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PubMed2026

Identification and in-depth characterization of clinical isolates of Peribacillus frigoritolerans.

UNLABELLED: Peribacillus frigoritolerans is a bacterial species commonly found in the environment and used as a plant-growth promoter and biocontrol agent in agriculture. Recent evidence has proven that Peribacillus spp. are also able to cause severe infections in humans, thus emerging as new human pathogens. In this study, for the first time, 10 P. frigoritolerans strains were isolated from human samples (both superficial and sterile deep body sites) and characterized in terms of morphology, lifestyle, genetics, and virulence. The molecular identification by MALDI-TOF mass spectrometry and 16S rRNA gene sequencing was inconclusive, while whole-genome sequencing was effective in properly identifying isolates within the species P. frigoritolerans. The pangenome analysis provided an overview of the virulence potential of P. frigoritolerans, revealing the presence of genes involved in antibiotic resistance and toxin/exoenzyme production. Phenotypically, the strains displayed different features and behaviors, indicating strain-specific properties and high intra-species variability. A part of the strains exhibited virulence factors, being able to swim and swarm, form biofilms, and produce enzymes and toxins. Antibiotic susceptibility testing revealed resistance to ampicillin for all strains and resistance to erythromycin and clindamycin for some of them. Antimicrobial activity against Gram-positive bacteria and fungi was demonstrated, further corroborating the presence of putative bacteriocin/antimicrobial peptide-encoding genes. An association between the overall virulence potential and infection site/severity was hypothesized. Altogether, these findings highlight the extreme diversity within the species, reveal the strain-dependent pathogenic potential of P. frigoritolerans, and support its role as a candidate human pathogen. IMPORTANCE: This study provides insights into the infectious role of Peribacillus frigoritolerans, an almost unknown bacterial species with agrobiotechnological potential but no history of human infections. This is the first report of P. frigoritolerans isolation from human clinical samples. Ten P. frigorit-olerans strains were herein characterized for their morphology, lifestyle, genetics, and virulence, highlighting an extreme intra-species variability and the potential to act as pathogens in humans. Importantly, this study points out the need for unconventional methods for proper identification of this species, since traditional techniques result inconclusive. Resistance to commonly prescribed antibiotics was also evidenced, confirming the importance of antimicrobial testing on clinical iso-lates. This study lays the foundation for a more in-depth characterization of Peribacillus spp. in the clinical context.

Humans↗
PubMed2024

Dual diagnosis of achondroplasia and mandibulofacial dysostosis with microcephaly.

BACKGROUND: Achondroplasia and mandibulofacial dysostosis with microcephaly (MFDM) are rare monogenic, dominant disorders, caused by gain-of-function fibroblast growth factor receptor 3 (FGFR3) gene variants and loss-of-function elongation factor Tu GTP binding domain-containing 2 (EFTUD2) gene variants, respectively. The coexistence of two distinct Mendelian disorders in a single individual is uncommon and challenges the traditional paradigm of a single genetic disorder explaining a patient's symptoms, opening new avenues for diagnosis and management. CASE PRESENTATION: We present a case of a female patient initially diagnosed with achondroplasia due to a maternally inherited pathogenic FGFR3 variant. She was referred to our genetic department due to her unusually small head circumference and short stature, which were both significantly below the expected range for achondroplasia. Additional features included distinctive facial characteristics, significant speech delay, conductive hearing loss, and epilepsy. Given the complexity of her phenotype, she was recruited to the DDD (Deciphering Developmental Disorders) study and the 100,000 Genomes project for further investigation. Subsequent identification of a complex EFTUD2 intragenic rearrangement confirmed an additional diagnosis of mandibulofacial dysostosis with microcephaly (MFDM). CONCLUSION: This report presents the first case of a dual molecular diagnosis of achondroplasia and mandibulofacial dysostosis with microcephaly in the same patient. This case underscores the complexity of genetic diagnoses and the potential for coexistence of multiple genetic syndromes in a single patient. This case expands our understanding of the molecular basis of dual Mendelian disorders and highlights the importance of considering the possibility of dual molecular diagnoses in patients with phenotypic features that are not fully accounted for by their primary diagnosis.

