A New Species of the γ-Proteobacterium Francisella, F. adeliensis Sp. Nov., Endocytobiont in an Antarctic Marine Ciliate and Potential Evolutionary Forerunner of Pathogenic Species.

The study of the draft genome of an Antarctic marine ciliate, Euplotes petzi, revealed foreign sequences of bacterial origin belonging to the γ-proteobacterium Francisella that includes pathogenic and environmental species. TEM and FISH analyses confirmed the presence of a Francisella endocytobiont in E. petzi. This endocytobiont was isolated and found to be a new species, named F. adeliensis sp. nov.. F. adeliensis grows well at wide ranges of temperature, salinity, and carbon dioxide concentrations implying that it may colonize new organisms living in deeply diversified habitats. The F. adeliensis genome includes the igl and pdp gene sets (pdpC and pdpE excepted) of the Francisella pathogenicity island needed for intracellular growth. Consistently with an F. adeliensis ancient symbiotic lifestyle, it also contains a single insertion-sequence element. Instead, it lacks genes for the biosynthesis of essential amino acids such as cysteine, lysine, methionine, and tyrosine. In a genome-based phylogenetic tree, F. adeliensis forms a new early branching clade, basal to the evolution of pathogenic species. The correlations of this clade with the other clades raise doubts about a genuine free-living nature of the environmental Francisella species isolated from natural and man-made environments, and suggest to look at F. adeliensis as a pioneer in the Francisella colonization of eukaryotic organisms.

Molecular Diagnosis of Hemorrhagic Fever with Renal Syndrome Caused by Puumala Virus.

Rodent-borne hantaviruses cause two severe acute diseases: hemorrhagic fever with renal syndrome (HFRS) in Eurasia, and hantavirus pulmonary syndrome (HPS; also called hantavirus cardiopulmonary syndrome [HCPS]) in the Americas. Puumala virus (PUUV) is the most common causative agent of HFRS in Europe. Current routine diagnostic methods are based on serological analyses and can yield inconclusive results. Hantavirus-infected patients are viremic during the early phase of disease; therefore, detection of viral RNA genomes can be a valuable complement to existing serological methods. However, the high genomic sequence diversity of PUUV has hampered the development of molecular diagnostics, and currently no real-time reverse transcription-quantitative (RT)-PCR assay is available for routine diagnosis of HFRS. Here, we present a novel PUUV RT-PCR assay. The assay was validated for routine diagnosis of HFRS on samples collected in Sweden during the winter season from 2013 to 2014. The assay allowed detection of PUUV RNA in 98.7% of confirmed clinical HFRS samples collected within 8 days after symptomatic onset. In summary, this study shows that real-time RT-PCR can be a reliable alternative to serological tests during the early phase of HFRS.

Humic substances cause fluorescence inhibition in real-time polymerase chain reaction.

Real-time polymerase chain reaction (qPCR) is the cornerstone of DNA analysis, enabling detection and quantification of minute nucleic acid amounts. However, PCR-based analysis is limited, in part, by the presence of inhibitors in the samples. PCR inhibition has been viewed solely as failure to efficiently generate amplicons, that is, amplification inhibition. Humic substances (HS) are well-known inhibitors of PCR amplification. Here we show that HS from environmental samples, specifically humic acid (HA), are very potent detection inhibitors, that is, quench the fluorescence signal of double-stranded DNA (dsDNA) binding dyes. HA quenched the fluorescence of the commonly used qPCR dyes EvaGreen, ResoLight, SYBR Green I, and SYTO 82, generating lowered amplification plots, although amplicon production was unaffected. For EvaGreen, 500 ng of HA quenched nearly all fluorescence, whereas 1000 ng of HA completely inhibited amplification when applying Immolase DNA polymerase with bovine serum albumin (BSA). Fluorescence spectroscopy measurements showed that HA quenching was either static or collisional and indicated that HA bound directly to the dye. Fulvic acid did not act as a qPCR detection inhibitor but inhibited amplification similarly to HA. Hydrolysis probe fluorescence was not quenched by HA. Detection inhibition is an overlooked phenomenon that needs to be considered to allow for development of optimal qPCR assays.

