Clinical variants paired with phenotype: A rich resource for brain gene curation.

PURPOSE: Clinically ascertained variants are under-utilized in neurodevelopmental disorder research. We established the Brain Gene Registry (BGR) to coregister clinically identified variants in putative brain genes with participant phenotypes. Here, we report 179 genetic variants in the first 179 BGR registrants and analyze the proportion that were novel to ClinVar at the time of entry and those that were absent in other disease databases. METHODS: From 10 academically affiliated institutions, 179 individuals with 179 variants were enrolled into the BGR. Variants were cross-referenced for previous presence in ClinVar and for presence in 6 other genetic databases. RESULTS: Of 179 variants in 76 genes, 76 (42.5%) were novel to ClinVar, and 62 (34.6%) were absent from all databases analyzed. Of the 103 variants present in ClinVar, 37 (35.9%) were uncertain (ClinVar aggregate classification of variant of uncertain significance or conflicting classifications). For 5 variants, the aggregate ClinVar classification was inconsistent with the interpretation from the BGR site-provided classification. CONCLUSION: A significant proportion of clinical variants that are novel or uncertain are not shared, limiting the evidence base for new gene-disease relationships. Registration of paired clinical genetic test results with phenotype has the potential to advance knowledge of the relationships between genes and neurodevelopmental disorders.

Redefining piscine lactococcosis.

Piscine lactococcosis is a significant threat to cultured and wild fish populations worldwide. The disease typically presents as a per-acute to acute hemorrhagic septicemia causing high morbidity and mortality, recalcitrant to antimicrobial treatment or management interventions. Historically, the disease was attributed to the gram-positive pathogen Lactococcus garvieae. However, recent work has revealed three distinct lactococcosis-causing bacteria (LCB)-L. garvieae, L. petauri, and L. formosensis-which are phenotypically and genetically similar, leading to widespread misidentification. An update on our understanding of lactococcosis and improved methods for identification are urgently needed. To this end, we used representative isolates from each of the three LCB species to compare currently available and recently developed molecular and phenotypic typing assays, including whole-genome sequencing (WGS), end-point and quantitative PCR (qPCR) assays, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), API 20 Strep and Biolog systems, fatty acid methyl ester analysis (FAME), and Sensititre antimicrobial profiling. Apart from WGS, sequencing of the gyrB gene was the only method capable of consistent and accurate identification to the species and strain level. A qPCR assay based on a putative glycosyltransferase gene was also able to distinguish L. petauri from L. garvieae/formosensis. Biochemical tests and MALDI-TOF MS showed some species-specific patterns in sugar and fatty acid metabolism or protein profiles but should be complemented by additional analyses. The LCB demonstrated overlap in host and geographic range, but there were relevant differences in host specificity, regional prevalence, and antimicrobial susceptibility impacting disease treatment and prevention. IMPORTANCE: Lactococcosis affects a broad range of host species, including fish from cold, temperate, and warm freshwater or marine environments, as well as several terrestrial animals, including humans. As such, lactococcosis is a disease of concern for animal and ecosystem health. The disease is endemic in European and Asian aquaculture but is rapidly encroaching on ecologically and economically important fish populations across the Americas. Piscine lactococcosis is difficult to manage, with issues of vaccine escape, ineffective antimicrobial treatment, and the development of carrier fish or biofilms leading to recurrent outbreaks. Our understanding of the disease is also widely outdated. The accepted etiologic agent of lactococcosis is Lactococcus garvieae. However, historical misidentification has masked contributions from two additional species, L. petauri and L. formosensis, which are indistinguishable from L. garvieae by common diagnostic methods. This work is the first comprehensive characterization of all three agents and provides direct recommendations for species-specific diagnosis and management.

A single shell protein plays a major role in choline transport across the shell of the choline utilization microcompartment of Escherichia coli 536.