Humans↗
PubMed2003

Phylogenetic analyses among octocorals (Cnidaria): mitochondrial and nuclear DNA sequences (lsu-rRNA, 16S and ssu-rRNA, 18S) support two convergent clades of branching gorgonians.

Gorgonian octocorals lack corroborated hypotheses of phylogeny. This study reconstructs genealogical relationships among some octocoral species based on published DNA sequences from the large ribosomal subunit of the mitochondrial RNA (lsu-rRNA, 16S: 524bp and 21 species) and the small subunit of the nuclear RNA (ssu-rRNA, 18S: 1815bp and 13 spp) using information from insertions-deletions (INDELS) and the predicted secondary structure of the lsu-rRNA (16S). There were seven short (3-10bp) INDELS in the 18S with consistent phylogenetic information. The INDELS in the 16S corresponded to informative signature sequences homologous to the G13 helix found in Escherichia coli. We found two main groups of gorgonian octocorals using a maximum parsimony analysis of the two genes. One group corresponds to deep-water taxa including species from the suborders Calcaxonia and Scleraxonia characterized by an enlargement of the G13 helix. The second group has species from Alcyoniina, Holaxonia and again Scleraxonia characterized by insertions in the 18S. Gorgonian corals, branching colonies with a gorgonin-containing flexible multilayered axis (Holaxonia and Calcaxonia), do not form a monophyletic group. These corroborated results from maternally inherited (16S) and biparentally inherited (18S) genes support a hypothesis of independent evolution of branching in the two octocoral clades.

Animals↗
PubMed2000

Phylogeography and population structure of an ecotonal marsupial, Bettongia tropica, determined using mtDNA and microsatellites.

The northern bettong, Bettongia tropica, is an endangered species of Potoroidae with a restricted distribution in the wet tropics of north Queensland, Australia. The species is only found within a thin strip of sclerophyll forest along the western margin of rainforest. This tight association with rainforest boundaries is predicted to have resulted in population isolation as rainforest contracted during the Pleistocene, though some have proposed that the northern bettong was not present in the wet tropics until the late Pleistocene. The dispersal ability of the species, and of the family, is not known. This study examined gene flow among populations within areas of continuous habitat complemented by a broader analysis of phylogeography. Individuals trapped at each of the four known regions (one region was subsampled at three different sites), were sequenced for 547 base pairs of the mitochondrial DNA (mtDNA) control region and typed for seven microsatellite loci. The mtDNA phylogeny showed congruence with a biogeographical hypothesis, a relatively deep split suggesting historical isolation in separate northern and southern refugia. The two divergent clades were both present within the Lamb Range, indicating an expansion from these refuges and subsequent admixture at one site. mtDNA allele frequencies indicated relatively limited gene flow within the Lamb Range over distances as short as nine km. Tests of population divergence using microsatellites (FST and assignment tests) strongly supported this result. A molecular signal indicative of a recent bottleneck was unexpectedly detected in one of the Lamb Range subpopulations. This lead us to examine the behaviour of the statistics used in this bottleneck test under a linear stepping-stone model with varying migration rates. We found that it may be more difficult to detect molecular signatures for recent bottlenecks under conditions of very low migration rates than for isolated populations and, conversely, that 'false' bottleneck signatures may be observed at higher migration rates. The Lamb Range FST estimate clearly fell within the category of potentially 'false' bottleneck signals. Despite relatively limited gene flow, evidence for asymmetric dispersal suggests more complicated population dynamics than a simple linear stepping-stone model.

Animals↗
PubMed2006

Expression and variability of molecular chaperones in the sugarcane expressome.

