Molecular and phenotypic characterization of a mouse model of oculopharyngeal muscular dystrophy reveals severe muscular atrophy restricted to fast glycolytic fibres

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by ptosis, dysphagia and proximal limb weakness. Autosomal-dominant OPMD is caused by a short (GCG)8–13 expansions within the first exon of the poly(A)-binding protein nuclear 1 gene (PABPN1), leading to an expanded polyalanine tract in the mutated protein. Expanded PABPN1 forms insoluble aggregates in the nuclei of skeletal muscle fibres. In order to gain insight into the different physiological processes affected in OPMD muscles, we have used a transgenic mouse model of OPMD (A17.1) and performed transcriptomic studies combined with a detailed phenotypic characterization of this model at three time points. The transcriptomic analysis revealed a massive gene deregulation in the A17.1 mice, among which we identified a significant deregulation of pathways associated with muscle atrophy. Using a mathematical model for progression, we have identified that one-third of the progressive genes were also associated with muscle atrophy. Functional and histological analysis of the skeletal muscle of this mouse model confirmed a severe and progressive muscular atrophy associated with a reduction in muscle strength. Moreover, muscle atrophy in the A17.1 mice was restricted to fast glycolytic fibres, containing a large number of intranuclear inclusions (INIs). The soleus muscle and, in particular, oxidative fibres were spared, even though they contained INIs albeit to a lesser degree. These results demonstrate a fibre-type specificity of muscle atrophy in this OPMD model. This study improves our understanding of the biological pathways modified in OPMD to identify potential biomarkers and new therapeutic targets.

Dynamics and diversity of the 'Atopobium cluster' in the human faecal microbiota, and phenotypic characterization of 'Atopobium cluster' isolates

This study monitored the dynamics and diversity of the human faecal 'Atopobium cluster' over a 3-month period using a polyphasic approach. Fresh faecal samples were collected fortnightly from 13 healthy donors (6 males and 7 females) aged between 26 and 61 years. Fluorescence in situ hybridization was used to enumerate total (EUB338mix) and 'Atopobium cluster' (ATO291) bacteria, with counts ranging between 1.12 × 1011 and 9.95 × 1011, and 1.03 × 109 and 1.16 × 1011 cells (g dry weight faeces)-1, respectively. The 'Atopobium cluster' population represented 0.2-22 % of the total bacteria, with proportions donor-dependent. Denaturing gradient gel electrophoresis (DGGE) using 'Atopobium cluster'-specific primers demonstrated faecal populations of these bacteria were relatively stable, with bands identified as Collinsella aerofaciens, Collinsella intestinalis/Collinsella stercoris, Collinsella tanakaei, Coriobacteriaceae sp. PEAV3-3, Eggerthella lenta, Gordonibacter pamelaeae, Olsenella profusa, Olsenella uli and Paraeggerthella hongkongensis in the DGGE profiles of individuals. Colony PCR was used to identify 'Atopobium cluster' bacteria isolated from faeces (n = 224 isolates). 16S rRNA gene sequence analysis of isolates demonstrated Collinsella aerofaciens represented the predominant (88 % of isolates) member of the 'Atopobium cluster' found in human faeces, being found in nine individuals. Eggerthella lenta was identified in three individuals (3.6 % of isolates). Isolates of Collinsella tanakaei, an 'Enorma' sp. and representatives of novel species belonging to the 'Atopobium cluster' were also identified in the study. Phenotypic characterization of the isolates demonstrated their highly saccharolytic nature and heterogeneous phenotypic profiles, and 97 % of the isolates displayed lipase activity.

Molecular and phenotypic characterization of the alternative seasonal growth habit and flowering time in barley (Hordeum vulgare ssp. vulgare L.)

