Microbial Ecology

Bacterial Community of the Digestive Tract of the European Medicinal Leech ( Hirudo verbana ) from the Danube River The original published version of this article had mistakes in figure legends. Correct figure legends are presented below.

" Candidatus Hafkinia simulans" gen. nov., sp. nov., a Novel Holospora -Like Bacterium from the Macronucleus of the Rare Brackish Water Ciliate Frontonia salmastra (Oligohymenophorea, Ciliophora): Multidisciplinary Characterization of the New Endosymbiont and Its Host Abstract We characterized a novel Holospora-like bacterium (HLB) (Alphaproteobacteria, Holosporales) living in the macronucleus of the brackish water ciliate Frontonia salmastra. This bacterium was morphologically and ultrastructurally investigated, and its life cycle and infection capabilities were described. We also obtained its 16S rRNA gene sequence and performed in situ hybridization experiments with a specifically-designed probe. A new taxon, "Candidatus Hafkinia simulans", was established for this HLB. The phylogeny of the family Holosporaceae based on 16S rRNA gene sequences was inferred, adding to the already available data both the sequence of the novel bacterium and those of other Holospora and HLB species recently characterized. Our phylogenetic analysis provided molecular support for the monophyly of HLBs and placed the new endosymbiont as the sister genus of Holospora. Additionally, the host ciliate F. salmastra, recorded in Europe for the first time, was concurrently described through a multidisciplinary study. Frontonia salmastra's phylogenetic position in the subclass Peniculia and the genus Frontonia was assessed according to 18S rRNA gene sequencing. Comments on the biodiversity of this genus were added according to past and recent literature.

Soil Properties and Multi-Pollution Affect Taxonomic and Functional Bacterial Diversity in a Range of French Soils Displaying an Anthropisation Gradient Abstract The intensive industrial activities of the twentieth century have left behind highly contaminated wasteland soils. It is well known that soil parameters and the presence of pollutants shape microbial communities. But in such industrial waste sites, the soil multi-contamination with organic (polycyclic aromatic hydrocarbons, PAH) and metallic (Zn, Pb, Cd) pollutants and long-term exposure may induce a selection pressure on microbial communities that may modify soil functioning. The aim of our study was to evaluate the impact of long-term multi-contamination and soil characteristics on bacterial taxonomic and functional diversity as related to the carbon cycle. We worked on 10 soils from northeast of France distributed into three groups (low anthropised controls, slag heaps, and settling ponds) based on their physico-chemical properties (texture, C, N) and pollution level. We assessed bacterial taxonomic diversity by 16S rDNA Illumina sequencing, and functional diversity using Biolog® and MicroResp™ microtiter plate tools. Although taxonomic diversity at the phylum level was not different among the soil groups, many operational taxonomic units were influenced by metal or PAH pollution, and by soil texture and total nitrogen content. Functional diversity was not influenced by PAH contamination while metal pollution selected microbial communities with reduced metabolic functional diversity but more tolerant to zinc. Limited microbial utilisation of carbon substrates in metal-polluted soils was mainly due to the nitrogen content. Based on these two observations, we hypothesised that reduced microbial activity and lower carbon cycle–related functional diversity may have contributed to the accumulation of organic matter in the soils that exhibited the highest levels of metal pollution.

Bacterial Community of the Digestive Tract of the European Medicinal Leech ( Hirudo verbana ) from the Danube River Abstract The digestive tract of medicinal leeches from commercial suppliers has been investigated previously and comprises of a relatively simple bacterial community. However, the microbiome of medicinal leeches collected directly from the natural habitat has not been examined. In this study, we characterized the bacterial community in the digestive tract (anterior crop, posterior crop, and intestine) of the European medicinal leech, Hirudo verbana, collected from the Danube river using culture-independent and culture-dependent approaches. Culture-independent approach confirmed that the digestive tract of H. verbana carries a relatively simple bacterial community with species richness in the individual samples ranging from 43 to164. The dominant bacterial taxon was Mucinivorans sp. (49.7% of total reads), followed by Aeromonas sp. (18.7% of total reads). Several low abundance taxa, new for H. verbana, such as Phreatobacter, Taibaiella, Fluviicola, Aquabacterium, Burkholderia, Hydrogenophaga, Wolinella, and unidentified Chitinophagia, were also detected. The aerobic culturing approach showed Aeromonas veronii (Proteobacteria), the known leech symbiont, as the most dominant taxon followed by several Pseudomonas and Acidovorax spp. No significant differences in the bacterial community composition were detected among different parts of the digestive tract of individual leeches. However, the overall composition of the bacterial community among individual specimen varied significantly and this is possibly due to differences in leech age, feeding status, and blood source. Our results showed that the core bacterial community of H. verbana collected from the natural habitat is similar to that reported from the digestive tract of commercially supplied leeches maintained in the laboratory.