Molecular chaperones perform folding assistance in newly synthesized polypeptides preventing aggregation processes, recovering proteins from aggregates, among other important cellular functions. Thus their study presents great biotechnological importance. The present work discusses the mining for chaperone-related sequences within the sugarcane EST genome project database, which resulted in approximately 300 different sequences. Since molecular chaperones are highly conserved in most organisms studied so far, the number of sequences related to these proteins in sugarcane was very similar to the number found in the Arabidopsis thaliana genome. The Hsp70 family was the main molecular chaperone system present in the sugarcane expressome. However, many other relevant molecular chaperones systems were also present. A digital RNA blot analysis showed that 5'ESTs from all molecular chaperones were found in every sugarcane library, despite their heterogeneous expression profiles. The results presented here suggest the importance of molecular chaperones to polypeptide metabolism in sugarcane cells, based on their abundance and variability. Finally, these data have being used to guide more in deep analysis, permitting the choice of specific targets to study.

Arabidopsis↗
PubMed2006

The origin and diversification of eukaryotes: problems with molecular phylogenetics and molecular clock estimation.

Determining the relationships among and divergence times for the major eukaryotic lineages remains one of the most important and controversial outstanding problems in evolutionary biology. The sequencing and phylogenetic analyses of ribosomal RNA (rRNA) genes led to the first nearly comprehensive phylogenies of eukaryotes in the late 1980s, and supported a view where cellular complexity was acquired during the divergence of extant unicellular eukaryote lineages. More recently, however, refinements in analytical methods coupled with the availability of many additional genes for phylogenetic analysis showed that much of the deep structure of early rRNA trees was artefactual. Recent phylogenetic analyses of a multiple genes and the discovery of important molecular and ultrastructural phylogenetic characters have resolved eukaryotic diversity into six major hypothetical groups. Yet relationships among these groups remain poorly understood because of saturation of sequence changes on the billion-year time-scale, possible rapid radiations of major lineages, phylogenetic artefacts and endosymbiotic or lateral gene transfer among eukaryotes. Estimating the divergence dates between the major eukaryote lineages using molecular analyses is even more difficult than phylogenetic estimation. Error in such analyses comes from a myriad of sources including: (i) calibration fossil dates, (ii) the assumed phylogenetic tree, (iii) the nucleotide or amino acid substitution model, (iv) substitution number (branch length) estimates, (v) the model of how rates of evolution change over the tree, (vi) error inherent in the time estimates for a given model and (vii) how multiple gene data are treated. By reanalysing datasets from recently published molecular clock studies, we show that when errors from these various sources are properly accounted for, the confidence intervals on inferred dates can be very large. Furthermore, estimated dates of divergence vary hugely depending on the methods used and their assumptions. Accurate dating of divergence times among the major eukaryote lineages will require a robust tree of eukaryotes, a much richer Proterozoic fossil record of microbial eukaryotes assignable to extant groups for calibration, more sophisticated relaxed molecular clock methods and many more genes sampled from the full diversity of microbial eukaryotes.

Eukaryotic Cells↗
PubMed2001

Structure of the Rho-activating domain of Escherichia coli cytotoxic necrotizing factor 1.

Certain uropathogenic and neonatal meningitis-causing strains of Escherichia coli express a 114 kDa protein toxin called cytotoxic necrotizing factor 1 (CNF1). The toxin causes alteration of the host cell actin cytoskeleton and promotes bacterial invasion of blood-brain barrier endothelial cells. CNF1 belongs to a unique group of large cytotoxins that cause constitutive activation of Rho guanosine triphosphatases (GTPases), which are key regulators of the actin cytoskeleton. This group also includes E. coli cytotoxic necrotizing factor 2 (CNF2, 114 kDa) and dermonecrotic toxins (DNT, 159 kDa) of Bordetella spp. with related sequences occurring in Yersinia spp. Here we show that the catalytic region of CNF1 exhibits a novel protein fold as determined by its 1.83 A resolution crystal structure. The structure reveals that CNF1 has a Cys-His-main chain oxygen catalytic triad reminiscent of enzymes belonging to the catalytic triad superfamily. The position of the catalytic Cys residue at the base of a deep pocket restricts access to potential substrates and helps explain the high specificity of this and related toxins.

