Metabolic trade-offs constrain the cell size ratio in a nitrogen-fixing symbiosis.

Biological dinitrogen (N2) fixation is a key metabolic process exclusively performed by prokaryotes, some of which are symbiotic with eukaryotes. Species of the marine haptophyte algae Braarudosphaera bigelowii harbor the N2-fixing endosymbiotic cyanobacteria UCYN-A, which might be evolving organelle-like characteristics. We found that the size ratio between UCYN-A and their hosts is strikingly conserved across sublineages/species, which is consistent with the size relationships of organelles in this symbiosis and other species. Metabolic modeling showed that this size relationship maximizes the coordinated growth rate based on trade-offs between resource acquisition and exchange. Our findings show that the size relationships of N2-fixing endosymbionts and organelles in unicellular eukaryotes are constrained by predictable metabolic underpinnings and that UCYN-A is, in many regards, functioning like a hypothetical N2-fixing organelle (or nitroplast).

A murine model for the del(GJB6-D13S1830) deletion recapitulating the phenotype of human DFNB1 hearing impairment: generation and functional and histopathological study.

Inherited hearing impairment is a remarkably heterogeneous monogenic condition, involving hundreds of genes, most of them with very small (< 1%) epidemiological contributions. The exception is GJB2, the gene encoding connexin-26 and underlying DFNB1, which is the most frequent type of autosomal recessive non-syndromic hearing impairment (ARNSHI) in most populations (up to 40% of ARNSHI cases). DFNB1 is caused by different types of pathogenic variants in GJB2, but also by large deletions that keep the gene intact but remove an upstream regulatory element that is essential for its expression. Such large deletions, found in most populations, behave as complete loss-of-function variants, usually associated with a profound hearing impairment. By using CRISPR-Cas9 genetic edition, we have generated a murine model (Dfnb1em274) that reproduces the most frequent of those deletions, del(GJB6-D13S1830). Dfnb1em274 homozygous mice are viable, bypassing the embryonic lethality of the Gjb2 knockout, and present a phenotype of profound hearing loss (> 90 dB SPL) that correlates with specific structural abnormalities in the cochlea. We show that Gjb2 expression is nearly abolished and its protein product, Cx26, is nearly absent all throughout the cochlea, unlike previous conditional knockouts in which Gjb2 ablation was not obtained in all cell types. The Dfnb1em274 model recapitulates the clinical presentation of patients harbouring the del(GJB6-D13S1830) variant and thus it is a valuable tool to study the pathological mechanisms of DFNB1 and to assay therapies for this most frequent type of human ARNSHI.

The impact of continuous lenalidomide maintenance treatment on people living with multiple myeloma-a single-centre, qualitative service evaluation study.

PURPOSE: Continuous lenalidomide maintenance treatment after autologous stem cell transplantation delivers improvement in progression free and overall survival among newly diagnosed multiple myeloma patients and has been the standard of care in the UK since March 2021. However, there is scant information about its impact on patients' day-to-day lives. This service evaluation aimed to qualitatively assess patients receiving lenalidomide treatment at a cancer centre in London, in order that the service might better align with needs and expectations of patients. METHODS: We conducted 20 semi-structured interviews among myeloma patients who were on continuous lenalidomide maintenance treatment at a specialist cancer centre in London. Members of the clinical team identified potentially eligible participants to take part, and convenience sampling was used to select 10 male and 10 female patients, median age of 58 (range, 45-71). The median treatment duration was 11 months (range, 1-60 months). Participants were qualitatively interviewed following the same semi-structured interview guide, which was designed to explore patient experience and insights of lenalidomide. Reflexive thematic analysis was used for data analysis. RESULTS: Four overarching themes were as follows: (i) lenalidomide: understanding its role and rationale; (ii) reframing the loss of a treatment-free period to a return to normal life; (iii) the reality of being on lenalidomide: balancing hopes with hurdles; (iv) gratitude and grievances: exploring mixed perceptions of care and communication. Results will be used to enhance clinical services by tailoring communication to better meet patients' preferences when making treatment decisions. CONCLUSION: This study highlights that most patients feel gratitude for being offered continuous lenalidomide and perceive it as alleviating some fears concerning relapse. It reveals variations in side effects in different age groups; younger patients reported no/negligible side effects, whilst several older patients with comorbidities described significant symptom burden, occasionally leading to treatment discontinuation which caused distress at the perceived loss of prolonged remission. Future research should prioritise understanding the unique needs of younger patients living with multiple myeloma.