Experimental Testing of Dispersal Limitation in Soil Bacterial Communities with a Propagule Addition Approach Abstract The role of dispersal in the assembly of microbial communities remains contentious. This study tested the importance of dispersal limitation for the structuring of local soil bacterial communities using an experimental approach of propagule addition. Microbes extracted from soil pooled from samples collected at 20 localities across ~ 400 km in a temperate steppe were added to microcosms of local soils at three sites; the microcosms were then incubated in situ for 3 months. We then assessed the composition and diversity of bacterial taxa in the soils using 16S rRNA gene amplicon sequencing. The addition of the regional microbial pool did not cause significant changes in the overall composition or diversity of the total bacterial community, although a very small number of individual taxa may have been affected by the addition treatment. Our results suggest a negligible role of dispersal limitation in structuring soil bacterial communities in our study area.

Early Changes in Nutritional Conditions Affect Formation of Synthetic Mutualism Between Chlorella sorokiniana and the Bacterium Azospirillum brasilense Abstract The effect of three different nutritional conditions during the initial 12 h of interaction between the microalgae Chlorella sorokiniana UTEX 2714 and the plant growth–promoting bacterium Azospirillum brasilense Cd on formation of synthetic mutualism was assessed by changes in population growth, production of signal molecules tryptophan and indole-3-acetic acid, starch accumulation, and patterns of cell aggregation. When the interaction was supported by a nutrient-rich medium, production of both signal molecules was detected, but not when this interaction began with nitrogen-free (N-free) or carbon-free (C-free) media. Overall, populations of bacteria and microalgae were larger when co-immobilized. However, the highest starch production was measured in C. sorokiniana immobilized alone and growing continuously in a C-free mineral medium. In this interaction, the initial nutritional condition influenced the time at which the highest accumulation of starch occurred in Chlorella, where the N-free medium induced faster starch production and the richer medium delayed its accumulation. Formation of aggregates made of microalgae and bacteria occurred in all nutritional conditions, with maximum at 83 h in mineral medium, and coincided with declining starch content. This study demonstrates that synthetic mutualism between C. sorokiniana and A. brasilense can be modulated by the initial nutritional condition, mainly by the presence or absence of nitrogen and carbon in the medium in which they are interacting.

Genetic and Genome Analyses Reveal Genetically Distinct Populations of the Bee Pathogen Nosema ceranae from Thailand Abstract The recent global decline in Western honeybee (Apis mellifera) populations is of great concern for pollination and honey production worldwide. Declining honeybee populations are frequently infected by the microsporidian pathogen Nosema ceranae. This species was originally described in the Asiatic honeybee (Apis cerana), and its identification in global A. mellifera hives could result from a recent host transfer. Recent genome studies have found that global populations of this parasite are polyploid and that humans may have fueled their global expansion. To better understand N. ceranae biology, we investigated its genetic diversity within part of their native range (Thailand) and among different hosts (A. mellifera, A. cerana) using both PCR and genome-based methods. We find that Thai N. ceranae populations share many SNPs with other global populations and appear to be clonal. However, in stark contrast with previous studies, we found that these populations also carry many SNPs not found elsewhere, indicating that these populations have evolved in their current geographic location for some time. Our genome analyses also indicate the potential presence of diploidy within Thai populations of N. ceranae.