Amino Acid Sequence↗
PubMed2005

Public health importance of Brachiola algerae (Microsporidia)--an emerging pathogen of humans.

Brachiola algerae, a parasite of Anopheles mosquitoes, has also been isolated from a human cornea, a cutaneous nodule and deep muscle tissue. All three human isolates of B. algerae are morphologically, serologically, and genetically similar to the mosquito-derived isolates including the original isolate of Vavra and Undeen. All of these isolates grew well in mammalian cell cultures at 37 degrees C and produced spores. Transmission electron microscopy revealed that all developmental stages including meronts, sporoblasts and spores were diplokaryotic and developed in direct contact with the host cell cytoplasm, a feature characteristic of the genus Brachiola. Spores of all isolates reacted well, in the immunofluorescence assay, with the rabbit anti-B. algerae serum. In the immunoblot assay, although the overall banding patterns of the human and mosquito isolates were similar, minor differences could be discerned. Sequencing of the PCR products of the amplified SSU rRNA gene revealed the existence of two distinct genotypes; the original mosquito (Undeen) isolate belonged to genotype 1 and the isolate from cornea and that from the deep muscle biopsy to genotype 2, whereas the isolates from a mosquito and one of the other two human isolates (one from skin abscess) had both genotypes, 1 and 2. It is known that spores of mosquito-derived B. algerae can not only proliferate in mammalian cell cultures at 37 degrees C but also can infect mice when injected into footpads or deposited on the corneal surface. These observations indicate that the spores have potential to be a risk factor for humans, especially those with immunodeficiency.

Animals↗
PubMed1991

[Atrial natriuretic factor: one of the mechanisms of action of the phlebology bath at Barbotan].

UNLABELLED: The effect of thermal baths on oedema of the lower limbs might be explained by physical mechanisms of hydrostatic pressure resulting from the use of a deep bath and a centripetal underwater jet, by which the veins and lymph ducts are drained every day. The purpose of this experiment is to demonstrate the existence of hormonal mechanisms which would account for the diuretic effect of thermal baths. One of the effects observed with hydrotherapy is the physiological diuresis that follows each bath, this diuresis would appear to depend at least in part on the atrial natriuretic factor (ANF). The criteria by which assessment can be made essentially biological: ANF level and its biological effects on blood and urine; aldosterone level; plasma renin activity (PRA); creatinine clearance; hematocrit; proteinemia; and blood and urine electrolyte balance. The inclusion criteria are: subjects selected at random and willing cooperate. The criteria for exclusion are disease states which modify ANF kinesis: congestive heart failure, cardiac rhythm disorders, decompensated cirrhosis of liver, obesity, treatment antihypertensive drugs. METHODS: Thirty patients were put through the same experimental sequence, as follows: emptying of the bladder and ingestion of 200 cc of water; seated rest fort 30 mn, after which (to): blood sample; urine sample; ingestion of 200 cc of water; deep bath for 20 mn, i.e. the basic hydro treatment in phlebology at Barbotan. The deep bath is specific to Barbotan and the patient is subjected to maximum immersion in water at a mesothermal temperature of 34.5 degrees C, followed by (t1): blood and urine sample; ingestion of 200 cc of water; supine rest for 90 mn, followed by (t2): blood and urine sample. RESULTS: Data from twenty-eight patients were usable. In this protocol, we use variance analysis with repeated measurements and a 95% confidence limit. The mean value of the principal parameters studies are set out in the following table; these value are accompanied by the degree of significance of the modification at (t1) and (t2). Our experimentation with thirty patients showed that the big thermal bath at Barbotan produces a highly significant increase in ANF secretion, resulting in the diuresis observed after the use of the bath. The antagonist effect of AFN on the renin--angiotensin-aldosterone system was corroborated: we found decreased aldosterone, PRA and creatinine clearance, and increased diuresis and natriuresis. The renal and cardiovascular effects observed after extended immersion in the Barbotan bath (increased diuresis, tachycardia and hypotension, transitory venous vasoplegia and ephemeral vasodilatation of the surface capillaries) are the result of increased ANF secretion. [formula: see text] Supine rest immediately after the bath is essential. This sustains the enhanced ANF and thus reinforces its renal effects, while reducing adverse cardiovascular effects such as the orthostatic hypotension and venous vasoplegia that are normally observed after use of the bath. Moreover, by reducing venular and lymphatic pressure, clinostatism facilitate interstitial to intravascular tissue fluid exchanges and thus helps to drain oedema from the legs. It is striking to note that the hydrotherapy prescribed at Barbotan les Thermes has always included the three most potent factors for ANF release: deep immersion in the big bath, immediate supine rest, and walking. Physiological diuresis has thus been induced empirically as an essential part of the treatment of lower limb phlebopathies.