Wrestling with a ghost: facing an opponent I can neither see nor clinch - the experience of professional wrestlers who have suffered an ACL injury.

This study explored professional wrestlers' experiences of the consequences of an anterior cruciate ligament (ACL) injury and their perception of whether the ACL injury could have been prevented. We interviewed 10 professional wrestlers (60% women, age range 21-34) treated with ACL reconstruction with semistructured interviews. Transcripts were analysed using qualitative content analysis: One major theme, 'Wrestling with a ghost: facing an opponent I can neither see nor clinch', supported by five main categories, emerged from the collected data. The five main categories were: My ACL injury: bad luck or bad planning?; The way back: a fight to return to sport; Only performance counts; The injury's impact on life: a wrestling with emotions; In hindsight, personal growth. Professional wrestlers who experienced an ACL injury expressed that not only the injury itself but also the subsequent recovery posed major challenges that they did not know how to deal with and that, in some cases, ended the athletes' wrestling careers. Professional wrestlers attributed their ACL injuries to bad luck or large training loads and wished that they had more support from the wrestling community when injured.

Cerebrospinal fluid cathepsin B and S.

Cathepsins are increased in the brain of elderly animals. We investigate the presence of cathepsin B and S in human cerebrospinal fluid (CSF) plasma and the associations with cystatin C, age and sex. We measured cathepsin B and S concentrations in CSFs from 118 persons, undergoing elective surgical procedures, with ELISA. Both cathepsin B and cathepsin S were positively correlated with age. No correlation was observed between cathepsin B or S and length, height or body mass index. Both cathepsin B and S were positively correlated to the cystatin C concentration in CSF. Calculated reference intervals were 4,893-17,636 pg/mL for cathepsin B and 2,681-11,459 pg/mL for cathepsin S. Elderly individuals had significantly higher levels of both cathepsin B (r s = 0.38, p = 0.00002) and cathepsin S (r s = 0.35, p = 0.0001) in CSF.

"I See What You Mean"-A Case Study of the Interactional Foundation of Building a Working Alliance in Care Decisions Involving an Older Couple Living with Cognitive Decline.

BACKGROUND: Social workers have a key role in needs assessment meetings with families dealing with dementia, providing information, support, and advocacy, while also assessing needs and making decisions about care services for several parties. These contacts are especially important during the introduction of home care services, where often the person has previously relied on informal support from relatives. The needs assessment process entails the involvement of all present parties, with the aim to reach a mutual agreement, a working alliance, regarding which services to apply for. PURPOSE: The aim of this case study is to explore how the participants, by means of different conversational practices, jointly create a working alliance between the different parties in one family. The study provides insights into the process of co-constructing a working alliance in the needs assessment process for elder care services. METHODS: This article addresses the process by which social workers build a working alliance in a multi-party conversation with a family living with cognitive decline; a meeting that lasted 50 min. In this case study, we benefit from an inductive and detailed conversation analytic methodology. The theoretical framework of working alliances in institutional interaction has informed the analysis. RESULTS: The findings illustrate how the social worker in this case study involves all parties in the decision regarding care services and explores the use of the conversational practices of mitigations, positive framing, adding information, and positioning, as a "we" achieve mutual agreement toward the end of several sequences. CONCLUSIONS: Drawing on the results of this case study, we argue that multi-party interaction involving relatives enables diversity in role-taking, where the professional, for instance, can pursue a more empathic role. Also, our results indicate that minimal agreement to a proposal is sufficient in a multi-party interaction involving clients with cognitive decline.

Excitatory inputs to four types of spinocerebellar tract neurons in the cat and the rat thoraco-lumbar spinal cord.