Barley can be classified into three major agronomic types, based on its seasonal growth habit (SGH): spring, winter and alternative. Winter varieties require exposure to vernalization to promote subsequent flowering and are autumn-sown. Spring varieties proceed to flowering in the absence of vernalization and are sown in the spring. The ‘alternative’ (also known as ‘facultative’) SGH is only loosely defined and can be sown in autumn or spring. Here, we investigate the molecular genetic basis of alternative barley. Analysis of the major barley vernalization (VRN-H1, VRN-H2) and photoperiod (PPD-H1, PPD-H2) response genes in a collection of 386 varieties found alternative SGH to be characterized by specific allelic combinations. Spring varieties possessed spring loci at one or both of the vernalization response loci, combined with long-day non-responsive ppd-H1 alleles and wild-type alleles at the short-day photoperiod response locus, PPD-H2. Winter varieties possessed winter alleles at both vernalization loci, in combination with the mutant ppd-H2 allele conferring delayed flowering under short-day photoperiods. In contrast, all alternative varieties investigated possessed a single spring allele (either at VRN-H1 or at VRN-H2) combined with mutant ppd-H2 alleles. This allelic combination is found only in alternative types and is diagnostic for alternative SGH in the collection studied. Analysis of flowering time under controlled environment found alternative varieties flowered later than spring control lines, with the difference most pronounced under short-day photoperiods. This work provides genetic characterization of the alternative SGH phenotype, allowing precise manipulation of SGH and flowering time within breeding programmes, and provides the molecular tools for classification of all three SGH categories within national variety registration processes.

Genetic and phenotypic characterization of complex hereditary spastic paraplegia

The hereditary spastic paraplegias are a heterogeneous group of degenerative disorders that are clinically classified as either pure with predominant lower limb spasticity, or complex where spastic paraplegia is complicated with additional neurological features, and are inherited in autosomal dominant, autosomal recessive or X-linked patterns. Genetic defects have been identified in over 40 different genes, with more than 70 loci in total. Complex recessive spastic paraplegias have in the past been frequently associated with mutations in SPG11 (spatacsin), ZFYVE26/SPG15, SPG7 (paraplegin) and a handful of other rare genes, but many cases remain genetically undefined. The overlap with other neurodegenerative disorders has been implied in a small number of reports, but not in larger disease series. This deficiency has been largely due to the lack of suitable high throughput techniques to investigate the genetic basis of disease, but the recent availability of next generation sequencing can facilitate the identification of disease- causing mutations even in extremely heterogeneous disorders. We investigated a series of 97 index cases with complex spastic paraplegia referred to a tertiary referral neurology centre in London for diagnosis or management. The mean age of onset was 16 years (range 3 to 39). The SPG11 gene was first analysed, revealing homozygous or compound heterozygous mutations in 30/97 (30.9%) of probands, the largest SPG11 series reported to date, and by far the most common cause of complex spastic paraplegia in the UK, with severe and progressive clinical features and other neurological manifestations, linked with magnetic resonance imaging defects. Given the high frequency of SPG11 mutations, we studied the autophagic response to starvation in eight affected SPG11 cases and control fibroblast cell lines, but in our restricted study we did not observe correlations between disease status and autophagic or lysosomal markers. In the remaining cases, next generation sequencing was carried out revealing variants in a number of other known complex spastic paraplegia genes, including five in SPG7 (5/97), four in FA2H (also known as SPG35) (4/97) and two in ZFYVE26/SPG15. Variants were identified in genes usually associated with pure spastic paraplegia and also in the Parkinson’s disease-associated gene ATP13A2, neuronal ceroid lipofuscinosis gene TPP1 and the hereditary motor and sensory neuropathy DNMT1 gene, highlighting the genetic heterogeneity of spastic paraplegia. No plausible genetic cause was identified in 51% of probands, likely indicating the existence of as yet unidentified genes.