Population and Culture Age Influence the Microbiome Profiles of House Dust Mites Abstract Interactions with microorganisms might enable house dust mites (HDMs) to derive nutrients from difficult-to-digest structural proteins and to flourish in human houses. We tested this hypothesis by investigating the effects of changes in the mite culture growth and population of two HDM species on HDM microbiome composition and fitness. Growing cultures of laboratory and industrial allergen-producing populations of Dermatophagoides farinae (DFL and DFT, respectively) and Dermatophagoides pteronyssinus (DPL and DPT, respectively) were sampled at four time points. The symbiotic microorganisms of the mites were characterized by DNA barcode sequencing and quantified by qPCR using universal/specific primers. The population growth of mites and nutrient contents of mite bodies were measured and correlated with the changes in bacteria in the HDM microbiome. The results showed that both the population and culture age significantly influenced the microbiome profiles. Cardinium formed 93% and 32% of the total sequences of the DFL and DFT bacterial microbiomes, respectively, but this bacterial species was less abundant in the DPL and DPT microbiomes. Staphylococcus abundance was positively correlated with increased glycogen contents in the bodies of mites, and increased abundances of Aspergillus, Candida, and Kocuria were correlated with increased lipid contents in the bodies of mites. The xerophilic fungus Wallemia accounted for 39% of the fungal sequences in the DPL microbiome, but its abundance was low in the DPT, DFL, and DFT microbiomes. With respect to the mite culture age, we made three important observations: the mite population growth from young cultures was 5–8-fold higher than that from old cultures; specimens from old cultures had greater abundances of fungi and bacteria in their bodies; and yeasts predominated in the gut contents of specimens from young cultures, whereas filamentous mycelium prevailed in specimens from old cultures. Our results are consistent with the hypothesis that mites derive nutrients through associations with microorganisms.

Geographic and Temporal Variation of Distinct Intracellular Endosymbiont Strains of Wolbachia sp. in the Grasshopper Chorthippus parallelus : a Frequency-Dependent Mechanism? Abstract Wolbachia is an intracellular endosymbiont that can produce a range of effects on host fitness, but the temporal dynamics of Wolbachia strains have rarely been experimentally evaluated. We compare interannual strain frequencies along a geographical region for understanding the forces that shape Wolbachia strain frequency in natural populations of its host, Chorthippus parallelus (Orthoptera, Acrididae). General linear models show that strain frequency changes significantly across geographical and temporal scales. Computer simulation allows to reject the compatibility of the observed patterns with either genetic drift or sampling errors. We use consecutive years to estimate total Wolbachia strain fitness. Our estimation of Wolbachia fitness is significant in most cases, within locality and between consecutive years, following a negatively frequency-dependent trend. Wolbachia spp. B and F strains show a temporal pattern of variation that is compatible with a negative frequency-dependent natural selection mechanism. Our results suggest that such a mechanism should be at least considered in future experimental and theoretical research strategies that attempt to understand Wolbachia biodiversity.

Titanium Ions Inhibit the Bacteria in Vase Solutions of Freshly Cut Gerbera jamesonii and Extend the Flower Longevity Abstract Titanium ions significantly promote plant growth, but the mechanism is still unclear. Cut flowers are ideal materials for the study of plant growth and senescence. In this study, freshly cut Gerbera jamesonii were used to study the effects of titanium ions (8 mg/L) on the flower longevity. Flowering observation showed that the gerbera vase life was significantly prolonged in the presence of titanium ions. Plate colony counts showed that the amounts of bacteria in the vase solution of the control group were approximately 1700 times more than that of titanium ion treatment group. High-throughput sequencing was used to determine the sequences of 16S rRNA gene V3-V4 variable regions of the vase solutions to analyze bacterial species, their average proportions, and absolute abundance. The results showed that the titanium ions reduced the entire bacterial counts as well as altered the absolute abundance of different bacterial species in the vase solution. The most prevalent bacteria were mainly Enterobacteriaceae, Pseudomonas veronii, Pseudomonas sp., Delftia sp., Agrobacterium sp., Sphingobacterium multivorum, Acinetobacter johnsonii, and Clostridiaceae. In combination with plate colony counts, we demonstrated that all the bacterial growths were significantly inhibited by titanium ions, regardless of their average proportions increased or decreased. These results showed that titanium ions could extend effectively the longevity of gerberas and possess the broad-spectrum antibacterial properties. This study provides a basis for further mechanism exploration of titanium ions action and its applications in cut flower preservation and agricultural production.