Genotyping of the rs1800440 Polymorphism in CYP1B1 Gene and the rs9258883 Polymorphism in HLA-B Gene in a Spanish Cohort of 223 Patients with Frontal Fibrosing Alopecia.

The pathogenesis of frontal fibrosing alopecia has been linked to specific genetic variants. CYP1B1 codes for a component of the cytochrome p450 machinery that is involved in the metabolism of xenobiotic oestrogens. The study of the prevalence of polymorphisms in this gene may help to understand their role in the development of frontal fibrosing alopecia. The aim of this study is to describe the frequency of genetic variations in the alleles HLA-B*07:02 and CYP1B1 in patients with frontal fibrosing alopecia. A cross-sectional study was designed to evaluate blood samples from patients with frontal fibrosing alopecia who attended the Dermatology Department at University Hospital Ramón y Cajal (Madrid, Spain), in search of the polymorphisms rs9258883 and rs1800440 in the alleles HLA-B*07:02 and CYP1B1, respectively. A total of 223 patients were included in the study. Among the 83.8% of patients who carried the rs9258883 polymorphism in HLA-B*07:02, 58.7% were heterozygous for this variant and it was not present in 14.8% of the cases. The majority of patients with frontal fibrosing alopecia lacked the protective rs1800440 polymorphism in CYP1B1 (75.2%). This suggests a relevant role of this variant in development of frontal fibrosing alopecia. The genetic approach to this condition might influence patient prognosis and therapy options.

Functional foods modulating inflammation and metabolism in chronic diseases: a systematic review.

Chronic diseases are responsible for approximately 71% global deaths. These are characterized by chronic low-grade inflammation and metabolic alterations. "Functional foods" have been attributed with anti-inflammatory properties, demonstrated in cell lines and murine models; however, studies in humans are inconclusive. The purpose of this systematic review is to identify clinical trials that analyzed changes in inflammatory and metabolic mediators, in response to consumption of specific functional foods. A total of 3581 trials were screened and 88 were included for this review. Foods identified to regulate inflammation included cranberries, grapes, pomegranate, strawberries, wheat, whole grain products, low fat dairy products, yogurt, green tea, cardamom, turmeric, soy foods, almonds, chia seeds, flaxseed, pistachios, algae oil, flaxseed oil and grape seed oil. Clinical trials that focus on a dietary pattern rich in functional foods are necessary to explore if the additive effect of these foods lead to more clinically relevant outcomes.

A Randomized Controlled Trial on the Effect of Local Insulin Glargine on Venous Ulcer Healing.

INTRODUCTION: To determine whether the local administration of insulin glargine compared with placebo in nondiabetic patients with venous ulcers (VUs) leads to increased wound healing. METHODS: A randomized controlled trial using a split-plot design was performed in 36 adults with leg VUs >25 cm2 and more than 3 mo of evolution. Each hemi-wound received either 10 UI insulin glargine or saline solution once a day for 7 d. Size of the wounds, thermal asymmetry, the number of blood vessels, and the percentage area of collagen content in wound biopsies were assessed at baseline and after 7 d of treatment. Blood capillary glucose was monitored once a day after the insulin injection. RESULTS: After 7 d of treatment, the hemi-wounds treated with insulin glargine were significantly smaller, had less thermal asymmetry, more blood vessels, and more collagen content than the saline-treated side. Correlation between thermal asymmetry and the number of blood vessels was also found (r2 = 66.2, P < 0.001). No patient presented capillary glucose levels ≤3.3 mmol/L nor any adverse effects. CONCLUSIONS: In nondiabetic patients with chronic VUs, the topical administration of insulin glargine seems to be safe and promotes wound healing and tissue repair after 7 d of treatment.