Bacterial microcompartments (MCPs) are widespread protein-based organelles that play important roles in the global carbon cycle and in the physiology of diverse bacteria, including a number of pathogens. MCPs consist of metabolic enzymes encapsulated within a protein shell. The main roles of MCPs are to concentrate enzymes together with their substrates (to increase reaction rates) and to sequester harmful metabolic intermediates. Prior studies indicate that MCPs have a selectively permeable protein shell, but the mechanisms that allow selective transport across the shell are not fully understood. Here we examine transport across the shell of the choline utilization (Cut) MCP of Escherichia coli 536, which has not been studied before. The shell of the Cut MCP is unusual in consisting of one pentameric and four hexameric bacterial microcompartment (BMC) domain proteins. It lacks trimeric shell proteins, which are thought to be required for the transport of larger substrates and enzymatic cofactors. In addition, its four hexameric BMC domain proteins are very similar in amino acid sequence. This raises questions about how the Cut MCP mediates the selective transport of the substrate, products and cofactors of choline metabolism. In this report, site-directed mutagenesis is used to modify the central pores (the main transport channels) of all four Cut BMC hexamers to assess their transport roles. Our findings indicate that a single shell protein, CmcB, plays the major role in choline transport across the shell of the Cut MCP and that the electrostatic properties of the CmcB pore also impact choline transport. The implications of these findings with regard to the higher-order structure of MCPs are discussed.

Temperature-associated virulence, species susceptibility and interspecies transmission of a Lactococcus petauri strain from rainbow trout.

Lactococcus petauri is an important emergent aquaculture pathogen in the USA. To better understand environmental conditions conducive to piscine lactococcosis and the susceptibility of fish species, laboratory-controlled challenges were used as models of infection. Rainbow trout Oncorhynchus mykiss maintained at 13 or 18°C were challenged by intracoelomic (ICe) injection with 101, 103 or 105 colony-forming units per fish (CFU fish-1) and monitored for 21 d. At 13°C, trout experienced mortalities of 7, 7 and 0%, and bacterial persistence of 0, 20 and 0% in survivors, respectively. When exposed to the same bacterial doses, trout maintained at 18°C experienced mortalities of 59, 84 and 91%, and bacterial persistence of 60, 66 and 0% in survivors, confirming a significant role of temperature in the pathogenesis of lactococcosis. Additionally, the susceptibility of rainbow trout, Chinook salmon Oncorhynchus tshawytscha, white sturgeon Acipenser transmontanus, Nile tilapia Oreochromis niloticus, and koi Cyprinus carpio to infection by L. petauri was compared using ICe challenges at 18°C. Trout and salmon experienced 96 and 56% cumulative mortality, respectively, and 17% of surviving salmon remained persistently infected. There were no mortalities in the other fish species, and no culturable bacteria recovered at the end of the challenge. However, when surviving fish were used in further cohabitation trials, naïve trout housed with previously exposed tilapia exhibited 6% mortality, demonstrating that non-salmonids can become sub-clinical carriers of this pathogen. The data obtained provide useful information regarding temperature-associated virulence, fish species susceptibility, and potential carrier transmission of L. petauri that can be used in the development of better management practices to protect against piscine lactococcosis.

Concurrent infections of Streptococcus iniae and Aeromonas veronii in farmed Giant snakehead (Channa micropeltes).

The giant snakehead, Channa micropeltes, is an increasingly important economic freshwater fish in Thailand and other regions of Asia. Presently, giant snakehead are cultured under intensive aquaculture conditions, leading to high stress and conditions favouring disease. In this study, we reported a disease outbreak in farmed giant snakehead with a cumulative mortality of 52.5%, continuing for 2 months. The affected fish exhibited signs of lethargy, anorexia and haemorrhage of the skin and eyes. Further bacterial isolations revealed two different types of colonies on tryptic soy agar: small white, punctate colonies of gram-positive cocci and cream-coloured, round and convex colonies of rod-shaped gram-negative bacteria. Additional biochemical and species-specific PCR analysis based on 16S rRNA confirmed the isolates as Streptococcus iniae and Aeromonas veronii. Multilocus sequence analysis (MLSA) placed the S. iniae isolate into a large clade of strains from clinically infected fish worldwide. Gross necropsy findings showed liver congestion, pericarditis and white nodules in the kidney and liver. Histologically, the affected fish showed focal to multifocal granulomas with inflammatory cell infiltration in kidney and liver, enlarged blood vessels with mild congestion within the meninges of the brain and severe necrotizing and suppurative pericarditis with myocardial infarction. Antibiotic susceptibility tests revealed that S. iniae was sensitive to amoxicillin, erythromycin, enrofloxacin, oxytetracycline, doxycycline and resistant to sulfamethoxazole-trimethoprim, while the A. veronii was susceptible to erythromycin, enrofloxacin, oxytetracycline, doxycycline, sulfamethoxazole-trimethoprim and resistant to amoxicillin. Conclusively, our findings highlighted the natural concurrent bacterial infections in cultured giant snakehead, which support the implementation of appropriate treatment and control strategies.