Identification of components of the Sigma B Regulon in Listeria monocytogenes that contribute to acid and salt tolerance

Sigma B (σB) is an alternative sigma factor that controls the transcriptional response to stress in Listeria monocytogenes and is also known to play a role in the virulence of this human pathogen. In the present study we investigated the impact of a sigB deletion on the proteome of L. monocytogenes grown in a chemically defined medium both in the presence and in the absence of osmotic stress (0.5 M NaCl). Two new phenotypes associated with the sigB deletion were identified using this medium. (i) Unexpectedly, the strain with the ΔsigB deletion was found to grow faster than the parent strain in the growth medium, but only when 0.5 M NaCl was present. This phenomenon was independent of the carbon source provided in the medium. (ii) The ΔsigB mutant was found to have unusual Gram staining properties compared to the parent, suggesting that σB contributes to the maintenance of an intact cell wall. A proteomic analysis was performed by two-dimensional gel electrophoresis, using cells growing in the exponential and stationary phases. Overall, 11 proteins were found to be differentially expressed in the wild type and the ΔsigB mutant; 10 of these proteins were expressed at lower levels in the mutant, and 1 was overexpressed in the mutant. All 11 proteins were identified by tandem mass spectrometry, and putative functions were assigned based on homology to proteins from other bacteria. Five proteins had putative functions related to carbon utilization (Lmo0539, Lmo0783, Lmo0913, Lmo1830, and Lmo2696), while three proteins were similar to proteins whose functions are unknown but that are known to be stress inducible (Lmo0796, Lmo2391, and Lmo2748). To gain further insight into the role of σB in L. monocytogenes, we deleted the genes encoding four of the proteins, lmo0796, lmo0913, lmo2391, and lmo2748. Phenotypic characterization of the mutants revealed that Lmo2748 plays a role in osmotolerance, while Lmo0796, Lmo0913, and Lmo2391 were all implicated in acid stress tolerance to various degrees. Invasion assays performed with Caco-2 cells indicated that none of the four genes was required for mammalian cell invasion. Microscopic analysis suggested that loss of Lmo2748 might contribute to the cell wall defect observed in the ΔsigB mutant. Overall, this study highlighted two new phenotypes associated with the loss of σB. It also demonstrated clear roles for σB in both osmotic and low-pH stress tolerance and identified specific components of the σB regulon that contribute to the responses observed.

Phenotypic and phylogenetic characterization of a new Corynebacterium species from dogs: description of Corynebacterium auriscanis sp. nov.

Six strains of a previously undescribed catalase-positive coryneform bacterium isolated from clinical specimens from dogs were characterized by phenotypic and molecular genetic methods. Biochemical and chemotaxonomic studies revealed that the unknown bacterium belonged to the genus Corynebacterium sensu stricto. Comparative 16S rRNA gene sequencing showed that the six strains were genealogically highly related and constitute a new subline within the genus Corynebacterium; this subline is close to but distinct from C. falsenii, C. jeikeium, and C. urealyticum. The unknown bacterium from dogs was distinguished from all currently validated Corynebacterium species by phenotypic tests including electrophoretic analysis of whole-cell proteins. On the basis of phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as a new species, Corynebacterium auriscanis. The type strain of C. auriscanis is CCUG 39938T.

Phenotypic and proteomic characterization of multiply antibiotic-resistant variants of Salmonella entetica serovar typhimurium selected following exposure to disinfectants

In previous work, Salmonella enterica serovar Typhimurium strain SL1344 was exposed to sublethal concentrations of three widely used farm disinfectants in daily serial passages for 7 days in an attempt to investigate possible links between the use of disinfectants and antimicrobial resistance. Stable variants OXCR1, QACFGR2, and TOPR2 were obtained following treatment with an oxidizing compound blend, a quaternary ammonium disinfectant containing formaldehyde and glutaraldehyde, and a tar acid-based disinfectant, respectively. All variants exhibited ca. fourfold-reduced susceptibility to ciprofloxacin, chloramphenicol, tetracycline, and ampicillin. This coincided with reduced levels of outer membrane proteins for all strains and high levels of AcrAB-To1C for OXCR1 and QACFGR2, as demonstrated by two-dimensional high-performance liquid chromatography-mass spectrometry. The protein profiles of OXCR1 and QACFGR2 were similar, but they were different from that of TOPR2. An array of different proteins protecting against oxidants, nitroaromatics, disulfides, and peroxides were overexpressed in all strains. The growth and motility of variants were reduced compared to the growth and motility of the parent strain, the expression of several virulence proteins was altered, and the invasiveness in an enteric epithelial cell line was reduced. The colony morphology of OXCR1 and QACFGR2 was smooth, and both variants exhibited a loss of modal distribution of the lipopolysaccharide O-antigen chain length, favoring the production of short O-antigen chain molecules. Metabolic changes were also detected, suggesting that there was increased protein synthesis and a shift from oxidative phosphorylation to substrate level phosphorylation. In this study, we obtained evidence that farm disinfectants can select for strains with reduced susceptibility to antibiotics, and here we describe changes in protein expression in such strains.