Hopanoid lipids may facilitate aerobic nitrogen fixation in the ocean.

Cyanobacterial diazotrophs are considered to be the most important source of fixed N2 in the open ocean. Biological N2 fixation is catalyzed by the extremely O2-sensitive nitrogenase enzyme. In cyanobacteria without specialized N2-fixing cells (heterocysts), mechanisms such as decoupling photosynthesis from N2 fixation in space or time are involved in protecting nitrogenase from the intracellular O2 evolved by photosynthesis. However, it is not known how cyanobacterial cells limit O2 diffusion across their membranes to protect nitrogenase in ambient O2-saturated surface ocean waters. Here, we explored all known genomes of the major marine cyanobacterial lineages for the presence of hopanoid synthesis genes, since hopanoids are a class of lipids that might act as an O2 diffusion barrier. We found that, whereas all non-heterocyst-forming cyanobacterial diazotrophs had hopanoid synthesis genes, none of the marine Synechococcus, Prochlorococcus (non-N2-fixing), and marine heterocyst-forming (N2-fixing) cyanobacteria did. Finally, we conclude that hopanoid-enriched membranes are a conserved trait in non-heterocyst-forming cyanobacterial diazotrophs that might lower the permeability to extracellular O2 This membrane property coupled with high respiration rates to decrease intracellular O2 concentration may therefore explain how non-heterocyst-forming cyanobacterial diazotrophs can fix N2 in the fully oxic surface ocean.

Discovery of New Phenylacetone Monooxygenase Variants for the Development of Substituted Indigoids through Biocatalysis.

Indigoids are natural pigments obtained from plants by ancient cultures. Romans used them mainly as dyes, whereas Asian cultures applied these compounds as treatment agents for several diseases. In the modern era, the chemical industry has made it possible to identify and develop synthetic routes to obtain them from petroleum derivatives. However, these processes require high temperatures and pressures and large amounts of solvents, acids, and alkali agents. Thus, enzyme engineering and the development of bacteria as whole-cell biocatalysts emerges as a promising green alternative to avoid the use of these hazardous materials and consequently prevent toxic waste generation. In this research, we obtained two novel variants of phenylacetone monooxygenase (PAMO) by iterative saturation mutagenesis. Heterologous expression of these two enzymes, called PAMOHPCD and PAMOHPED, in E. coli was serendipitously found to produce indigoids. These interesting results encourage us to characterize the thermal stability and enzyme kinetics of these new variants and to evaluate indigo and indirubin production in a whole-cell system by HPLC. The highest yields were obtained with PAMOHPCD supplemented with L-tryptophan, producing ~3000 mg/L indigo and ~130.0 mg/L indirubin. Additionally, both enzymes could oxidize and produce several indigo derivatives from substituted indoles, with PAMOHPCD being able to produce the well-known Tyrian purple. Our results indicate that the PAMO variants described herein have potential application in the textile, pharmaceutics, and semiconductors industries, prompting the use of environmentally friendly strategies to obtain a diverse variety of indigoids.

Innovative Three-Step Microwave-Promoted Synthesis of N-Propargyltetrahydroquinoline and 1,2,3-Triazole Derivatives as a Potential Factor Xa (FXa) Inhibitors: Drug Design, Synthesis, and Biological Evaluation.