Detection and virulence of Lactococcus garvieae and L. petauri from four lakes in southern California.

OBJECTIVE: The first objective of the study aimed to detect the presence of Lactococcus petauri, L. garvieae, and L. formosensis in fish (n = 359) and environmental (n = 161) samples from four lakes near an affected fish farm in California during an outbreak in 2020. The second objective was to compare the virulence of the Lactococcus spp. in Rainbow Trout Oncorhynchus mykiss and Largemouth Bass Micropterus salmoides. METHODS: Standard bacterial culture methods were used to isolate Lactococcus spp. from brain and posterior kidney of sampled fish from the four lakes. Quantitative PCR (qPCR) was utilized to detect Lactococcus spp. DNA in fish tissues and environmental samples from the four lakes. Laboratory controlled challenges were conducted by injecting fish intracoelomically with representative isolates of L. petauri (n = 17), L. garvieae (n = 2), or L. formosensis (n = 4), and monitored for 14 days postchallenge (dpc). RESULT: Lactococcus garvieae was isolated from the brains of two Largemouth Bass in one of the lakes. Lactococcus spp. were detected in 14 fish (8 Bluegills Lepomis macrochirus and 6 Largemouth Bass) from 3 out of the 4 lakes using a qPCR assay. Of the collected environmental samples, all 4 lakes tested positive for Lactococcus spp. in the soil samples, while 2 of the 4 lakes tested positive in the water samples through qPCR. Challenged Largemouth Bass did not show any signs of infection postinjection throughout the challenge period. Rainbow Trout infected with L. petauri showed clinical signs within 3 dpc and presented a significantly higher cumulative mortality (62.4%; p < 0.0001) at 14 dpc when compared to L. garvieae (0%) and L. formosensis (7.5%) treatments. CONCLUSION: The study suggests that qPCR can be used for environmental DNA monitoring of Lactococcus spp. and demonstrates virulence diversity between the etiological agents of piscine lactococcosis.

Clinical validity assessment of genes frequently tested on intellectual disability/autism sequencing panels.

PURPOSE: Neurodevelopmental disorders (NDDs), such as intellectual disability (ID) and autism spectrum disorder (ASD), exhibit genetic and phenotypic heterogeneity, making them difficult to differentiate without a molecular diagnosis. The Clinical Genome Resource Intellectual Disability/Autism Gene Curation Expert Panel (GCEP) uses systematic curation to distinguish ID/ASD genes that are appropriate for clinical testing (ie, with substantial evidence supporting their relationship to disease) from those that are not. METHODS: Using the Clinical Genome Resource gene-disease validity curation framework, the ID/Autism GCEP classified genes frequently included on clinical ID/ASD testing panels as Definitive, Strong, Moderate, Limited, Disputed, Refuted, or No Known Disease Relationship. RESULTS: As of September 2021, 156 gene-disease pairs have been evaluated. Although most (75%) were determined to have definitive roles in NDDs, 22 (14%) genes evaluated had either Limited or Disputed evidence. Such genes are currently not recommended for use in clinical testing owing to the limited ability to assess the effect of identified variants. CONCLUSION: Our understanding of gene-disease relationships evolves over time; new relationships are discovered and previously-held conclusions may be questioned. Without periodic re-examination, inaccurate gene-disease claims may be perpetuated. The ID/Autism GCEP will continue to evaluate these claims to improve diagnosis and clinical care for NDDs.

Development and efficacy of Streptococcus iniae live-attenuated vaccines in Nile tilapia, Oreochromis niloticus.