Aldosterone↗
PubMed2001

Aspergillus nidulans DigA, a potential homolog of Saccharomyces cerevisiae Pep3 (Vps18), is required for nuclear migration, mitochondrial morphology and polarized growth.

The fungal vacuole is an acidic organelle that is involved in a variety of physiological processes, such as protein turnover, ion and pH homeostasis and osmoregulation. The function of the vacuole largely depends on vesicle transport providing the organelle with enzymes and substrates. The process of vesicle transportation has been studied best in Saccharomyces cerevisiae, where several proteins that are crucial for intracellular vesicle sorting have been identified. One such protein is Pep3 (Vps18). In pep3 mutants vacuole function and vacuole morphology are affected. We cloned the gene for a potential homolog of Pep3 from the filamentous fungus Aspergillus nidulans. The gene, digA (for dichotomous growth), was identified in a screen for nuclear migration mutants. A. nidulans digA encodes a protein of 108.3 kDa, which includes a 122-amino acid clathrin repeat motif, two short coiled-coil regions, and a RING finger Zn-binding motif at the C-terminus. All three sequence motifs suggest interaction of DigA with other proteins. DigA is 25% identical to a homolog from Schizosaccharomyces pombe, 23% to a protein from human, 21% to the product of the Drosophila melanogaster gene deep orange (dor) and 18% to S. cerevisiae Pep3 (Vps18). We localized DigA as a HA-epitope-tagged protein in the cytoplasm of A. nidulans vegetative cells, by secondary immunofluorescence. A digA mutant displays a pleiotropic phenotype with clustered mitochondria, clustered nuclei and a defect in polarization of the actin cytoskeleton. These results suggest that vacuolar functions are required for organelle positioning and polarized growth.

Adaptor Proteins, Vesicular Transport↗
PubMed1999

Taxonomic studies of extremely barophilic bacteria isolated from the Mariana Trench and description of Moritella yayanosii sp. nov., a new barophilic bacterial isolate.

We have isolated two strains of extremely barophilic bacteria from sediment collected from the world's deepest ocean floor in the Mariana Trench, Challenger Deep, at a depth of 10898m [Kato C, Li L, Nogi Y, Nakamura Y, Tamaoka J, Horikoshi K (1998) Appl Environ Microbiol 64:1510-1513]. One strain, DB21MT-2, was identified as a strain of Shewanella benthica, and the other strain, DB21MT-5, is closely affiliated with members of the genus Moritella on the basis of 16S rDNA sequence analysis. The hybridization values for DNA-DNA relatedness between DB21MT-5 and the Moritella reference strains were significantly lower than that accepted as the phylogenetic definition of a species. Based on this and other taxonomic differences, strain DB21MT-5 appears to represent a novel obligately barophilic deep-sea Moritella species. The name Moritella yaynanosii (JCM 10263) is proposed. This is the first proposed species of obligately barophilic bacteria of the genus Moritella.

Bacteria↗
PubMed1999

Appearance of normal brain maturation on fluid-attenuated inversion-recovery (FLAIR) MR images.