The coagulation cascade is the process of the conversion of soluble fibrinogen to insoluble fibrin that terminates in production of a clot. Factor Xa (FXa) is a serine protease involved in the blood coagulation cascade. Moreover, FXa plays a vital role in the enzymatic sequence which ends with the thrombus production. Thrombosis is a common causal pathology for three widespread cardiovascular syndromes: acute coronary syndrome (ACS), venous thromboembolism (VTE), and strokes. In this research a series of N-propargyltetrahydroquinoline and 1,2,3-triazole derivatives as a potential factor Xa (FXa) inhibitor were designed, synthesized, and evaluated for their FXa inhibitor activity, cytotoxicity activity and coagulation parameters. Rational design for the desired novel molecules was performed through protein-ligand complexes selection and ligand clustering. The microwave-assisted synthetic strategy of selected compounds was carried out by using Ullmann-Goldberg, N-propargylation, Mannich addition, Friedel-Crafts, and 1,3-dipolar cycloaddition type reactions under microwave irradiation. The microwave methodology proved to be an efficient way to obtain all novel compounds in high yields (73-93%). Furthermore, a thermochemical analysis, optimization and reactivity indexes such as electronic chemical potential (µ), chemical hardness (η), and electrophilicity (ω) were performed to understand the relationship between the structure and the energetic behavior of all the series. Then, in vitro analysis showed that compounds 27, 29-31, and 34 exhibited inhibitory activity against FXa and the corresponding half maximal inhibitory concentration (IC50) values were calculated. Next, a cell viability assay in HEK293 and HepG2 cell lines, and coagulation parameters (anti FXa, Prothrombin time (PT), activated Partial Thromboplastin Time (aPTT)) of the most active novel molecules were performed to determine the corresponding cytotoxicity and possible action on clotting pathways. The obtained results suggest that compounds 27 and 29 inhibited FXa targeting through coagulation factors in the intrinsic and extrinsic pathways. However, compound 34 may target coagulation FXa mainly by the extrinsic and common pathway. Interestingly, the most active compounds in relation to the inhibition activity against FXa and coagulation parameters did not show toxicity at the performed coagulation assay concentrations. Finally, docking studies confirmed the preferential binding mode of N-propargyltetrahydroquinoline and 1,2,3-triazole derivatives inside the active site of FXa.

UCYN-A3, a newly characterized open ocean sublineage of the symbiotic N2 -fixing cyanobacterium Candidatus Atelocyanobacterium thalassa.

The symbiotic unicellular cyanobacterium Candidatus Atelocyanobacterium thalassa (UCYN-A) is one of the most abundant and widespread nitrogen (N2 )-fixing cyanobacteria in the ocean. Although it remains uncultivated, multiple sublineages have been detected based on partial nitrogenase (nifH) gene sequences, including the four most commonly detected sublineages UCYN-A1, UCYN-A2, UCYN-A3 and UCYN-A4. However, very little is known about UCYN-A3 beyond the nifH sequences from nifH gene diversity surveys. In this study, single cell sorting, DNA sequencing, qPCR and CARD-FISH assays revealed discrepancies involving the identification of sublineages, which led to new information on the diversity of the UCYN-A symbiosis. 16S rRNA and nifH gene sequencing on single sorted cells allowed us to identify the 16S rRNA gene of the uncharacterized UCYN-A3 sublineage. We designed new CARD-FISH probes that allowed us to distinguish and observe UCYN-A2 in a coastal location (SIO Pier; San Diego) and UCYN-A3 in an open ocean location (Station ALOHA; Hawaii). Moreover, we reconstructed about 13% of the UCYN-A3 genome from Tara Oceans metagenomic data. Finally, our findings unveil the UCYN-A3 symbiosis in open ocean waters suggesting that the different UCYN-A sublineages are distributed along different size fractions of the plankton defined by the cell-size ranges of their prymnesiophyte hosts.

Primer Design for an Accurate View of Picocyanobacterial Community Structure by Using High-Throughput Sequencing.