Streptococcus iniae is a re-emerging bacterial pathogen in freshwater and marine aquaculture worldwide. There are no commercial vaccines available for S. iniae in the United States, and autogenous vaccines are restricted to inactivated whole-cell preparations with limited protection against heterogenous strains. Live-attenuated vaccines (LAV) represent an advantageous alternative to these bacterins, as they induce robust cellular and humoral immunity, and may provide longer lasting protection through less stressful routes of administration. We investigated whether accumulation of mutations in S. iniae by serial passage in the presence of rifampin can generate immunogenic LAV conferring protection against challenge with heterologous wild-type (WT) S. iniae strains in Nile tilapia (Oreochromis niloticus). Three lineages of rifampin-resistant S. iniae strains were generated from three genetically distinct parent strains (n = 9) by multiple passages in increments of Rifamycin SV sodium salt. Growth in liquid media, extent of capsulation, antimicrobial susceptibility, survival in Nile tilapia whole blood, and cytotoxicity in an O. mossambicus endothelial cell line were compared between the passaged and WT strains. Nile tilapia challenges were used to assess strain virulence, generation of anti-S. iniae IgM, and the protection conferred by LAV candidates against virulent S. iniae. Rifampin-resistant strains demonstrated changes in growth rate and cytotoxicity in endothelial cells, as well as significant reductions in whole blood survival (p < 0.05). Selected strains also showed attenuated virulence in the Nile tilapia challenge model, and anti-S. iniae IgM generated against these strains demonstrated cross-reactivity against heterologous bacteria. Immunization by intracoelomic injection induced protection against a virulent WT strain of S. iniae, with relative percent survival up to 95.05%.

Development of a quantitative polymerase chain reaction assay for detection of the aetiological agents of piscine lactococcosis.

Piscine lactococcosis is an emergent bacterial disease that is associated with high economic losses in many farmed and wild aquatic species worldwide. Early and accurate detection of the causative agent of piscine lactococcosis is essential for management of the disease in fish farms. In this study, a TaqMan quantitative polymerase chain reaction (qPCR) targeting the 16S-23S rRNA internal transcribed spacer region was developed and validated. Validation of the qPCR was performed with DNA of previously typed L. petauri and L. garvieae recovered from different aquatic hosts from distinct geographical locations, closely related bacterial species and common pathogens in trout aquaculture. Further diagnostic sensitivity and specificity was investigated by screening of fish, water and faecal samples. The developed qPCR assay showed high specificity, sensitivity and accuracy in detection of L. petauri and L. garvieae with lack of signals from non-target pathogens, and in screening of rainbow trout (Oncorhynchus mykiss) posterior kidney and environmental samples. The detection limit of the qPCR was four amplicon copies. Moreover, the sensitivity of the qPCR assay was not affected by presence of non-target DNA from either fish or environmental samples. The robustness, specificity and sensitivity of the developed qPCR will facilitate fast and accurate diagnosis of piscine lactococcosis to establish appropriate control measures in fish farms and aquaria.

Nitric oxide (NO) elicits aminoglycoside tolerance in Escherichia coli but antibiotic resistance gene carriage and NO sensitivity have not co-evolved.

The spread of multidrug-resistance in Gram-negative bacterial pathogens presents a major clinical challenge, and new approaches are required to combat these organisms. Nitric oxide (NO) is a well-known antimicrobial that is produced by the immune system in response to infection, and numerous studies have demonstrated that NO is a respiratory inhibitor with both bacteriostatic and bactericidal properties. However, given that loss of aerobic respiratory complexes is known to diminish antibiotic efficacy, it was hypothesised that the potent respiratory inhibitor NO would elicit similar effects. Indeed, the current work demonstrates that pre-exposure to NO-releasers elicits a > tenfold increase in IC50 for gentamicin against pathogenic E. coli (i.e. a huge decrease in lethality). It was therefore hypothesised that hyper-sensitivity to NO may have arisen in bacterial pathogens and that this trait could promote the acquisition of antibiotic-resistance mechanisms through enabling cells to persist in the presence of toxic levels of antibiotic. To test this hypothesis, genomics and microbiological approaches were used to screen a collection of E. coli clinical isolates for antibiotic susceptibility and NO tolerance, although the data did not support a correlation between increased carriage of antibiotic resistance genes and NO tolerance. However, the current work has important implications for how antibiotic susceptibility might be measured in future (i.e. ± NO) and underlines the evolutionary advantage for bacterial pathogens to maintain tolerance to toxic levels of NO.

Contemporary procedure characteristics and outcomes of accessory atrioventricular pathway ablations in an integrated community-based health care system using a tiered approach.