The cerebellum receives information from the hindlimbs through several populations of spinocerebellar tract neurons. Although the role of these neurons has been established in electrophysiological experiments, the relative contribution of afferent fibres and central neurons to their excitatory input has only been estimated approximately so far. Taking advantage of differences in the immunohistochemistry of glutamatergic terminals of peripheral afferents and of central neurons (with vesicular glutamate transporters VGLUT1 or VGLUT2, respectively), we compared sources of excitatory input to four populations of spinocerebellar neurons in the thoraco-lumbar spinal cord: dorsal spinocerebellar tract neurons located in Clarke's column (ccDSCT) and in the dorsal horn (dhDSCT) and ventral spinocerebellar tract (VSCT) neurons including spinal border (SB) neurons. This was done on 22 electrophysiologically identified intracellularly labelled neurons in cats and on 80 neurons labelled by retrograde transport of cholera toxin b subunit injected into the cerebellum of rats. In both species distribution of antibodies against VGLUT1 and VGLUT2 on SB neurons (which have dominating inhibitory input from limb muscles), revealed very few VGLUT1 contacts and remarkably high numbers of VGLUT2 contacts. In VSCT neurons with excitatory afferent input, the number of VGLUT1 contacts was relatively high although VGLUT2 contacts likewise dominated, while the proportions of VGLUT1 and VGLUT2 immunoreactive terminals were the reverse on the two populations of DSCT neurons. These findings provide morphological evidence that SB neurons principally receive excitatory inputs from central neurons and provide the cerebellum with information regarding central neuronal activity.

Framing dementia experiences in a positive light: Conversational practices in one couple living with dementia.

The general approach to a life with dementia is negatively charged, and alternative views are rarely found in research or in media coverage. This case-study explores conversational practices for framing dementia in a more positive light, employed by a husband of a wife with dementia. Framing regards the structured experiences of dementia, drawing on Goffman's 'Frame Analysis'. Benefitting from conversation analysis, this article presents principal results of four conversational practices used by the spouse without dementia: mitigating trouble, normalising trouble, justifying trouble, and praising. The conclusions drawn are that the practices contribute to the challenging of the dominant negative framework of the dementia experience, as they facilitate talk which emphasises the wife with dementia's positive progression and skills in managing the household chores. Despite a positive framing of dementia, this couple still embed their talk in the overall negative framework of loss and decreased cognitive competence. The visualisation of a positive framing could add to a broadened view of dementia, which in turn could contribute to greater well-being for those affected. However, the results may also imply a risk of one spouse's conversational practices of normalising and mitigating trouble being dominant in interaction and thereby neglecting the other spouse's experience of the situation.

Bilateral postsynaptic actions of pyramidal tract and reticulospinal neurons on feline erector spinae motoneurons.

Trunk muscles are important for postural adjustments associated with voluntary movements but little has been done to analyze mechanisms of supraspinal control of these muscles at a cellular level. The present study therefore aimed to investigate the input from pyramidal tract (PT) neurons to motoneurons of the musculus longissimus lumborum of the erector spinae and to analyze to what extent it is relayed by reticulospinal (RS) neurons. Intracellular records from motoneurons were used to evaluate effects of electrical stimulation of medullary pyramids and of axons of RS neurons descending in the medial longitudinal fasciculus (MLF). The results revealed that similar synaptic actions were evoked from the ipsilateral and contralateral PTs, including disynaptic and trisynaptic EPSPs and trisynaptic IPSPs. Stimulation of the MLF-evoked monosynaptic and disynaptic EPSPs and disynaptic or trisynaptic IPSPs in the same motoneurons. All short-latency PSPs of PT origin were abolished by transection of the MLF, while they remained after transection of PT fibers at a spinal level. Hence, RS neurons might serve as the main relay neurons of the most direct PT actions on musculus (m.) longissimus. However, longer-latency IPSPs remaining after MLF or PT spinal lesions and after ipsilateral or contralateral hemisection of spinal cord indicate that PT actions are also mediated by ipsilaterally and/or contralaterally located spinal interneurons. The bilateral effects of PT stimulation thereby provide an explanation why trunk movements after unilateral injuries of PT neurons (e.g., stroke) are impaired to a lesser degree than movements of the extremities.

A novel polypeptide derived from human lactoferrin in sodium hyaluronate prevents postsurgical adhesion formation in the rat.

