More than just an eagle killer: The freshwater cyanobacterium Aetokthonos hydrillicola produces highly toxic dolastatin derivatives.

Cyanobacteria are infamous producers of toxins. While the toxic potential of planktonic cyanobacterial blooms is well documented, the ecosystem level effects of toxigenic benthic and epiphytic cyanobacteria are an understudied threat. The freshwater epiphytic cyanobacterium Aetokthonos hydrillicola has recently been shown to produce the "eagle killer" neurotoxin aetokthonotoxin (AETX) causing the fatal neurological disease vacuolar myelinopathy. The disease affects a wide array of wildlife in the southeastern United States, most notably waterfowl and birds of prey, including the bald eagle. In an assay for cytotoxicity, we found the crude extract of the cyanobacterium to be much more potent than pure AETX, prompting further investigation. Here, we describe the isolation and structure elucidation of the aetokthonostatins (AESTs), linear peptides belonging to the dolastatin compound family, featuring a unique modification of the C-terminal phenylalanine-derived moiety. Using immunofluorescence microscopy and molecular modeling, we confirmed that AEST potently impacts microtubule dynamics and can bind to tubulin in a similar matter as dolastatin 10. We also show that AEST inhibits reproduction of the nematode Caenorhabditis elegans. Bioinformatic analysis revealed the AEST biosynthetic gene cluster encoding a nonribosomal peptide synthetase/polyketide synthase accompanied by a unique tailoring machinery. The biosynthetic activity of a specific N-terminal methyltransferase was confirmed by in vitro biochemical studies, establishing a mechanistic link between the gene cluster and its product.

Prognostic relevance of the C-X-C motif chemokine ligand 13 and interleukin-8 in predicting the transition from clinically isolated syndrome to multiple sclerosis.

The initial phase of multiple sclerosis (MS), often known as clinically isolated syndrome (CIS), is a critical period for identifying individuals at high risk of progressing to full-blown MS and initiating timely treatment. In this study, we aimed to evaluate the prognostic value of C-X-C motif chemokine ligand 13 (CXCL13) and interleukin-8 (IL-8) as potential markers for CIS patients' conversion to MS. Our study encompassed patients with CIS, those with relapsing-remitting MS (RRMS), and control subjects, with sample analysis conducted on both cerebrospinal fluid (CSF) and serum. Patients were categorized into four groups: CIS-CIS (no MS development within 2 years), CIS-RRMS (conversion to RRMS within 2 years), RRMS (already diagnosed), and a control group (CG) with noninflammatory central nervous system disorders. Results showed significantly elevated levels of CXCL13 in CSF across all patient groups compared with the CG (p < 0.0001, Kruskal-Wallis test). Although CXCL13 concentrations were slightly higher in the CIS-RRMS group, statistical significance was not reached. Similarly, significantly higher levels of IL-8 were detected in CSF samples from all patient groups compared with the CG (p < 0.0001, Kruskal-Wallis test). Receiver operating characteristic analysis in the CIS-RRMS group identified both CXCL13 (area under receiver operating characteristic curve = .959) and IL-8 (area under receiver operating characteristic curve = .939) in CSF as significant predictors of CIS to RRMS conversion. In conclusion, our study suggests a trend towards elevated CSF IL-8 and CSF CXCL13 levels in CIS patients progressing to RRMS. These findings emphasize the importance of identifying prognostic markers to guide appropriate treatment strategies for individuals in the early stages of MS.

A thylakoid biogenesis BtpA protein is required for the initial step of tetrapyrrole biosynthesis in cyanobacteria.