BACKGROUND: Since the early descriptions of large series of accessory atrioventricular pathway ablations in adults and adolescents over 20 years ago, there have been limited published reports based on more recent experiences of large referral centers. We aimed to characterize accessory pathway distribution and features in a large community-based population that influence ablation outcomes using a tiered approach to ablation. METHODS: Retrospective analysis of 289 patients (age 14-81) who underwent accessory ablation from 2015-2019 was performed. Pathways were categorized into anteroseptal, left freewall, posteroseptal, and right freewall locations. We analyzed patient and pathway features to identify factors associated with prolonged procedure time parameters. RESULTS: Initial ablation success rate was 94.7% with long-term success rate of 93.4% and median follow-up of 931 days. Accessory pathways were in left freewall (61.6%), posteroseptal (24.6%), right freewall (9.6%), and anteroseptal (4.3%) locations. Procedure outcome was dependent on pathway location. Acute success was highest for left freewall pathways (97.1%) with lowest case times (144 ± 68 min) and fluoroscopy times (15 ± 19 min). Longest procedure time parameters were seen with anteroseptal, left anterolateral, epicardial-coronary sinus, and right anterolateral pathway ablations. CONCLUSIONS: In this community-based adult and adolescent population, majority of the accessory pathways are in the left freewall and posteroseptal region and tend to be more easily ablated. A tiered approach with initial use of standard ablation equipment before the deployment of more advance tools, such as irrigated tips and 3D mapping, is cost effective without sacrificing overall efficacy.

The association of multimorbidity to mortality in older adults after permanent pacemaker placement.

BACKGROUND: In the United States 2018 bradycardia guideline, the current class III recommendation that patients with permanent pacemaker (PPM) indications and high multimorbidity burden may not have meaningful clinical benefit from PPM therapy is based on limited data. METHODS: Observational study (January 1, 2008-December 31, 2015) of adults ≥65 years (N = 16,678) who underwent PPM implantation. Exposure variable: Elixhauser comorbidity number (ECN, 29 well-validated conditions). PRIMARY OUTCOME: ≤1-year mortality; secondary outcome: > 1-year mortality. RESULTS: Those who died ≤1-year were older, had a lower body mass index (BMI), and higher ECN (p < .001). Cumulative survival at 1-year was 92.3% (95% confidence interval [CI]: 91.9-92.7). One-year survival decreased by increasing ECN-with a difference at 1-year between lowest and highest ECN category of 17.3% (ECN 0-1: 97.1% [95% CI: 96.3-97.7]; ECN ≥8: 79.8% [95% CI: 77.9-81.5]). For those who survived the first year, cumulative survival at 8-years was 51.2% (95% CI = 49.8-52.6) with a difference between ECN 0-1 and ≥8 of 43.4%. Increasing ECN was associated equally with ≤1-year (HR 1.28 [95% CI: 1.25-1.30]) and >1-year (HR 1.19 [95% CI: 1.17-1.20]) mortality. A predictive model including age, sex, BMI, PPM type, race, and ECN had greater discriminative ability (p < .0001) than a bedside model (age, sex) for the primary outcome. CONCLUSION: Across the heterogeneity of indications for PPM placement, multimorbidity is increasingly common. The association of multimorbidity to mortality (≤1-year, >1-year) should be routinely discussed during the shared decision-making process as an important prognostic geriatric domain variable.

Genetic characterization of Flavobacterium columnare isolates from the Pacific Northwest, USA.

Flavobacterium columnare is the causative agent of columnaris disease. Previous work has demonstrated a high degree of genetic variability among F. columnare isolates, identifying 4 genetic groups (GGs) with some host associations. Herein, a total of 49 F. columnare isolates were characterized, the majority of which were collected from 15 different locations throughout the US Pacific Northwest. Most isolates were collected from 2015-2018 and originated from disease outbreaks in salmonid hatcheries and rearing ponds, sturgeon hatcheries and ornamental fish. Other isolates were part of collections recovered from 1980-2018. Initial identification was confirmed by F. columnare species-specific qPCR. Study isolates were further characterized using a multiplex PCR that differentiates between the 4 currently recognized F. columnare GGs. Multiplex PCR results were supported by repetitive sequence-mediated PCR fingerprinting and gyrB sequence analysis. F. columnare GG1 was the most prevalent (83.7%, n = 41/49), represented by isolates from salmonids (n = 32), white sturgeon (n = 2), channel catfish (n = 1), ornamental goldfish (n = 1), koi (n = 3), wild sunfish (n = 1) and 1 unknown host. Six isolates (12.2%, n = 6/49) were identified as GG3, which were cultured from rainbow trout (n = 3) and steelhead trout (n = 3). Two isolates were identified as GG2 (4.1%, n = 2/49) and were from ornamental fish. No GG4 isolates were cultured in this study. The biological significance of this genetic variability remains unclear, but this variation could have significant implications for fish health management. The results from this study provide baseline data for future work developing strategies to ameliorate columnaris-related losses in the US Pacific Northwest.