Cellular and Molecular

From neural crest cells to melanocytes: cellular plasticity during development and beyond Abstract Here, we review melanocyte development and how the embryonic melanoblast, although specified to become a melanocyte, is prone to cellular plasticity and is not fully committed to the melanocyte lineage. Even fully differentiated and pigment-producing melanocytes do not always have a stable phenotype. The gradual lineage restriction of neural crest cells toward the melanocyte lineage is determined by both cell-intrinsic and extracellular signals in which differentiation and pathfinding ability reciprocally influence each other. These signals are leveraged by subtle differences in timing and axial positioning. The most extensively studied migration route is the dorsolateral path between the dermomyotome and the prospective epidermis, restricted to melanoblasts. In addition, the embryonic origin of the skin dermis through which neural crest derivatives migrate may also affect the segregation between melanogenic and neurogenic cells in embryos. It is widely accepted that, irrespective of the model organism studied, the immediate precursor of both melanoblast and neurogenic populations is a glial-melanogenic bipotent progenitor. Upon exposure to different conditions, melanoblasts may differentiate into other neural crest-derived lineages such as neuronal cells and vice versa. Key factors that regulate melanoblast migration and patterning will regulate melanocyte homeostasis during different stages of hair cycling in postnatal hair follicles.

N -acetylglucosaminyltransferases and nucleotide sugar transporters form multi-enzyme–multi-transporter assemblies in golgi membranes in vivo Abstract Branching and processing of N-glycans in the medial-Golgi rely both on the transport of the donor UDP-N-acetylglucosamine (UDP-GlcNAc) to the Golgi lumen by the SLC35A3 nucleotide sugar transporter (NST) as well as on the addition of the GlcNAc residue to terminal mannoses in nascent N-glycans by several linkage-specific N-acetyl-glucosaminyltransferases (MGAT1-MGAT5). Previous data indicate that the MGATs and NSTs both form higher order assemblies in the Golgi membranes. Here, we investigate their specific and mutual interactions using high-throughput FRET- and BiFC-based interaction screens. We show that MGAT1, MGAT2, MGAT3, MGAT4B (but not MGAT5) and Golgi alpha-mannosidase IIX (MAN2A2) form several distinct molecular assemblies with each other and that the MAN2A2 acts as a central hub for the interactions. Similar assemblies were also detected between the NSTs SLC35A2, SLC35A3, and SLC35A4. Using in vivo BiFC-based FRET interaction screens, we also identified novel ternary complexes between the MGATs themselves or between the MGATs and the NSTs. These findings suggest that the MGATs and the NSTs self-assemble into multi-enzyme/multi-transporter complexes in the Golgi membranes in vivo to facilitate efficient synthesis of complex N-glycans.

Dgcr8 knockout approaches to understand microRNA functions in vitro and in vivo Abstract Biologic function of the majority of microRNAs (miRNAs) is still unknown. Uncovering the function of miRNAs is hurdled by redundancy among different miRNAs. The deletion of Dgcr8 leads to the deficiency in producing all canonical miRNAs, therefore, overcoming the redundancy issue. Dgcr8 knockout strategy has been instrumental in understanding the function of miRNAs in a variety of cells in vitro and in vivo. In this review, we will first give a brief introduction about miRNAs, miRNA biogenesis pathway and the role of Dgcr8 in miRNA biogenesis. We will then summarize studies performed with Dgcr8 knockout cell models with a focus on embryonic stem cells. After that, we will summarize results from various in vivo Dgcr8 knockout models. Given significant phenotypic differences in various tissues between Dgcr8 and Dicer knockout, we will also briefly review current progresses on understanding miRNA-independent functions of miRNA biogenesis factors. Finally, we will discuss the potential use of a new strategy to stably express miRNAs in Dgcr8 knockout cells. In future, Dgcr8knockout approaches coupled with innovations in miRNA rescue strategy may provide further insights into miRNA functions in vitro and in vivo.