Biogenesis of the photosynthetic apparatus requires complicated molecular machinery, individual components of which are either poorly characterized or unknown. The BtpA protein has been described as a factor required for the stability of photosystem I (PSI) in cyanobacteria; however, how the BtpA stabilized PSI remains unexplained. To clarify the role of BtpA, we constructed and characterized the btpA-null mutant (ΔbtpA) in the cyanobacterium Synechocystis sp. PCC 6803. The mutant contained only c. 1% of chlorophyll and nearly no thylakoid membranes. However, this strain, growing only in the presence of glucose, was genetically unstable and readily generated suppressor mutations that restore the photoautotrophy. Two suppressor mutations were mapped into the hemA gene encoding glutamyl-tRNA reductase (GluTR) - the first enzyme of tetrapyrrole biosynthesis. Indeed, the GluTR was not detectable in the ΔbtpA mutant and the suppressor mutations restored biosynthesis of tetrapyrroles and photoautotrophy by increased GluTR expression or by improved GluTR stability/processivity. We further demonstrated that GluTR associates with a large BtpA oligomer and that BtpA is required for the stability of GluTR. Our results show that the BtpA protein is involved in the biogenesis of photosystems at the level of regulation of tetrapyrrole biosynthesis.

Crocosphaera watsonii - A widespread nitrogen-fixing unicellular marine cyanobacterium.

Crocosphaera watsonii is a unicellular N2-fixing (diazotrophic) cyanobacterium observed in tropical and subtropical oligotrophic oceans. As a diazotroph, it can be a source of bioavailable nitrogen (N) to the microbial community in N-limited environments, and this may fuel primary production in the regions where it occurs. Crocosphaera watsonii has been the subject of intense study, both in culture and in field populations. Here, we summarize the current understanding of the phylogenetic and physiological diversity of C. watsonii, its distribution, and its ecological niche. Analysis of the relationships among the individual Crocosphaera species and related free-living and symbiotic lineages of diazotrophs based on the nifH gene have shown that the C. watsonii group holds a basal position and that its sequence is more similar to Rippkaea and Zehria than to other Crocosphaera species. This finding warrants further scrutiny to determine if the placement is related to a horizontal gene transfer event. Here, the nifH UCYN-B gene copy number from a recent synthesis effort was used as a proxy for relative C. watsonii abundance to examine patterns of C. watsonii distribution as a function of environmental factors, like iron and phosphorus concentration, and complimented with a synthesis of C. watsonii physiology. Furthermore, we have summarized the current knowledge of C. watsonii with regards to N2 fixation, photosynthesis, and quantitative modeling of physiology. Because N availability can limit primary production, C. watsonii is widely recognized for its importance to carbon and N cycling in ocean ecosystems, and we conclude this review by highlighting important topics for further research on this important species.

Evolution of Ycf54-independent chlorophyll biosynthesis in cyanobacteria.

Chlorophylls (Chls) are essential cofactors for photosynthesis. One of the least understood steps of Chl biosynthesis is formation of the fifth (E) ring, where the red substrate, magnesium protoporphyrin IX monomethyl ester, is converted to the green product, 3,8-divinyl protochlorophyllide a In oxygenic phototrophs, this reaction is catalyzed by an oxygen-dependent cyclase, consisting of a catalytic subunit (AcsF/CycI) and an auxiliary protein, Ycf54. Deletion of Ycf54 impairs cyclase activity and results in severe Chl deficiency, but its exact role is not clear. Here, we used a Δycf54 mutant of the model cyanobacterium Synechocystis sp. PCC 6803 to generate suppressor mutations that restore normal levels of Chl. Sequencing Δycf54 revertants identified a single D219G amino acid substitution in CycI and frameshifts in slr1916, which encodes a putative esterase. Introduction of these mutations to the original Δycf54 mutant validated the suppressor effect, especially in combination. However, comprehensive analysis of the Δycf54 suppressor strains revealed that the D219G-substituted CycI is only partially active and its accumulation is misregulated, suggesting that Ycf54 controls both the level and activity of CycI. We also show that Slr1916 has Chl dephytylase activity in vitro and its inactivation up-regulates the entire Chl biosynthetic pathway, resulting in improved cyclase activity. Finally, large-scale bioinformatic analysis indicates that our laboratory evolution of Ycf54-independent CycI mimics natural evolution of AcsF in low-light-adapted ecotypes of the oceanic cyanobacteria Prochlorococcus, which lack Ycf54, providing insight into the evolutionary history of the cyclase enzyme.