Development of clinical competence - a longitudinal survey of nurse practitioner students.

BACKGROUND: In order to achieve a sustainable standard of advanced clinical competence for nurse practitioners leading to a credible role, it is important to investigate the development of clinical competence among nurse practitioner students. AIM: The aim of the present study is to analyse the development of nurse practitioner students' self-assessed clinical competence from the beginning of their education to after completion of their clinical studies. DESIGN: The study involved the application of a longitudinal survey design adhering to STROBE guidelines. METHODS: The participants consisted of 36 registered nurses from a nurse practitioner programme at a Norwegian university. The Professional Nurse Self-Assessment Scale II was used for data collection during the period August 2015 to May 2020. RESULTS: The students developed their clinical competence the most for direct clinical practice. Our findings are inconclusive in terms of whether the students developed clinical competence regarding consultation, coaching and guidance, and collaboration. However, they do indicate a lack of development in some aspects of clinical leadership. The students with the lowest level of clinical competence developed their clinical competence regarding direct clinical practice significantly more than the students with the highest level of clinical competence. The differences between students with high and low levels of clinical competence were levelled out during their education. Thus, the students as a whole became a more homogenous group after completion of their clinical studies. Previous work experience in primary healthcare was a statistically significant, yet minor, predictor of the development of clinical competence. CONCLUSION: Our findings indicate that the students developed their clinical competence for direct clinical practice in accordance with the intended learning outcomes of the university's Master's programme and international standards for nurse practitioners. It is imperative that the clinical field supports nurse practitioners by facilitating extended work-task fits that are appropriate to their newly developed clinical competence. We refrain from concluding with a recommendation that prior clinical work experience should be an entry requirement for nurse practitioner programmes. However, we recommend an evaluation of the nurse practitioner education programme with the aim of investigating whether the curriculum meets the academic standards of clinical leadership expected in advanced level of nursing.

Streptococcus dysgalactiae: A Pathogen of Feral Populations of Silver Carp from a Fish Kill Event.

In August 2018, a series of large fish kills involving only Silver Carp Hypophthalmichthys molitrix occurred on the Mississippi River in northern Louisiana. Clinical signs observed in moribund animals included erratic swimming behavior, such as spiraling and spinning at the surface. A moribund specimen was captured by dip net near the surface at Lake Providence Landing in East Carroll Parish, northern Louisiana, and was submitted for analysis. An aseptic necropsy was performed, and diagnostic procedures, including bacteriology, parasitology, histopathology, virology, and electron microscopy, revealed that a gram-positive coccus was the primary pathogen. Pure cultures of the organism were obtained from the brain, and it was the predominant colony type isolated from the spleen, kidney, and liver. Bacterial sepsis caused by the gram-positive coccus and involving multiple organ systems was diagnosed histologically. Bacterial colonization and necrotic lesions were seen in the spleen, liver, kidney, heart, eye, and brain. Numerous cocci were observed dividing intracellularly in phagocytic cells of the kidney and brain by transmission electron microscopy. The organism was identified as Streptococcus dysgalactiae ssp. dysgalactiae by conventional biochemical methods and subsequently by the API 20 Strep system. The identity of the pathogen was later confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and sequencing of the 16S ribosomal RNA gene. Multilocus sequence analysis clustered this isolate along with two other S. dysgalactiae isolates from fish in a divergent phyletic group that was separate from other S. dysgalactiae ssp. dysgalactiae isolates from terrestrial animals, implying a possible novel clade that is pathogenic for fish.