OBJECTIVE: The objective of the study was to evaluate whether a peptide derived from human lactoferrin, PXL01 could act safely to reduce the formation of peritoneal adhesions in the rat model and to map the molecular mechanisms of its action. SUMMARY BACKGROUND DATA: Adhesion formation is a significant problem within every surgical discipline causing suffering for the patients and major cost for the society. For many decades, attempts have been made to reduce postsurgical adhesions by reducing surgical trauma. It is now believed that major improvements in adhesion prevention will only be reached by developing dedicated antiscarring products, which are administrated in connection to the surgical intervention. METHODS: Anti-inflammatory as well as fibrinolytic activities of PXL01 were studied in relevant human cell lines. Using the sidewall defect-cecum abrasion model in the rat, the adhesion prevention properties of PXL01 formulated in sodium hyaluronate were evaluated. Large bowel anastomosis healing model in the rat was applied to study if PXL01 would have any negative effects on intestine healing. RESULTS: PXL01 exhibits an inhibitory effect on the most important hallmarks of scar formation by reducing infections, prohibiting inflammation, and promoting fibrinolysis. PXL01 formulated in sodium hyaluronate markedly reduced formation of peritoneal adhesions in rat without any adverse effects on wound healing. CONCLUSIONS: A new class of synthetically derived water soluble low molecular weight peptide compound, PXL01 showed marked reduction of peritoneal adhesion formation in an animal model without any negative effects on healing. On the basis of these data, a comprehensive adhesion prevention regimen in clinical situation is expected.

Quantitative analysis of polypeptide pharmaceuticals by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

An accurate method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been developed for quantitative analysis of calcitonin and insulin in different commercially available pharmaceutical products. Tryptic peptides derived from these polypeptides were chemically modified at their C-terminal lysine-residues with 2-methoxy-4,5-dihydro-imidazole (light tagging) as standard and deuterated 2-methoxy-4,5-dihydro-imidazole (heavy tagging) as internal standard (IS). The heavy modified tryptic peptides (4D-Lys tag), differed by four atomic mass units from the corresponding light labelled counterparts (4H-Lys tag). The normalized peak areas (the ratio between the light and heavy tagged peptides) were used to construct a standard curve to determine the concentration of the analytes. The concentrations of calcitonin and insulin content of the analyzed pharmaceutical products were accurately determined, and less than 5% error was obtained between the present method and the manufacturer specified values. It was also found that the cysteine residues in CSNLSTCVLGK from tryptic calcitonin were converted to lanthionine by the loss of one sulfhydryl group during the labelling procedure.

More than 10 years' continuous oral treatment with specific immunoglobulin Y for the prevention of Pseudomonas aeruginosa infections: a case report.

Immunotherapy with specific antibodies is an alternative to antibiotics for the prevention of infections in humans and animals. We have used orally administered immunoglobulin Y (IgY) preparations, purified from eggs of hens immunized with Pseudomonas aeruginosa bacteria, to prevent pulmonary P. aeruginosa infections in a group of patients with cystic fibrosis (CF). Respiratory infections are major problems for CF patients because of the thick mucus in the airways, and chronic P. aeruginosa lung infections occur in virtually all CF patients and cause morbidity and mortality. The IgY-treated group had only 2.5 P. aeruginosa-positive sputum cultures per 100 months, and none of the IgY-treated patients became chronically colonized with P. aeruginosa. In the control group, 13.7 of the cultures per 100 months were positive for P. aeruginosa, and 24% of patients became chronically colonized with P. aeruginosa. The first enrolled patient in this study has now been treated continuously for more than 10 years. During the first 8 years she only had four P. aeruginosa-positive cultures. After 8 years she became chronically infected, but still after 10 years the bacteria have not turned mucoid. No negative side effects of IgY treatment have been noted during these 10 years. To our knowledge this is the longest treatment with specific yolk antibodies for therapeutic purposes.

Adverse effects of psychological therapy: An exploratory study of practitioners' experiences from child and adolescent psychiatry.

The scientific knowledge about adverse effects of psychological therapies and how such effects should be detected is limited. It is possible that children and adolescents are particularly vulnerable and need specific support in order to express adverse effects. In this exploratory study, we used a qualitative approach to explore practitioners' experiences of this phenomenon. Fourteen practitioners providing psychological therapy within the Child and Adolescent Psychiatric Service were interviewed. Qualitative content analysis was applied to the data. Four overarching categories brought up by the practitioners were identified: vagueness of the concept (reflecting that the concept was novel and hard to define), psychotherapist-client interaction (encompassing aspects of the interaction possibly related to adverse effects), consequences for the young person (including a range of emotional, behavioural and social consequences) and family effects (e.g. professional complications and decreased autonomy for the parent). Professional discussions on these issues could improve psychological therapy for children and adolescents. Based on our findings and previous research, we propose three basic aspects to consider when adverse effects are detected and managed in this context: typology (form, severity and duration), aetiology (hypothesis about the causes) and perspective (adverse effects seen from the points of view of different interested parties).

