. All these results were compared with the corresponding data previously reported for the cambialistic SOD from the taxonomically related S. mutans. In S. thermophilus the regulation of the enzyme functions by the Mn content appears less relevant with respect to S. mutans.     

Rapid identification of monospecific monoclonal antibodies using a human proteome microarray

To broaden the range of tools available for proteomic research, we generated a library of 16,368 unique full-length human ORFs that are expressible as N-terminal GST-His(6) fusion proteins. Following expression in yeast, these proteins were then individually purified and used to construct a human proteome microarray. To demonstrate the usefulness of this reagent, we developed a streamlined strategy for the production of monospecific monoclonal antibodies that used immunization with live human cells and microarray-based analysis of antibody specificity as its central components. We showed that microarray-based analysis of antibody specificity can be performed efficiently using a two-dimensional pooling strategy. We also demonstrated that our immunization and selection strategies result in a large fraction of monospecific monoclonal antibodies that are both immunoblot and immunoprecipitation grade. Our data indicate that the pipeline provides a robust platform for the generation of monoclonal antibodies of exceptional specificity.     

In vivo and laccase-catalysed decolourization of xenobiotic azo dyes by a basidiomycetous fungus: characterization of its ligninolytic system

Bioremediation is considered a promising eco-efficient alternative for industrial wastewater treatment. Particular attention is currently being given to biological degradation of synthetic dyes and more specifically to colour removal by fungi. This work looks at the extracellular enzymatic system of strain Euc-1. Its ability to decolourize 14 xenobiotic azo dyes was evaluated and compared with the well-known species Phanerochaete chrysosporium. Strain Euc-1 is a mesophilic white-rot basidiomycete, the main secreted ligninolytic enzyme being laccase (0.38 U ml^–1). Although low manganese-dependent peroxidase activity (0.05 U ml^–1) was also detected, neither lignin peroxidase nor aryl alcohol oxidase could be found in batch culture. Optimum pH values of 4.0 and 5.0 were obtained in the laccase-catalysed oxidation of guaiacol and syringaldazine, respectively. Laccase activity increased with the temperature rise up to 50–60 °C and remarkable thermal stability was observed at 50 °C with a half-life of 12 h and no deactivation within the first 2 h. Solid-plate decolourization studies showed that basidiomycete Euc-1 decolourized 11 azo dyes whereas P. chrysosporium only two. Moreover, it is shown that purified laccase from basidiomycete Euc-1 efficiently decolourizes the azo dye acid red 88.     

Traffic-related air pollution and respiratory symptoms among asthmatic children,resident in Mexico City: the EVA cohort study

Background

Taffic-related air pollution has been related to adverse respiratory outcomes; however, there is still uncertainty concerning the type of vehicle emission causing most deleterious effects.

Methods

A panel study was conducted among 147 asthmatic and 50 healthy children, who were followed up for an average of 22 weeks. Incidence density of coughing, wheezing and breathing difficulty was assessed by referring to daily records of symptoms and child''s medication. The association between exposure to pollutants and occurrence of symptoms was evaluated using mixed-effect models with binary response and poisson regression.

Results

Wheezing was found to relate significantly to air pollutants: an increase of 17.4 μg/m³ (IQR) of PM_2.5 (24-h average) was associated with an 8.8% increase (95% CI: 2.4% to 15.5%); an increase of 34 ppb (IQR) of NO₂ (1-h maximum) was associated with an 9.1% increase (95% CI: 2.3% to16.4%) and an increase of 48 ppb (IQR) in O₃ levels (1 hr maximum) to an increase of 10% (95% CI: 3.2% to 17.3%). Diesel-fueled motor vehicles were significantly associated with wheezing and bronchodilator use (IRR = 1.29; 95% CI: 1.03 to 1.62, and IRR = 1.32; 95% CI: 0.99 to 1.77, respectively, for an increase of 130 vehicles hourly, above the 24-hour average).

Conclusion

Respiratory symptoms in asthmatic children were significantly associated with exposure to traffic exhaust, especially from natural gas and diesel-fueled vehicles.     

A closer look at intraspecific variation of Cerataphis brasiliensis (Hempel) (Hemiptera: Hormaphididae)

The aim of our study was to characterize Cerataphis brasiliensis Hempel, an aphid of native palm tree of tropical savannas (cerrado), and to record intraspecific variation. We found two parthenogenetic forms of apterous viviparae females of C. brasiliensis. We differentiate the two forms through measurable and qualitative characters: (i) length of cephalic horns, (ii) number of antennal segments, (iii) presence or absence of wax glands, (iv) location of siphunculi.     

The Arabidopsis kinase-associated protein phosphatase regulates adaptation to Na+ stress

The kinase-associated protein phosphatase (KAPP) is a regulator of the receptor-like kinase (RLK) signaling pathway. Loss-of-function mutations rag1-1 (root attenuated growth1-1) and rag1-2, in the locus encoding KAPP, cause NaCl hypersensitivity in Arabidopsis thaliana. The NaCl hypersensitive phenotype exhibited by rag1 seedlings includes reduced shoot and primary root growth, root tip swelling, and increased lateral root formation. The phenotype exhibited by rag1-1 seedlings is associated with a specific response to Na(+) toxicity. The sensitivity to Na(+) is Ca(2+) independent and is not due to altered intracellular K(+)/Na(+). Analysis of the genetic interaction between rag1-1 and salt overly sensitive1 (sos1-14) revealed that KAPP is not a component of the SOS signal transduction pathway, the only Na(+) homeostasis signaling pathway identified so far in plants. All together, these results implicate KAPP as a functional component of the RLK signaling pathway, which also mediates adaptation to Na(+) stress. RLK pathway components, known to be modulated by NaCl at the messenger RNA level, are constitutively down-regulated in rag1-1 mutant plants. The effect of NaCl on their expression is not altered by the rag1-1 mutation.