USE OF TOPICAL TREATMENTS AND EFFECTS OF WATER TEMPERATURE ON WOUND HEALING IN COMMON CARP (CYPRINUS CARPIO).

Skin lesions are frequently diagnosed in fish medicine. Although systemic fish treatments exist, little is known about the efficacy of topical drugs on fish skin lesions. This study aimed to investigate the efficacy of medical-grade honey and silver sulfadiazine on skin lesions using common carp (Cyprinus carpio) as a model. Additionally, the effect of temperature on the wound healing process was evaluated. Punch biopsies were generated on six fish per treatment group under anesthesia. Treatment groups received one of the following topical medications after wounding: Dr. Nordyke's Wound Honey, MicroLyte Ag Vet, or SilvaSorb Gel. Nontreated positive control groups were similarly wounded but did not receive topical treatment. Fish were housed at 10°C to 13°C or 18°C to 21°C for 29 days. Macroscopic evaluation and image collection of wounds were performed on days 0, 4, 8, 12, 21, and 29 after wounding to compare changes in wound areas and inflammation over time. On day 29, tissue samples were collected for histologic analysis. From day 12 after wounding onward, wounds in positive controls maintained at 18°C to 21°C were significantly smaller (days 12, 21, and 29: P < 0.0001) compared with positive controls kept at 10°C to 13°C. There was an overall improvement in macroscopic appearance in honey-treated groups compared with positive controls on day 12 after wounding at 18°C to 21°C (P = 0.001), whereas with the use of Microlyte and Silvasorb, wounds had increased inflammation grades (P < 0.0001 and P < 0.0001, respectively) with enlarged wound areas (P < 0.0001 and P < 0.001, respectively) in comparison with positive controls on day 12 after wounding at 18°C to 21°C. This study suggests that topical use of medical-grade honey produces positive effects on wound healing in the carp model and higher water temperatures enhance the effects, whereas the use of silver sulfadiazine and lower water temperatures delays or worsens the wound healing process.

Genetic Characterization of a Glycyl Radical Microcompartment Used for 1,2-Propanediol Fermentation by Uropathogenic Escherichia coli CFT073.

Bacterial microcompartments (MCPs) are widespread protein-based organelles composed of metabolic enzymes encapsulated within a protein shell. The function of MCPs is to optimize metabolic pathways by confining toxic and/or volatile pathway intermediates. A major class of MCPs known as glycyl radical MCPs has only been partially characterized. Here, we show that uropathogenic Escherichia coli CFT073 uses a glycyl radical MCP for 1,2-propanediol (1,2-PD) fermentation. Bioinformatic analyses identified a large gene cluster (named grp for glycyl radical propanediol) that encodes homologs of a glycyl radical diol dehydratase, other 1,2-PD catabolic enzymes, and MCP shell proteins. Growth studies showed that E. coli CFT073 grows on 1,2-PD under anaerobic conditions but not under aerobic conditions. All 19 grp genes were individually deleted, and 8/19 were required for 1,2-PD fermentation. Electron microscopy and genetic studies showed that a bacterial MCP is involved. Bioinformatics combined with genetic analyses support a proposed pathway of 1,2-PD degradation and suggest that enzymatic cofactors are recycled internally within the Grp MCP. A two-component system (grpP and grpQ) is shown to mediate induction of the grp locus by 1,2-PD. Tests of the E. coli Reference (ECOR) collection indicate that >10% of E. coli strains ferment 1,2-PD using a glycyl radical MCP. In contrast to other MCP systems, individual deletions of MCP shell genes (grpE, grpH, and grpI) eliminated 1,2-PD catabolism, suggesting significant functional differences with known MCPs. Overall, the studies presented here are the first comprehensive genetic analysis of a Grp-type MCP.IMPORTANCE Bacterial MCPs have a number of potential biotechnology applications and have been linked to bacterial pathogenesis, cancer, and heart disease. Glycyl radical MCPs are a large but understudied class of bacterial MCPs. Here, we show that uropathogenic E. coli CFT073 uses a glycyl radical MCP for 1,2-PD fermentation, and we conduct a comprehensive genetic analysis of the genes involved. Studies suggest significant functional differences between the glycyl radical MCP of E. coli CFT073 and better-studied MCPs. They also provide a foundation for building a deeper general understanding of glycyl radical MCPs in an organism where sophisticated genetic methods are available.