BACKGROUND AND PURPOSE: Fluid-attenuated inversion-recovery (FLAIR) MR imaging is widely accepted for brain diagnoses, though to our knowledge no description of the MR FLAIR appearance of the normal infantile brain has been published. The purpose of this study was to investigate the appearance of normal infantile brain maturation on FLAIR MR images. METHODS: FLAIR images were obtained in 52 children between the ages of 1 day and 4 years who had clinically suspected brain disease but no neurologic abnormality or growth retardation. T1- and T2-weighted images were also obtained in all the children, and these images were compared with the FLAIR sequences for the appearance of brain maturation. A grading system for the differences in signal intensity between gray and white matter on FLAIR images was introduced to make detailed profiles of maturation in each brain region, including the posterior limb of the internal capsule, the cerebellar peduncle, the frontal deep white matter, the occipital deep white matter, and the centrum semiovale. These grades were plotted against patients' ages. RESULTS: On the FLAIR images, the myelinated white matter, including the cerebellar peduncle and the posterior limb of the internal capsule, showed high signal intensity relative to gray matter at birth. Thereafter, the white matter lost signal intensity with time and showed low signal intensity at 50 weeks and beyond. The unmyelinated white matter, including the frontal deep white matter, the occipital deep white matter, and the centrum semiovale, showed low signal intensity at birth. The white matter showed high signal intensity at 20 to 30 weeks, and low signal intensity again at 100 to 160 weeks and after. CONCLUSION: The dynamics of brain myelination can be accurately delineated and evaluated on FLAIR images without other spin-echo (SE) sequences. The FLAIR appearance of infantile white matter can be divided into two phases, reflecting development of the myelination process: the first phase is similar to that seen on SE T1-weighted images and the second phase is similar to that seen on SE T2-weighted images.

Brain↗
PubMed2025

Unraveling the complex genetic landscape of OTOF-related hearing loss: a deep dive into cryptic variants and haplotype phasing.

BACKGROUND: Pathogenic variants in OTOF are a major cause of auditory synaptopathy. However, challenges remain in interpreting OTOF variants, including difficulties in confirming haplotype phasing using traditional short-read sequencing (SRS) due to the large gene size, the potential incomplete penetrance of certain variants, and difficulties in assessing variants at non-canonical splice sites. This study aims to revisit the genetic landscape of OTOF variants in a Taiwanese non-syndromic auditory neuropathy spectrum disorder (ANSD) cohort using a combination of sequencing technologies, predictive tools, and experimental validations. METHODS: We performed SRS to analyze OTOF variants in 65 unrelated Taiwanese patients diagnosed with non-syndromic ANSD, complemented by long-read sequencing (LRS) for haplotype phasing. A prediction-to-validation pipeline was implemented to assess the pathogenicity of cryptic variants using SpliceAI software and minigene assays. RESULTS: Biallelic pathogenic OTOF variants were identified in 33 patients (50.8%), while monoallelic variants were found in five patients. Three novel variants, c.3864G > A (p.Ala1288 =), c.4501G > A (p.Ala1501Thr), and c.5813 + 2T > C, were detected. The pathogenicity of two non-canonical mis-splicing variants, c.3894 + 5G > C and c.3864G > A (p.Ala1288 =), was confirmed by minigene assays. LRS-based haplotype phasing revealed that the common missense variant c.5098G > C (p.Glu1700Gln) and the novel variant c.5975A > G (p.Lys1992Arg) are in cis and form a founder pathogenic allele in the Taiwanese population. CONCLUSIONS: Our study highlights the genetic heterogeneity of DFNB9 and emphasizes the importance of population-specific variant interpretation. The integration of advanced sequencing technologies, predictive algorithms, and functional validation assays will improve the accuracy of molecular diagnosis and inform personalized treatment strategies for individuals with DFNB9.

Humans↗
PubMed1996

Isolation of Geobacter species from diverse sedimentary environments.