High-throughput sequencing (HTS) of the 16S rRNA gene has been used successfully to describe the structure and dynamics of microbial communities. Picocyanobacteria are important members of bacterioplankton communities, and, so far, they have predominantly been targeted using universal bacterial primers, providing a limited resolution of the picocyanobacterial community structure and dynamics. To increase such resolution, the study of a particular target group is best approached with the use of specific primers. Here, we aimed to design and evaluate specific primers for aquatic picocyanobacterial genera to be used with high-throughput sequencing. Since the various regions of the 16S rRNA gene have different degrees of conservation in different bacterial groups, we therefore first determined which hypervariable region of the 16S rRNA gene provides the highest taxonomic and phylogenetic resolution for the genera Synechococcus, Prochlorococcus, and Cyanobium An in silico analysis showed that the V5, V6, and V7 hypervariable regions appear to be the most informative for this group. We then designed primers flanking these hypervariable regions and tested them in natural marine and freshwater communities. We successfully detected that most (97%) of the obtained reads could be assigned to picocyanobacterial genera. We defined operational taxonomic units as exact sequence variants (zero-radius operational taxonomic units [zOTUs]), which allowed us to detect higher genetic diversity and infer ecologically relevant information about picocyanobacterial community composition and dynamics in different aquatic systems. Our results open the door to future studies investigating picocyanobacterial diversity in aquatic systems.IMPORTANCE The molecular diversity of the aquatic picocyanobacterial community cannot be accurately described using only the available universal 16S rRNA gene primers that target the whole bacterial and archaeal community. We show that the hypervariable regions V5, V6, and V7 of the 16S rRNA gene are better suited to study the diversity, community structure, and dynamics of picocyanobacterial communities at a fine scale using Illumina MiSeq sequencing. Due to its variability, it allows reconstructing phylogenies featuring topologies comparable to those generated when using the complete 16S rRNA gene sequence. Further, we successfully designed a new set of primers flanking the V5 to V7 region whose specificity for picocyanobacterial genera was tested in silico and validated in several freshwater and marine aquatic communities. This work represents a step forward for understanding the diversity and ecology of aquatic picocyanobacteria and sets the path for future studies on picocyanobacterial diversity.

Delineating ecologically significant taxonomic units from global patterns of marine picocyanobacteria.

Prochlorococcus and Synechococcus are the two most abundant and widespread phytoplankton in the global ocean. To better understand the factors controlling their biogeography, a reference database of the high-resolution taxonomic marker petB, encoding cytochrome b6, was used to recruit reads out of 109 metagenomes from the Tara Oceans expedition. An unsuspected novel genetic diversity was unveiled within both genera, even for the most abundant and well-characterized clades, and 136 divergent petB sequences were successfully assembled from metagenomic reads, significantly enriching the reference database. We then defined Ecologically Significant Taxonomic Units (ESTUs)-that is, organisms belonging to the same clade and occupying a common oceanic niche. Three major ESTU assemblages were identified along the cruise transect for Prochlorococcus and eight for Synechococcus Although Prochlorococcus HLIIIA and HLIVA ESTUs codominated in iron-depleted areas of the Pacific Ocean, CRD1 and the yet-to-be cultured EnvB were the prevalent Synechococcus clades in this area, with three different CRD1 and EnvB ESTUs occupying distinct ecological niches with regard to iron availability and temperature. Sharp community shifts were also observed over short geographic distances-for example, around the Marquesas Islands or between southern Indian and Atlantic Oceans-pointing to a tight correlation between ESTU assemblages and specific physico-chemical parameters. Together, this study demonstrates that there is a previously overlooked, ecologically meaningful, fine-scale diversity within some currently defined picocyanobacterial ecotypes, bringing novel insights into the ecology, diversity, and biology of the two most abundant phototrophs on Earth.

A genetically selected cyclic peptide inhibitor of BCL6 homodimerization.

We report an inhibitor of the homodimeric protein-protein interaction of the BCL6 oncoprotein, identified from a genetically encoded SICLOPPS library of 3.2 million cyclic hexapeptides in combination with a bacterial reverse two-hybrid system. This cyclic peptide is shown to bind the BTB domain of BCL6, disrupts its homodimerization, and subsequent binding of the SMRT2 corepressor peptide.