An 8-year Analysis of Magnesium Status in Elite International Track & Field Athletes.

Magnesium plays a critical role in athlete health and performance. It is involved in numerous physiological mechanisms that support energy production, immune function, pain modulation, muscle function and bone health. Athletes may be susceptible to magnesium deficiency due to an increased utilization during exercise.Objective: This study reports on the magnesium status of 192 Olympic and Paralympic athletes over the course of eight years.Methods: Athletes on the British Athletics world class performance plan undertook blood testing for Red Cell Magnesium status. Their history of tendon pain, muscle and bone injury, ethnicity, sporting event and gender were also recorded. 510 samples from 192 athletes were included in the study.Results: On at least one blood test during the study time, 22% of athletes were identified as clinically deficient (<1.19 mmol/L). The average red cell magnesium concentration was 1.34 nmol/L. Magnesium was significantly lower in female athletes and those with Black or Mixed-Race ethnicity and was higher in Throws athletes and Paralympians with Cerebral Palsy. Athletes with a history of achilles or patella tendon pain had significantly lower magnesium levels than average.Conclusions: This study highlights the importance of investigating magnesium within this population to identify deficiency and support athlete health. Several areas for future work are identified to explore the relationship between magnesium and gender, ethnicity and tendon pain and muscle injury in athletes. Furthermore, new guidelines for magnesium status within athletics populations are proposed.

Electrochemical detection of exogenously administered melatonin in the brain.

Melatonin (MT) is an important electroactive hormone that regulates different physiological actions in the brain, ranging from circadian clock to neurodegeneration. An impressive number of publications have highlighted the effectiveness of MT treatments in different types of sleep and neurological disorders, including Alzheimer's and Parkinson's disease. The ability to detect MT in different regions of the brain would provide further insights into the physiological roles and therapeutic effects of MT. While multiple electrochemical methods have been used to detect MT in biological samples, monitoring MT in the brain of live animals has not been demonstrated. Here, we optimized a square wave voltammetry (SWV) electroanalytical method to evaluate the MT detection performance at CFEs in vitro and in vivo. SWV was able to sensitively detect the MT oxidation peak at 0.7 V, and discriminate MT from most common interferents in vitro. More importantly, using the optimized SWV, CFEs successfully detected and reliably quantified MT concentrations in the visual cortex of anesthetized mice after intraperitoneal injections of different MT doses, offering stable MT signals for up to 40 minutes. To the best of our knowledge, this is the first electrochemical measurement of exogenously administered MT in vivo. This electrochemical MT sensing technique will provide a powerful tool for further understanding MT's action in the brain.

Multilocus sequence analysis of diverse Streptococcus iniae isolates indicates an underlying genetic basis for phenotypic heterogeneity.

Streptococcus iniae is a Gram-positive, opportunistically zoonotic bacterium infective to a wide variety of farmed and wild fish species worldwide. Outbreaks in wild fish can have detrimental environmental and cultural impacts, and mortality events in aquaculture can result in significant economic losses. As an emerging or re-emerging pathogen of global significance, understanding the coalescing factors contributing to piscine streptococcosis is crucial for developing strategies to control infections. Intraspecific antigenic and genetic variability of S. iniae has made development of autogenous vaccines a challenge, particularly where the diversity of locally endemic S. iniae strains is unknown. This study genetically and phenotypically characterized 11 S. iniae isolates from diseased wild and farmed fish from North America, Central America, and the Caribbean. A multilocus sequence analysis (MLSA) scheme was developed to phylogenetically compare these isolates to 84 other strains of Streptococcus spp. relevant to aquaculture. MLSA generated phylogenies comparable to established genotyping methods, and isolates formed distinct clades related to phenotype and host species. The endothelial Oreochromis mossambicus bulbus arteriosus cell line and whole blood from rainbow trout Oncorhynchus mykiss, Nile tilapia Oreochromis niloticus, and white sturgeon Acipenser transmontanus were used to investigate the persistence and virulence of the 11 isolates using in vitro assays. In vivo challenges using an O. niloticus model were used to evaluate virulence by the intragastric route of infection. Isolates showed significant differences (p < 0.05) in virulence and persistence, with some correlation to genogroup, establishing a basis for further work uncovering genetic factors leading to increased pathogenicity.