Phenotypic and genotypic characterization of avian Escherichia coli O86:K61 isolates possessing a gamma-like intimin

Escherichia coli O86:K61 has long been associated with outbreaks of infantile diarrhea in humans and with diarrheal disease in many animal species. Studies in the late 1990s identified E. coli 086:K61 as the cause of mortality in a variety of wild birds, and in this study, 34 E. coli 086:K61 isolates were examined. All of the isolates were nonmotile, but most elaborated at least two morphologically distinct surface appendages that were confirmed to be type I and curli fimbriae. Thirty-three isolates were positive for the eaeA gene encoding a gamma type of intimin. No phenotypic or genotypic evidence was obtained for elaboration of Shiga-like toxins, but most isolates possessed the gene coding for the cytolethal distending toxin. Five isolates were selected for adherence assays performed with tissue explants and HEp-2 cells, and four of these strains produced attaching and effacing lesions on HEp-2 cells and invaded the cells, as determined by transmission electron microscopy. Two of the five isolates were inoculated orally into 1-day-old specific-pathogen-free chicks, and both of these isolates colonized, invaded, and persisted well in this model. Neither isolate produced attaching and effacing lesions in chicks, although some pathology was evident in the alimentary tract. No deaths were recorded in inoculated chicks. These findings are discussed in light of the possibility that wild birds are potential zoonotic reservoirs of attaching and effacing E. coli.

Functional genomics in zebrafish permits rapid characterization of novel platelet membrane proteins

In this study, we demonstrate the suitability of the vertebrate Danio rerio (zebrafish) for functional screening of novel platelet genes in vivo by reverse genetics. Comparative transcript analysis of platelets and their precursor cell, the megakaryocyte, together with nucleated blood cell elements, endothelial cells, and erythroblasts, identified novel platelet membrane proteins with hitherto unknown roles in thrombus formation. We determined the phenotype induced by antisense morpholino oligonucleotide (MO)–based knockdown of 5 of these genes in a laser-induced arterial thrombosis model. To validate the model, the genes for platelet glycoprotein (GP) IIb and the coagulation protein factor VIII were targeted. MO-injected fish showed normal thrombus initiation but severely impaired thrombus growth, consistent with the mouse knockout phenotypes, and concomitant knockdown of both resulted in spontaneous bleeding. Knockdown of 4 of the 5 novel platelet proteins altered arterial thrombosis, as demonstrated by modified kinetics of thrombus initiation and/or development. We identified a putative role for BAMBI and LRRC32 in promotion and DCBLD2 and ESAM in inhibition of thrombus formation. We conclude that phenotypic analysis of MO-injected zebrafish is a fast and powerful method for initial screening of novel platelet proteins for function in thrombosis.