Endoluminal radiofrequency ablation in patients with malignant biliary obstruction: a randomised trial.

BACKGROUND: Endoluminal radiofrequency ablation (RFA) has been promoted as palliative treatment for patients with cholangiocarcinoma (CCA) and pancreatic ductal adenocarcinoma (PDAC) in order to improve biliary drainage and eventually prolong survival. No high level evidence is, however, available on this technique. DESIGN: In this randomised controlled study, we compared endoluminal RFA plus stenting with stenting alone (control group) in patients with malignant biliary obstruction; metal stents were primarily placed. Primary outcome was overall survival; secondary outcomes were stent patency, quality of life and adverse events. In a superiority design, survival was assumed to be doubled by RFA as compared with 6.4 months in the control group (n=280). RESULTS: A total of 161 patients (male:female 90:71, mean age 71±9 years) were randomised before recruitment was terminated for futility after an interim analysis. Eighty-five patients had CCA (73 hilar, 12 distal) and 76 had pancreatic cancer. There was no difference in survival in both subgroups: for patients with CCA, median survival was 10.5 months (95% CI 6.7 to 18.3) in the RFA group vs 10.6 months (95% CI 9.0 to 24.8), p=0.58)) in the control group. In the subgroup with pancreatic cancer, median survival was 6.4 months (95% CI 4.3 to 9.7) for the RFA vs 7.7 months (95% CI 5.6 to 11.3), p=0.73) for the control group. No benefit was seen in the RFA group with regard to stent patency (at 12 months 40% vs 36% in CCA and 66% vs 65% in PDAC), and quality of life was unchanged by either treatment and comparable between the groups. Adverse events occurred in seven patients in each groups. CONCLUSION: A combination of endoluminal RFA and stenting was not superior to stenting alone in prolonging survival or improving stent patency in patients with malignant biliary obstruction. TRIAL REGISTRATION NUMBER: NCT03166436.

MIR204 n.37C>T variant as a cause of chorioretinal dystrophy variably associated with iris coloboma, early-onset cataracts and congenital glaucoma.

Four members of a three-generation Czech family with early-onset chorioretinal dystrophy were shown to be heterozygous carriers of the n.37C>T in MIR204. The identification of this previously reported pathogenic variant confirms the existence of a distinct clinical entity caused by a sequence change in MIR204. Chorioretinal dystrophy was variably associated with iris coloboma, congenital glaucoma, and premature cataracts extending the phenotypic range of the condition. In silico analysis of the n.37C>T variant revealed 713 novel targets. Additionally, four family members were shown to be affected by albinism resulting from biallelic pathogenic OCA2 variants. Haplotype analysis excluded relatedness with the original family reported to harbour the n.37C>T variant in MIR204. Identification of a second independent family confirms the existence of a distinct MIR204-associated clinical entity and suggests that the phenotype may also involve congenital glaucoma.

An updated classification of cyanobacterial orders and families based on phylogenomic and polyphasic analysis.