Long-range dispersal moved Francisella tularensis into Western Europe from the East.

For many infections transmitting to humans from reservoirs in nature, disease dispersal patterns over space and time are largely unknown. Here, a reversed genomics approach helped us understand disease dispersal and yielded insight into evolution and biological properties of Francisella tularensis, the bacterium causing tularemia. We whole-genome sequenced 67 strains and characterized by single-nucleotide polymorphism assays 138 strains, collected from individuals infected 1947-2012 across Western Europe. We used the data for phylogenetic, population genetic and geographical network analyses. All strains (n=205) belonged to a monophyletic population of recent ancestry not found outside Western Europe. Most strains (n=195) throughout the study area were assigned to a star-like phylogenetic pattern indicating that colonization of Western Europe occurred via clonal expansion. In the East of the study area, strains were more diverse, consistent with a founder population spreading from east to west. The relationship of genetic and geographic distance within the F. tularensis population was complex and indicated multiple long-distance dispersal events. Mutation rate estimates based on year of isolation indicated null rates; in outbreak hotspots only, there was a rate of 0.4 mutations/genome/year. Patterns of nucleotide substitution showed marked AT mutational bias suggestive of genetic drift. These results demonstrate that tularemia has moved from east to west in Europe and that F. tularensis has a biology characterized by long-range geographical dispersal events and mostly slow, but variable, replication rates. The results indicate that mutation-driven evolution, a resting survival phase, genetic drift and long-distance geographical dispersal events have interacted to generate genetic diversity within this species.

Couples with dementia: Positioning the 'we'.

The aim of this article is to investigate how spouses in couples with dementia position themselves in relation to each other by analysing their use of pronouns, especially the we. The study uses joint interviews with 11 couples. Based on a quantitative analysis of pronoun use, it is argued that the pronoun we is used by all the spouses; however, it is used less frequently by the spouses with dementia in comparison with healthy spouses. A qualitative analysis of the use of the pronoun we shows that the spouses position, experience and consider themselves as a couple and that they position and experience themselves as individuals in relation to the couple. One of the challenges for couples with dementia is to be able to retain a we in face of the progression of the dementia disease. By positioning themselves in various ways, the spouses establish and negotiate quite a complex and emotionally charged web of relationships.

Efficacy and safety profile of the novel antimicrobial peptide PXL150 in a mouse model of infected burn wounds.

The urgent need to develop novel antimicrobial therapies has stimulated interest in antimicrobial peptides as therapeutic candidates for the treatment of infectious diseases. The aim of this study was to evaluate the anti-infectious effect of the synthetic antimicrobial peptide PXL150, formulated in hydroxypropyl cellulose (HPC) gel, on Pseudomonas aeruginosa in vitro and in an in vivo mouse model of infected burn wounds as well as to assess the in vivo safety profile of PXL150 in rats and rabbits. Minimal microbicidal concentration analysis showed prominent efficacy of PXL150 against P. aeruginosa in vitro, which was further enhanced in formulating the peptide in HPC gel. Application of 1.25, 2.5, 5, 10 and 20mg/g PXL150 in HPC gel twice daily for four consecutive days significantly reduced bacterial counts in the burn wounds compared with non-treated or placebo-treated controls. Continuous bioluminescence measurements of the bacteria revealed a pronounced anti-infective effect already at the first day post infection by PXL150 in concentrations of ≥2.5mg/g. In the non-clinical safety studies, PXL150 showed a favourable safety profile following repeated administration systemically and locally in rats and rabbits, respectively. In conclusion, these data support that PXL150 has the potential to be an effective and safe drug candidate for the treatment of infected burn wounds. The findings encourage the progression of PXL150 as a novel topical treatment of microbial infections.

Complete Genome Sequence of Francisella guangzhouensis Strain 08HL01032T, Isolated from Air-Conditioning Systems in China.