Characterization of some Actinomyces-like isolates from human clinical sources: description of Varibaculum cambriensis gen. nov., sp nov

Fifteen strains of an anaerobic, catalase-negative, gram-positive diphtheroid-shaped bacterium recovered from human sources were characterized by phenotypic and molecular chemical and molecular genetic methods. The unidentified bacterium showed some resemblance to Actinomyces species and related taxa, but biochemical testing, polyacrylamide gel electrophoresis analysis of whole-cell proteins, and amplified 16S ribosomal DNA restriction analysis indicated the strains were distinct from all currently named Actinomyces species and related taxa. Comparative 16S rRNA gene sequencing studies showed that the bacterium represents a hitherto-unknown phylogenetic line that is related to but distinct from Actinomyces, Actinobaculum, Arcanobacterium, and Mobiluncus. We propose, on the basis of phenotypic and phylogenetic evidence, that the unknown bacterium from human clinical specimens should be classified as a new genus and species, Varibaculum cambriensis gen. nov., sp. nov. The type strain of Varibaculum cambriensis sp. nov. is CCUG 44998(T) = CIP 107344(T).

Characterization of some strains from human clinical sources which resemble “Leptotrichia sanguinegens”: description of Sneathia sanguinegens sp. nov., gen. nov.

Three strains of a gram-negative, blood or serum requiring, rod-shaped bacterium recovered from human clinical specimens were characterised by phenotypic and molecular taxonomic methods. Comparative 16S rRNA gene sequencing showed the unknown rod-shaped strains are members of the same species as some fastidious isolates recovered from human blood specimens and previously designated "Leptotrichia sanguinegens". Based on phylogenetic and phenotypic evidence, it is proposed that the isolates from human sources be classified in a new genus Sneathia, as Sneathia sanguinegens gen. nov., sp. nov. The type strain of Sneathia sanguinegens is CCUG 41628T.

Characterization of a Gemella-like organism isolated from an abscess of a rabbit: description of Gemella cunicula sp. nov.

An unknown Gram-positive, catalase-negative, ovoid-shaped bacterium isolated from the submandibular abscess of a rabbit was subjected to a polyphasic taxonomic analysis. Comparative 16S rRNA gene sequencing demonstrated the unknown coccus represents a new subline within the genus Gemella. The unknown isolate was readily distinguished from other recognized members of the genus Gemella, namely Gemella haemolysans, Gemella bergeri, Gemella morbillorum, Gemella palaticanis and Gemella sanguinis, by biochemical tests and electrophoretic analysis of whole-cell proteins. Based on both phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium is classified in the genus Gemella as Gemella cuniculi sp. nov. The type strain is CCUG 42726T.

Characterization of Actinomyces isolates from infected root canals of teeth: description of Actinomyces radicidentis sp. nov.

Two strains of a previously undescribed Actinomyces-like bacterium were recovered in pure culture from infected root canals of teeth. Analysis by biochemical testing and polyacrylamide gel electrophoresis of whole-cell proteins indicated that the strains closely resembled each other phenotypically but were distinct from previously described Actinomyces and Arcanobacterium species. Comparative 16S rRNA gene-sequencing studies showed the bacterium to be a hitherto unknown subline within a group of Actinomyces species which includes Actinomyces bovis, the type species of the genus. Based on phylogenetic and phenotypic evidence, we propose that the unknown bacterium isolated from human clinical specimens be classified as Actinomyces radicidentis sp. nov. The type strain of Actinomyces radicidentis is CCUG 36733.

Characterization of Actinomyces isolates from samples from the human urogenital tract: description of Actinomyces urogenitalis sp. nov.

Three strains of a previously undescribed Actinomyces-like bacterium were isolated from human clinical sources (urine, urethra and vaginal secretion). Biochemical testing and PAGE analysis of whole-cell proteins indicated that the strains were phenotypically homogeneous and distinct from previously described Actinomyces and Arcanobacterium species. Comparative 16S rRNA gene sequencing studies showed the bacterium to be a hitherto unknown subline within a group of Actinomyces species which includes Actinomyces bovis, the type species of the genus. Based on phylogenetic and phenotypic evidence it is proposed that the unknown bacterium from humans be classified as Actinomyces urogenitalis sp. nov. The type strain of Actinomyces urogenitalis is CCUG 38702T (= CIP 106421T).