Cyanobacterial taxonomy is facing a period of rapid changes thanks to the ease of 16S rRNA gene sequencing and established workflows for description of new taxa. Since the last comprehensive review of the cyanobacterial system in 2014 until 2021, at least 273 species in 140 genera were newly described. These taxa were mainly placed into previously defined orders and families although several new families were proposed. However, the classification of most taxa still relied on hierarchical relationships inherited from the classical morphological taxonomy. Similarly, the obviously polyphyletic orders such as Synechococcales and Oscillatoriales were left unchanged. In this study, the rising number of genomic sequences of cyanobacteria and well-described reference strains allowed us to reconstruct a robust phylogenomic tree for taxonomic purposes. A less robust but better sampled 16S rRNA gene phylogeny was mapped to the phylogenomic backbone. Based on both these phylogenies, a polyphasic classification throughout the whole phylum of Cyanobacteria was created, with ten new orders and fifteen new families. The proposed system of cyanobacterial orders and families relied on a phylogenomic tree but still employed phenotypic apomorphies where possible to make it useful for professionals in the field. It was, however, confirmed that morphological convergence of phylogenetically distant taxa was a frequent phenomenon in cyanobacteria. Moreover, the limited phylogenetic informativeness of the 16S rRNA gene, resulting in ambiguous phylogenies above the genus level, emphasized the integration of genomic data as a prerequisite for the conclusive taxonomic placement of a vast number of cyanobacterial genera in the future.

Periprocedural safety profile of peroral endoscopic myotomy (POEM)-a retrospective analysis of adverse events according to two different classifications.

BACKGROUND: Peroral endoscopic myotomy (POEM) is nowadays a standard method for treatment of achalasia; nevertheless, it remains an invasive intervention with corresponding risk of adverse events (AEs). The classification and grading of AEs are still a matter of discussion. The aim of our retrospective study was to assess the occurrence of all "undesirable" events and "true" adverse events in patients undergoing POEM and to compare the outcomes when either Clavien-Dindo classification (CDC) or American Society of Gastrointestinal Endoscopy (ASGE) lexicon classification applied. METHODS: This was a retrospective analysis of prospectively managed database of all patients who had undergone POEM between December 2012 and August 2018. We assessed the pre-, peri-, and early-postoperative (up to patient's discharge) undesirable events (including those not fulfilling criteria for AEs) and "true" AEs according the definition in either of the classifications. RESULTS: A total of 231 patients have successfully undergone 244 POEM procedures (13 × re-POEM). Twenty-nine procedures (11.9%) passed uneventfully, while in 215 procedures (88.1%), a total of 440 undesirable events occurred. The CDC identified 27 AEs (17 minor, 10 major) occurring in 23/244 (9.4%) procedures. The ASGE lexicon identified identical 27 AEs (21 mild or moderate, 6 severe or fatal) resulting in the severity distribution of AEs being the only difference between the two classifications. Only the absence of previous treatment was found to be a risk factor [p = 0.047, OR with 95% CI: 4.55 (1.02; 20.25)] in the combined logistic regression model. CONCLUSION: Undesirable events are common in patients undergoing POEM but the incidence of true AEs is low according to both classifications. Severe adverse events are infrequent irrespective of the classification applied. CDC may be more appropriate than ASGE lexicon for classifying POEM-related AEs given a surgical nature of this procedure.

Long-term exposure to polycyclic musk tonalide - A potential threat to juvenile zebrafish (Danio rerio)?

Polycyclic musk compounds are commonly used in personal care products to replace expensive natural fragrances. Due to their huge consumption, they have become a part of the aquatic environment. In the present study, a two-month exposure effect of tonalide on juvenile zebrafish (Danio rerio) was investigated. We determined the vitellogenin concentration to define the potential endocrine-disrupting effect of tonalide and also analysed selected indices to evaluate the induction of oxidative stress. The environmentally relevant concentration of tonalide (i.e., 500 ng/l) caused a significant decrease in the catalase activity (P < 0.05) and a significant increase (P < 0.05) in the lipid peroxidation. An increasing lipid peroxidation was also recorded for the highest concentration group tested (i.e., 50 000 ng/l). On the other hand, no significant changes were recorded in vitellogenin in all the exposed groups. Thus, based on these results, we have not demonstrated the endocrine-disrupting activity of tonalide in zebrafish. The results of the oxidative stress indices showed a significant impairment of the antioxidant defence after the two-month tonalide exposure, which could indicate part of the adaptive response to the tonalide toxicity.