In an attempt to better understand the microorganisms responsible for Fe(III) reduction in sedimentary environments, Fe(III)-reducing microorganisms were enriched for and isolated from freshwater aquatic sediments, a pristine deep aquifer, and a petroleum-contaminated shallow aquifer. Enrichments were initiated with acetate or toluene as the electron donor and Fe(III) as the electron acceptor. Isolations were made with acetate or benzoate. Five new strains which could obtain energy for growth by dissimilatory Fe(III) reduction were isolated. All five isolates are gram-negative strict anaerobes which grow with acetate as the electron donor and Fe(III) as the electron acceptor. Analysis of the 16S rRNA sequence of the isolated organisms demonstrated that they all belonged to the genus Geobacter in the delta subdivision of the Proteobacteria. Unlike the type strain, Geobacter metallireducens, three of the five isolates could use H2 as an electron donor for Fe(III) reduction. The deep subsurface isolate is the first Fe(III) reducer shown to completely oxidize lactate to carbon dioxide, while one of the freshwater sediment isolates is only the second Fe(III) reducer known that can oxidize toluene. The isolation of these organisms demonstrates that Geobacter species are widely distributed in a diversity of sedimentary environments in which Fe(III) reduction is an important process.

Base Sequence↗
PubMed2025

Whole-genome sequencing, as a powerful diagnostic tool in hearing loss, reveals novel variants in PTPRQ missed by whole-exome sequencing.

BACKGROUND/OBJECTIVES: Hearing loss (HL) is one of the most common congenital disorders, affecting 1-2 in 1,000 newborns. Modern genetic diagnostics using large gene panels and/or whole exome analysis (WES) can identify disease-causing mutations in 25-50 % of patients, with higher solve rates in individuals with earlier onset. RESULTS: Here, we used whole-genome sequencing (WGS) to reanalyze 14 index patients/families who remained without genetic diagnosis by WES. We were able to identify the genetic cause of HL in 6 families (43 %). Two families were diagnosed with DFNB84A caused by compound heterozygous recessive mutations in PTPRQ. Three of the four underlying variants, including a structural variant, a deep intronic variant, and a splice variant, escaped detection by WES. Minigene assays confirmed the pathogenicity of the intronic and the splice variants. In addition, we used protein 3D structure prediction and rigid ligand docking to study the pathogenicity of variants that escape nonsense-mediated decay. CONCLUSION: In our study, we present four novel variants in PTPRQ, three of which were detected only by WGS. To our knowledge, we report here the first pathogenic deep intronic PTPRQ variant causing HL. Our results suggest that the mutational spectrum of PTPRQ is not well covered by standard WES and that PTPRQ-associated hearing loss may be more frequent than previously thought. WGS provides an additional layer of information in the diagnostics of HL.

Humans↗
PubMed1991

Engineered interdomain disulfide in the periplasmic receptor for sulfate transport reduces flexibility. Site-directed mutagenesis and ligand-binding studies.

Using recombinant DNA techniques, an Escherichia coli periplasmic sulfate receptor or sulfate-binding protein involved in active transport has been overexpressed and characterized. This protein is essentially identical in size, sequence, antigenicity, and ligand affinity and specificity to the sulfate receptor from Salmonella typhimurium whose crystal structure has been refined at 2 A resolution. The dehydrated sulfate is bound in the deep cleft between the two lobes of the bilobate protein. Using the structure of the S. typhimurium as a guide, three site-directed mutants (Ser129Cys, Gly46Cys, and Ser129Cys/Gly46Cys) have been made. In the Cys129/Cys46 mutant the disulfide has been successfully introduced across the opening of the ligand-binding site cleft of the E. coli sulfate-binding protein. The dissociation of sulfate from the double mutant protein is very slow under oxidizing conditions and increases more than 200-fold when reducing agent is added. This effect is attributed to a loss of interdomain structural flexibility in the presence of the disulfide, and underscores the importance of protein conformational change in binding protein function.

Amino Acid Sequence↗
PubMed2006

Deep and comparative analysis of the mycelium and appressorium transcriptomes of Magnaporthe grisea using MPSS, RL-SAGE, and oligoarray methods.