A Cyanide-Induced 3-Cyanoalanine Nitrilase in the Cyanide-Assimilating Bacterium Pseudomonas pseudoalcaligenes Strain CECT 5344.

Pseudomonas pseudoalcaligenes CECT 5344 is a bacterium able to assimilate cyanide as a sole nitrogen source. Under this growth condition, a 3-cyanoalanine nitrilase enzymatic activity was induced. This activity was encoded by nit4, one of the four nitrilase genes detected in the genome of this bacterium, and its expression in Escherichia coli enabled the recombinant strain to fully assimilate 3-cyanoalanine. P. pseudoalcaligenes CECT 5344 showed a weak growth level with 3-cyanoalanine as the N source, unless KCN was also added. Moreover, a nit4 knockout mutant of P. pseudoalcaligenes CECT 5344 became severely impaired in its ability to grow with 3-cyanoalanine and cyanide as nitrogen sources. The native enzyme expressed in E. coli was purified up to electrophoretic homogeneity and biochemically characterized. Nit4 seems to be specific for 3-cyanoalanine, and the amount of ammonium derived from the enzymatic activity doubled in the presence of exogenously added asparaginase activity, which demonstrated that the Nit4 enzyme had both 3-cyanoalanine nitrilase and hydratase activities. The nit4 gene is located downstream of the cyanide resistance transcriptional unit containing cio1 genes, whose expression levels are under the positive control of cyanide. Real-time PCR experiments revealed that nit4 expression was also positively regulated by cyanide in both minimal and LB media. These results suggest that this gene cluster including cio1 and nit4 could be involved both in cyanide resistance and in its assimilation by P. pseudoalcaligenes CECT 5344.IMPORTANCE Cyanide is a highly toxic molecule present in some industrial wastes due to its application in several manufacturing processes, such as gold mining and the electroplating industry. The biodegradation of cyanide from contaminated wastes could be an attractive alternative to physicochemical treatment. P. pseudoalcaligenes CECT 5344 is a bacterial strain able to assimilate cyanide under alkaline conditions, thus avoiding its volatilization as HCN. This paper describes and characterizes an enzyme (Nit4) induced by cyanide that is probably involved in cyanide assimilation. The biochemical characterization of Nit4 provides a segment for building a cyanide assimilation pathway in P. pseudoalcaligenes This information could be useful for understanding, and hopefully improving, the mechanisms involved in bacterial cyanide biodegradation and its application in the treatment of cyanide-containing wastes.

Persistence of a ST6 clone of Enterococcus faecalis genotype vanB2 in two Hospitals in Aragon (Spain). / Persistencia de un clon ST6 de Enterococcus faecalis con genotipo vanB2 en dos hospitales de Aragón.

INTRODUCTION: In order to study the evolution of the outbreak that occurred between 2009 and 2010 in 3 hospitals in Zaragoza, all vancomycin-resistant clinical Enterococcus faecalis isolates identified between 2011 and 2013 at these hospitals were characterised. METHODS: Molecular characterisation of the isolates and analysis of their clonal relationships was performed using pulsed field electrophoresis, along with a retrospective review of the patient records. RESULTS: A total of 79 vancomycin-resistant E.faecalis isolates with genotype vanB2 of 73 patients were recovered in 2 of the 3 hospitals, most of them from urine specimens. About 46% of the cases were nosocomial. Distribution of the isolates among hospital services demonstrated high variability, making it difficult to predict a common source of infection. All the strains were multiresistant (vancomycin, erythromycin, tetracycline, ciprofloxacin, streptomycin, gentamicin, kanamycin) and belonged to lineage ST6. Seventy-four isolates (93.7%) were identical or closely related to the dominant one in the origin of the outbreak. CONCLUSION: The outbreak remains constant over three years after being initially described, indicating the need to implement an active control in order to limit the emergence and spread of vancomycin-resistant clones.