Endoscopic pyloromyotomy for the treatment of severe and refractory gastroparesis: a pilot, randomised, sham-controlled trial.

OBJECTIVE: Endoscopic pyloromyotomy (G-POEM) is a minimally invasive treatment option with promising uncontrolled outcome results in patients with gastroparesis. DESIGN: In this prospective randomised trial, we compared G-POEM with a sham procedure in patients with severe gastroparesis. The primary outcome was the proportion of patients with treatment success (defined as a decrease in the Gastroparesis Cardinal Symptom Index (GCSI) by at least 50%) at 6 months. Patients randomised to the sham group with persistent symptoms were offered cross-over G-POEM. RESULTS: The enrolment was stopped after the interim analysis by the Data and Safety Monitoring Board prior to reaching the planned sample of 86 patients. A total of 41 patients (17 diabetic, 13 postsurgical, 11 idiopathic; 46% male) were randomised (21 G-POEM, 20-sham). Treatment success rate was 71% (95% CI 50 to 86) after G-POEM versus 22% (8-47) after sham (p=0.005). Treatment success in patients with diabetic, postsurgical and idiopathic gastroparesis was 89% (95% CI 56 to 98), 50% (18-82) and 67% (30-90) after G-POEM; the corresponding rates in the sham group were 17% (3-57), 29% (7-67) and 20% (3-67).Median gastric retention at 4 hours decreased from 22% (95% CI 17 to 31) to 12% (5-22) after G-POEM and did not change after sham: 26% (18-39) versus 24% (11-35). Twelve patients crossed over to G-POEM with 9 of them (75%) achieving treatment success. CONCLUSION: In severe gastroparesis, G-POEM is superior to a sham procedure for improving both symptoms and gastric emptying 6 months after the procedure. These results are not entirely conclusive in patients with idiopathic and postsurgical aetiologies. TRIAL REGISTRATION NUMBER: NCT03356067; ClinicalTrials.gov.

Validation of the names Cyanobacterium and Cyanobacterium stanieri, and proposal of Cyanobacteriota phyl. nov.

The decision by the International Committee on Systematics of Prokaryotes (ICSP) to place the rank of phylum under the rules of the International Code of Nomenclature of Prokaryotes (ICNP), with phylum names ending in -ota based on the name of a type genus, enables the valid publication of the phylum name Cyanobacteriota with Cyanobacterium as the type genus. The names Cyanobacterium and its type species Cyanobacterium stanieri were effectively published in 1983 by Rippka and Cohen-Bazire, but the names were not validly published under the rules of the ICNP (then named the International Code of Nomenclature of Bacteria) or the rules of the ICN (International Code of Nomenclature for algae, fungi, and plants, then named the International Code of Botanical Nomenclature). We here propose the names Cyanobacterium gen. nov and Cyanobacterium stanieri sp. nov. for valid publication under the provisions of the ICN. Upon validation these names are also validly published under the ICNP according to General Consideration 5 and Rule 30. We also propose the phylum name Cyanobacteriota phyl. nov. under the rules of the ICNP.

Cyanochelins, an Overlooked Class of Widely Distributed Cyanobacterial Siderophores, Discovered by Silent Gene Cluster Awakening.