BACKGROUND: Rice blast, caused by the fungal pathogen Magnaporthe grisea, is a devastating disease causing tremendous yield loss in rice production. The public availability of the complete genome sequence of M. grisea provides ample opportunities to understand the molecular mechanism of its pathogenesis on rice plants at the transcriptome level. To identify all the expressed genes encoded in the fungal genome, we have analyzed the mycelium and appressorium transcriptomes using massively parallel signature sequencing (MPSS), robust-long serial analysis of gene expression (RL-SAGE) and oligoarray methods. RESULTS: The MPSS analyses identified 12,531 and 12,927 distinct significant tags from mycelia and appressoria, respectively, while the RL-SAGE analysis identified 16,580 distinct significant tags from the mycelial library. When matching these 12,531 mycelial and 12,927 appressorial significant tags to the annotated CDS, 500 bp upstream and 500 bp downstream of CDS, 6,735 unique genes in mycelia and 7,686 unique genes in appressoria were identified. A total of 7,135 mycelium-specific and 7,531 appressorium-specific significant MPSS tags were identified, which correspond to 2,088 and 1,784 annotated genes, respectively, when matching to the same set of reference sequences. Nearly 85% of the significant MPSS tags from mycelia and appressoria and 65% of the significant tags from the RL-SAGE mycelium library matched to the M. grisea genome. MPSS and RL-SAGE methods supported the expression of more than 9,000 genes, representing over 80% of the predicted genes in M. grisea. About 40% of the MPSS tags and 55% of the RL-SAGE tags represent novel transcripts since they had no matches in the existing M. grisea EST collections. Over 19% of the annotated genes were found to produce both sense and antisense tags in the protein-coding region. The oligoarray analysis identified the expression of 3,793 mycelium-specific and 4,652 appressorium-specific genes. A total of 2,430 mycelial genes and 1,886 appressorial genes were identified by both MPSS and oligoarray. CONCLUSION: The comprehensive and deep transcriptome analysis by MPSS and RL-SAGE methods identified many novel sense and antisense transcripts in the M. grisea genome at two important growth stages. The differentially expressed transcripts that were identified, especially those specifically expressed in appressoria, represent a genomic resource useful for gaining a better understanding of the molecular basis of M. grisea pathogenicity. Further analysis of the novel antisense transcripts will provide new insights into the regulation and function of these genes in fungal growth, development and pathogenesis in the host plants.

DNA, Fungal↗
PubMed2006

The crystal structure of the transthyretin-like protein from Salmonella dublin, a prokaryote 5-hydroxyisourate hydrolase.

The mechanism of binding of thyroid hormones by the transport protein transthyretin (TTR) in vertebrates is structurally well characterised. However, a homologous family of transthyretin-like proteins (TLPs) present in bacteria as well as eukaryotes do not bind thyroid hormones, instead they are postulated to perform a role in the purine degradation pathway and function as 5-hydroxyisourate hydrolases. Here we describe the 2.5 Angstroms X-ray crystal structure of the TLP from the Gram-negative bacterium Salmonella dublin, and compare and contrast its structure with vertebrate TTRs. The overall architecture of the homotetramer is conserved and, despite low sequence homology with vertebrate TTRs, structural differences within the monomer are restricted to flexible loop regions. However, sequence variation at the dimer-dimer interface has profound consequences for the ligand binding site and provides a structural rationalisation for the absence of thyroid hormone binding affinity in bacterial TLPs: the deep, negatively charged thyroxine-binding pocket that characterises vertebrate TTR contrasts with a shallow and elongated, positively charged cleft in S. dublin TLP. We have demonstrated that Sdu_TLP is a 5-hydroxyisourate hydrolase. Furthermore, using site-directed mutagenesis, we have identified three conserved residues located in this cleft that are critical to the enzyme activity. Together our data reveal that the active site of Sdu_TLP corresponds to the thyroxine binding site in TTRs.

Amidohydrolases↗