Expression of Vascular Endothelial Growth Factor Receptors in Benign Vascular Lesions of the Orbit: A Case Series.

PURPOSE: Vascular lesions of the orbit, although not malignant, can cause morbidity because of their location near critical structures in the orbit. For the same reason, they can be challenging to remove surgically. Anti-vascular endothelial growth factor (VEGF) drugs are increasingly being used to treat diseases with prominent angiogenesis. Our study aimed to determine to what extent VEGF receptors and their subtypes are expressed on selected vascular lesions of the orbit. DESIGN: Retrospective case series of all orbital vascular lesions removed by one of the authors (JAG) at the Mayo Clinic. PARTICIPANTS: A total of 52 patients who underwent removal of vascular orbital lesions. METHODS: The pathology specimens from the patients were retrieved, their pathologic diagnosis was confirmed, demographic and clinical information were gathered, and sections from vascular tumors were stained with vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor type 1 (VEGFR1), vascular endothelial growth factor receptor type 2 (VEGFR2), and vascular endothelial growth factor receptor type 3 (VEGFR3). MAIN OUTCOME MEASURES: The existence and pattern of staining with VEGF and its subtypes on these lesions. RESULTS: There were 28 specimens of venous malformations, 4 capillary hemangiomas, 7 lymphatic malformations, and 6 lymphaticovenous malformations. All samples stained with VEGF, 55% stained with VEGFR1, 98% stained with VEGFR2, and 96% stained with VEGFR3. Most (94%) of the VEGFR2 staining was diffuse. CONCLUSIONS: Most orbital vascular lesions express VEGF receptors, which may suggest a future target for nonsurgical treatment.

Mutations of the gene encoding otogelin are a cause of autosomal-recessive nonsyndromic moderate hearing impairment.

Already 40 genes have been identified for autosomal-recessive nonsyndromic hearing impairment (arNSHI); however, many more genes are still to be identified. In a Dutch family segregating arNSHI, homozygosity mapping revealed a 2.4 Mb homozygous region on chromosome 11 in p15.1-15.2, which partially overlapped with the previously described DFNB18 locus. However, no putative pathogenic variants were found in USH1C, the gene mutated in DFNB18 hearing impairment. The homozygous region contained 12 additional annotated genes including OTOG, the gene encoding otogelin, a component of the tectorial membrane. It is thought that otogelin contributes to the stability and strength of this membrane through interaction or stabilization of its constituent fibers. The murine orthologous gene was already known to cause hearing loss when defective. Analysis of OTOG in the Dutch family revealed a homozygous 1 bp deletion, c.5508delC, which leads to a shift in the reading frame and a premature stop codon, p.Ala1838ProfsX31. Further screening of 60 unrelated probands from Spanish arNSHI families detected compound heterozygous OTOG mutations in one family, c.6347C>T (p.Pro2116Leu) and c. 6559C>T (p.Arg2187X). The missense mutation p.Pro2116Leu affects a highly conserved residue in the fourth von Willebrand factor type D domain of otogelin. The subjects with OTOG mutations have a moderate hearing impairment, which can be associated with vestibular dysfunction. The flat to shallow "U" or slightly downsloping shaped audiograms closely resembled audiograms of individuals with recessive mutations in the gene encoding α-tectorin, another component of the tectorial membrane. This distinctive phenotype may represent a clue to orientate the molecular diagnosis.

Validation of a new catalysed reporter deposition-fluorescence in situ hybridization probe for the accurate quantification of marine Bacteroidetes populations.