Cyanobacteria require iron for growth and often inhabit iron-limited habitats, yet only a few siderophores are known to be produced by them. We report that cyanobacterial genomes frequently encode polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) biosynthetic pathways for synthesis of lipopeptides featuring ß-hydroxyaspartate (ß-OH-Asp), a residue known to be involved in iron chelation. Iron starvation triggered the synthesis of ß-OH-Asp lipopeptides in the cyanobacteria Rivularia sp. strain PCC 7116, Leptolyngbya sp. strain NIES-3755, and Rubidibacter lacunae strain KORDI 51-2. The induced compounds were confirmed to bind iron by mass spectrometry (MS) and were capable of Fe3+ to Fe2+ photoreduction, accompanied by their cleavage, when exposed to sunlight. The siderophore from Rivularia, named cyanochelin A, was structurally characterized by MS and nuclear magnetic resonance (NMR) and found to contain a hydrophobic tail bound to phenolate and oxazole moieties followed by five amino acids, including two modified aspartate residues for iron chelation. Phylogenomic analysis revealed 26 additional cyanochelin-like gene clusters across a broad range of cyanobacterial lineages. Our data suggest that cyanochelins and related compounds are widespread ß-OH-Asp-featuring cyanobacterial siderophores produced by phylogenetically distant species upon iron starvation. Production of photolabile siderophores by phototrophic cyanobacteria raises questions about whether the compounds facilitate iron monopolization by the producer or, rather, provide Fe2+ for the whole microbial community via photoreduction. IMPORTANCE All living organisms depend on iron as an essential cofactor for indispensable enzymes. However, the sources of bioavailable iron are often limited. To face this problem, microorganisms synthesize low-molecular-weight metabolites capable of iron scavenging, i.e., the siderophores. Although cyanobacteria inhabit the majority of the Earth's ecosystems, their repertoire of known siderophores is remarkably poor. Their genomes are known to harbor a rich variety of gene clusters with unknown function. Here, we report the awakening of a widely distributed class of silent gene clusters by iron starvation to yield cyanochelins, ß-hydroxy aspartate lipopeptides involved in iron acquisition. Our results expand the limited arsenal of known cyanobacterial siderophores and propose products with ecological function for a number of previously orphan gene clusters.

Tenebriella gen. nov. - The dark twin of Oscillatoria.

Oscillatoria has long been known to be polyphyletic. After recent resequencing of the reference strain for this genus, many Oscillatoria-like groups phylogenetically distant from the type species O. princeps remained unresolved. Here we describe one of these groups as a new genus Tenebriella. Most of the studied strains originate from Central Europe, where they are able to form prominent microbial mats. Despite the overall Oscillatoria-like morphology, Tenebriella can be distinguished by darker trichomes and forms a separate monophyletic clade in phylogenies inferred from the 16S rRNA gene and two additional loci (rpoC1, rbcLX). Within Tenebriella we recognize two new species differing from each other by morphological and ecological characteristics. First species does not fit any known taxon description, and thus is described as a new species T. amphibia. The latter one corresponds with the information available for Oscillatoria curviceps Agardh ex Gomont, and thus new combination T. curviceps is proposed. The phylogenetic analyses of the 16S-23S ITS region together with the comparison of the hypothetical secondary structures confirmed recognition of these two species and additionally revealed presence of a morphologically cryptic species Tenebriella sp. The results corroborate frequent recurrence of convergent morphotypes in the evolution of cyanobacteria and justify further exploration even of the intensively studied European freshwaters using molecular phylogenetics to discover new and ecologically relevant taxa.

Substitution of citrate with tissue plasminogen activator (rt-PA) for catheter lock does not improve patency of tunnelled haemodialysis catheters in a randomised trial.