We present the complete genome sequence of Francisella guangzhouensis strain 08HL01032(T), which consists of one chromosome (1,658,482 bp) and one plasmid (3,045 bp) with G+C contents of 32.0% and 28.7%, respectively.

Complete Genome Sequence of Francisella endociliophora Strain FSC1006, Isolated from a Laboratory Culture of the Marine Ciliate Euplotes raikovi.

A strain of Francisella endociliophora was isolated from a laboratory culture of the marine ciliate Euplotes raikovi. Here, we report the complete genome sequence of the bacterial strain FSC1006 (Francisella Strain Collection, Swedish Defence Research Agency, Umeå, Sweden).

Mechanical compression and nucleus pulposus application on dorsal root Ganglia differentially modify evoked neuronal activity in the thalamus.

A combination of mechanical compression caused by a protruding disc and leakage of nucleus pulposus (NP) from the disc core is presumed to contribute to intervertebral disc hernia-related pain. Experimental models of disc hernia including both components have resulted in changes in neuronal activity at the level of the dorsal root ganglion (DRG) and spinal cord, but changes within the brain have been less well studied. However, acute application of NP to a DRG without mechanical compression rapidly increases neuronal activity in the thalamus, a major brain relay nucleus processing information from sensory pathways including ascending nociceptive tracts. The combination of mechanical compression and NP might therefore result in further increases in central neuronal activity. Using an experimental disc herniation rat model including both mechanical compression and NP the present study aimed to investigate changes in neuronal activity in the contralateral thalamic ventral posterior lateral nucleus in vivo. Measurements were obtained while electrically stimulating the ipsilateral sciatic nerve at Aδ fiber intensities. The L4 DRG was subjected to light mechanical compression and NP exposure, and acute changes in evoked thalamic responses were recorded for up to 40 min. In order to compare effects in naïve animals with effects following a longer period of NP exposure, animals that were either disc-punctured or sham-operated 24 h previously were also included. In all animals, light mechanical compression of the DRG depressed the number of evoked neuronal responses. Prior NP exposure resulted in less potent changes following mechanical compression (80% of baseline) than that observed in naïve animals (50%). During the subsequent NP application, the number of evoked responses compared to baseline increased in pre-exposed animals (to 87%) as well as in naïve animals (72%) in which the removal of the mechanical compression resulted in a further increase (106%). The contribution of acute DRG compression and disc material leakage to changes in transmission in central neuronal networks is likely to be complex and to involve both short-term and long-term effects. Since a light mechanical compression may reduce transmission in nociceptive pathways, it is possible that the presence or absence of NP is crucial for pain development in the acute phase of disc herniation.

Development and evaluation of a broad reacting SYBR-green based quantitative real-time PCR for the detection of different hantaviruses.

BACKGROUND: Hantaviruses are endemic in most parts of the world and cause hundreds of thousand human cases of hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) annually throughout Eurasia and the Americas. They are zoonotic viruses, most commonly transmitted to humans by aerosolized rodent excreta. New hantaviruses are frequently discovered in previously unknown reservoir species and geographic areas. Consequently, there is a need to improve hantavirus diagnostics. OBJECTIVES: This paper describes the design and evaluation of a rapid and robust quantitative real-time PCR (QRT-PCR) assay able to detect a wide range of hantaviruses. STUDY DESIGN: Primers with the potential to detect different hantaviruses were designed from conserved regions of different hantavirus L segments, as identified from multiple sequence alignments. RESULTS: By using SYBR-green-based QRT-PCR 100-1000 target molecules of in vitro produced RNA and less than 100 copies of hantavirus RNA from different hantavirus clades and regions of the world were detected. When using the assay on clinical samples from patients with acute HFRS, Puumala hantavirus (PUUV) RNA was confirmed in all previously positive samples. Notably, the broad reacting L-segment QRT-PCR also detected viral RNA in HFRS patient samples, previously negative by a QRT-PCR targeting the S segment of PUUV. CONCLUSIONS: This novel assay provides a powerful tool for diagnosis of hantaviruses from different clades and regions and may also be useful in surveys with the purpose of finding new hantaviruses in rodent or insectivore species.