Catalysed reporter deposition-fluorescence in situ hybridization (CARD-FISH) is a powerful approach to quantify bacterial taxa. In this study, we compare the performance of the widely used Bacteroidetes CF319a probe with the new CF968 probe. In silico analyses and tests with isolates demonstrate that CF319a hybridizes with non-Bacteroidetes sequences from the Rhodobacteraceae and Alteromonadaceae families. We test the probes' accuracy in 37 globally distributed marine samples and over two consecutive years at the Blanes Bay Microbial Observatory (NW Mediterranean). We also compared the CARD-FISH data with the Bacteroidetes 16S rRNA gene sequences retrieved from 27 marine metagenomes from the TARA Oceans expedition. We find no significant differences in abundances between both approaches, although CF319a targeted some unspecific sequences and both probes displayed different abundances of specific Bacteroidetes phylotypes. Our results demonstrate that quantitative estimations by using both probes are significantly different in certain oceanographic regions (Mediterranean Sea, Red Sea and Arabian Sea) and that CF968 shows seasonality within marine Bacteroidetes, notably large differences between summer and winter that is overlooked by CF319a. We propose CF968 as an alternative to CF319a for targeting the whole Bacteroidetes phylum since it has better coverage, greater specificity and overall better quantifies marine Bacteroidetes.

Closing the gaps on the viral photosystem-I†psaDCAB gene organization.

Marine photosynthesis is largely driven by cyanobacteria, namely Synechococcus and Prochlorococcus. Genes encoding for photosystem (PS) I and II reaction centre proteins are found in cyanophages and are believed to increase their fitness. Two viral PSI gene arrangements are known, psaJF→C→A→B→K→E→D and psaD→C→A→B. The shared genes between these gene cassettes and their encoded proteins are distinguished by %G + C and protein sequence respectively. The data on the psaD→C→A→B gene organization were reported from only two partial gene cassettes coming from Global Ocean Sampling stations in the Pacific and Indian oceans. Now we have extended our search to 370 marine stations from six metagenomic projects. Genes corresponding to both PSI gene arrangements were detected in the Pacific, Indian and Atlantic oceans, confined to a strip along the equator (30°N and 30°S). In addition, we found that the predicted structure of the viral PsaA protein from the psaD→C→A→B organization contains a lumenal loop conserved in PsaA proteins from Synechococcus, but is completely absent in viral PsaA proteins from the psaJF→C→A→B→K→E→D gene organization and most Prochlorococcus strains. This may indicate a co-evolutionary scenario where cyanophages containing either of these gene organizations infect cyanobacterial ecotypes biogeographically restricted to the 30°N and 30°S equatorial strip.

Sample dilution and bacterial community composition influence empirical leucine-to-carbon conversion factors in surface waters of the world's oceans.

The transformation of leucine incorporation rates to prokaryotic carbon production rates requires the use of either theoretical or empirically determined conversion factors. Empirical leucine-to-carbon conversion factors (eCFs) vary widely across environments, and little is known about their potential controlling factors. We conducted 10 surface seawater manipulation experiments across the world's oceans, where the growth of the natural prokaryotic assemblages was promoted by filtration (i.e., removal of grazers [F treatment]) or filtration combined with dilution (i.e., also relieving resource competition [FD treatment]). The impact of sunlight exposure was also evaluated in the FD treatments, and we did not find a significant effect on the eCFs. The eCFs varied from 0.09 to 1.47 kg C mol Leu(-1) and were significantly lower in the FD than in the F samples. Also, changes in bacterial community composition during the incubations, as assessed by automated ribosomal intergenic spacer analysis (ARISA), were more pronounced in the FD than in the F treatments, compared to unmanipulated controls. Thus, we discourage the common procedure of diluting samples (in addition to filtration) for eCF determination. The eCFs in the filtered treatment were negatively correlated with the initial chlorophyll a concentration, picocyanobacterial abundance (mostly Prochlorococcus), and the percentage of heterotrophic prokaryotes with high nucleic acid content (%HNA). The latter two variables explained 80% of the eCF variability in the F treatment, supporting the view that both Prochlorococcus and HNA prokaryotes incorporate leucine in substantial amounts, although this results in relatively low carbon production rates in the oligotrophic ocean.