BACKROUND: The study aim was to establish if substitution of citrate with rt-PA for catheter lock once weekly can reduce the incidence of catheter-related blood stream infections (CR-BSI) or improve patency of tunneled haemodialysis catheters. METHODS: All incident patients undergoing insertion of a tunneled haemodialysis catheter were screened and included except those suffering infection or using oral anticoagulation. Study participants were randomized into two arms according to the solution applied as catheter lock: receiving either trisodium citrate (Citra-LockTM 4%) only or rt-PA (Actilyse® 1 mg/ml) on the middle session each week with citrate used on the first and third sessions. The incidence of CR-BSI (confirmed by positive blood culture), catheter non-function (complete obstruction), and malfunction (blood flow < 250 ml/min) was recorded. Statistical significance was tested with ANOVA, post hoc analysis was performed by means of multiple linear regression. RESULTS: Totally, 18 patients were included and followed during 655 haemodialysis sessions. No episode of CR-BSI was detected while 6 catheter non-functions (0.9% sessions) and 101 malfunctions (15.4% sessions) were recorded. The incidence of both events was equal between the study arms: 4 non-functions and 55 malfunctions in the rt-PA arm and 2 non-functions and 46 malfunctions in the citrate arm (p = 0.47 and p = 0.24, respectively). Additionally, the mean blood flow achieved did not differ significantly between the arms: 326 ± 1,8 and 326 ± 1,9 ml/min (p = 0.95) in rt-PA and citrate arms, respectively. Post hoc analysis identified time elapsed since previous session (ß = 0.12, p = 0.005) and malfunction on previous session (ß = 0.25, p < 0.001) as significant factors affecting the occurrence of malfunction. By contrast, the study arm, rt-PA application on previous session, and catheter vintage did not enter the model. CONCLUSION: Substitution of citrate with rt-PA for catheter lock does not reduce the incidence of catheter malfunction neither does it affect the blood flow achieved during haemodialysis. Catheter patency is related rather to the time interval between sessions and to previous malfunction (thus probably reflecting undefined individual factors). The incidence of CR-BSI within pre-selected haemodialysis population is sporadic (less than 1 per 4.3 patient years in our sample). TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry, ACTRN12612000152820 . Retrospectively registered 03/02/2012.

Comparison of diagnostic methods for Tetracapsuloides bryosalmonae detection in salmonid fish.

Diagnostic accuracy of pathogen detection depends upon the selection of suitable tests. Problems can arise when the selected diagnostic test gives false-positive or false-negative results, which can affect control measures, with consequences for the population health. The aim of this study was to compare sensitivity of different diagnostic methods IHC, PCR and qPCR detecting Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease in salmonid fish and as a consequence differences in disease prevalence. We analysed tissue from 388 salmonid specimens sampled from a recirculating system and rivers in the Czech Republic. Overall prevalence of T. bryosalmonae was extremely high at 92.0%, based on positive results of at least one of the above-mentioned screening methods. IHC resulted in a much lower detection rate (30.2%) than both PCR methods (qPCR32: 65.4%, PCR: 81.9%). While qPCR32 produced a good match with IHC (60.8%), all other methods differed significantly (p < .001) in the proportion of samples determined positive. Both PCR methods showed similar sensitivity, though specificity (i.e., the proportion of non-diseased fish classified correctly) differed significantly (p < .05). Sample preservation method significantly (p < .05) influenced the results of PCR, with a much lower DNA yield extracted from paraffin-embedded samples. Use of different methods that differ in diagnostic sensitivity and specificity resulted in random and systematic diagnosis errors, illustrating the importance of interpreting the results of each method carefully.

Low-level pathogen transmission from wild to farmed salmonids in a flow-through fish farm.

While the potential effects of pathogens spread from farmed fish to wild populations have frequently been studied, evidence for the transmission of parasites from wild to farmed fish is scarce. In the present study, we evaluated natural bacterial and parasitic infections in brown trout (Salmo trutta m. fario) collected from the Cerná Opava river (Czech Republic) as a potential source of infections for rainbow trout (Oncorhynchus mykiss) reared in a flow-through farm system fed by the same river. The prevalence of bacterial and protozoan infections in farmed fish was comparable, or higher, than for riverine fish. Despite this, none of the infected farmed fish showed any signs of severe diseases. Substantial differences in metazoan parasite infections were observed between wild and farmed fish regarding monogeneans, adult trematodes, nematodes, the myxozoan Tetracapsuloides bryosalmonae found in riverine fish only, and larval eye-fluke trematodes sporadically found in farmed fish. The different distribution of metazoan parasites between brown and rainbow trout most probably reflects the availability of infected intermediate hosts in the two habitats. Despite the river being the main water source for the farm, there was no significant threat of parasite infection to the farmed fish from naturally infected riverine fish.

The antenna-like domain of the cyanobacterial ferrochelatase can bind chlorophyll and carotenoids in an energy-dissipative configuration.

Ferrochelatase (FeCh) is an essential enzyme catalyzing the synthesis of heme. Interestingly, in cyanobacteria, algae, and plants, FeCh possesses a conserved transmembrane chlorophyll a/b binding (CAB) domain that resembles the first and the third helix of light-harvesting complexes, including a chlorophyll-binding motif. Whether the FeCh CAB domain also binds chlorophyll is unknown. Here, using biochemical and radiolabeled precursor experiments, we found that partially inhibited activity of FeCh in the cyanobacterium Synechocystis PCC 6803 leads to overproduction of chlorophyll molecules that accumulate in the thylakoid membrane and, together with carotenoids, bind to FeCh. We observed that pigments bound to purified FeCh are organized in an energy-dissipative conformation and further show that FeCh can exist in vivo as a monomer or a dimer depending on its own activity. However, pigmented FeCh was purified exclusively as a dimer. Separately expressed and purified FeCH CAB domain contained a pigment composition similar to that of full-length FeCh and retained its quenching properties. Phylogenetic analysis suggested that the CAB domain was acquired by a fusion between FeCh and a single-helix, high light-inducible protein early in the evolution of cyanobacteria. Following this fusion, the FeCh CAB domain with a functional chlorophyll-binding motif was retained in all currently known cyanobacterial genomes except for a single lineage of endosymbiotic cyanobacteria. Our findings indicate that FeCh from Synechocystis exists mostly as a pigment-free monomer in cells but can dimerize, in which case its CAB domain creates a functional pigment-binding segment organized in an energy-dissipating configuration.

Field study indicating susceptibility differences between salmonid species and their lineages to proliferative kidney disease.

Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea) is the causative agent of proliferative kidney disease (PKD), which affects both wild and farmed salmonid fish. The objective of this study was to outline differences in susceptibility to PKD in different salmonid species, hybrids and breeding lineages. Susceptibility to T. bryosalmonae infection was established based on cumulative mortality, pathological findings and detection of T. bryosalmonae in the kidney using immunohistochemistry and molecular methods. Determination of pure and hybrid individuals of different species in the genus Salvelinus, and dissimilarity of rainbow trout lineages, was performed using traditional polymerase chain reaction (PCR) and microsatellite analyses. Rainbow trout displayed higher disease severity compared with brook trout and Alsatian charr. Moreover, the results indicated differences in infection susceptibility, not only among different salmonid species but also among different lineages of charr and rainbow trout. Our study indicated that some salmonid species and even different lineages of the same species are more suitable for farming under PKD pressure.

Advanced Statistical Analysis of 3D Kinect Data: Mimetic Muscle Rehabilitation Following Head and Neck Surgeries Causing Facial Paresis.

An advanced statistical analysis of patients' faces after specific surgical procedures that temporarily negatively affect the patient's mimetic muscles is presented. For effective planning of rehabilitation, which typically lasts several months, it is crucial to correctly evaluate the improvement of the mimetic muscle function. The current way of describing the development of rehabilitation depends on the subjective opinion and expertise of the clinician and is not very precise concerning when the most common classification (House-Brackmann scale) is used. Our system is based on a stereovision Kinect camera and an advanced mathematical approach that objectively quantifies the mimetic muscle function independently of the clinician's opinion. To effectively deal with the complexity of the 3D camera input data and uncertainty of the evaluation process, we designed a three-stage data-analytic procedure combining the calculation of indicators determined by clinicians with advanced statistical methods including functional data analysis and ordinal (multiple) logistic regression. We worked with a dataset of 93 distinct patients and 122 sets of measurements. In comparison to the classification with the House-Brackmann scale the developed system is able to automatically monitor reinnervation of mimetic muscles giving us opportunity to discriminate even small improvements during the course of rehabilitation.