Establishment and depletion of the ovarian reserve: physiology and impact of environmental chemicals Abstract The reproductive life span in women starts at puberty and ends at menopause, following the exhaustion of the follicle stockpile termed the ovarian reserve. Increasing data from experimental animal models and epidemiological studies indicate that exposure to a number of ubiquitously distributed reproductively toxic environmental chemicals (RTECs) can contribute to earlier menopause and even premature ovarian failure. However, the causative relationship between environmental chemical exposure and earlier menopause in women remains poorly understood. The present work, is an attempt to review the current evidence regarding the effects of RTECs on the main ovarian activities in mammals, focusing on how such compounds can affect the ovarian reserve at any stages of ovarian development. We found that in rodents, strong evidence exists that in utero, neonatal, prepubescent and even adult exposure to RTECs leads to impaired functioning of the ovary and a shortening of the reproductive lifespan. Regarding human, data from cross-sectional surveys suggest that human exposure to certain environmental chemicals can compromise a woman's reproductive health and in some cases, correlate with earlier menopause. In conclusion, evidences exist that exposure to RTECs can compromise a woman's reproductive health. However, human exposures may date back to the developmental stage, while the adverse effects are usually diagnosed decades later, thus making it difficult to determine the association between RTECs exposure and human reproductive health. Therefore, epidemiological surveys and more experimental investigation on humans, or alternatively primates, are needed to determine the direct and indirect effects caused by RTECs exposure on the ovary function, and to characterize their action mechanisms.

Exosomes: from carcinogenesis and metastasis to diagnosis and treatment of gastric cancer Abstract Exosomes represent an important group of extracellular vesicles with a defined size between 40 and 150 nm and cup-shaped construction which have a pivotal role in elimination of intracellular debris and intercellular signaling networks. A line of evidence revealed the impact of different types of exosomes in initiation, progression, and metastasis of gastric cancer (GC). These bioactive vesicles mediate tumor and stromal communication network through modulation of cell signaling for carcinogenesis and pre-metastatic niche formation in distant organs. Exosomes contain various cargos including DNAs (mitochondrial and genomic), proteins, transposable elements, and RNAs (coding and noncoding) with different compositions related to functional status of origin cells. In this review, we summarize the main roles of key exosomal cargos in induction of exosome-mediated signaling in cancer cells. Body fluids are employed frequently as the source of exosomes released by tumor cells with a potential role in early diagnosis of GC and chemoresistance. These vesicles as non-toxic and non-immunogenic carriers are also found to be applied for novel drug delivery systems.

Non-obesogenic doses of fatty acids modulate the functionality of the circadian clock in the liver Abstract Saturated fatty acids, such as palmitate, lead to circadian disruption in cell culture. Moreover, information regarding the effects of unsaturated fatty acids on circadian parameters is scarce. We aimed at studying the effects of low doses of saturated as well as unsaturated fatty acids on circadian metabolism in vivo and at deciphering the mechanism by which fatty acids convey their effect. Mice were fed non-obesogenic doses of palm or olive oil and hepatocytes were treated with palmitate and oleate. Mice fed non-obesogenic doses of palm oil showed increased signaling towards fatty acid synthesis, while olive oil increased signaling towards fatty acid oxidation. Low doses of palmitate and oleate were sufficient to alter circadian rhythms, due to changes in the expression and/or activity of key metabolic proteins. Palmitate, but not oleate, counteracted the reduction in lipid accumulation and BMAL1-induced expression of mitochondrial genes involved in fatty acid oxidation. Palmitate was also found to interfere with the transcriptional activity of CLOCK:BMAL1 by preventing BMAL1 deacetylation and activation. In addition, palmitate, but not oleate, reduced PER2-mediated transcriptional activation and increased REV-ERBα-mediated transcriptional inhibition of Bmal1. The inhibition of PER2-mediated transcriptional activation by palmitate was achieved by interfering with PER2 nuclear translocation. Indeed, PER2 reduced fat accumulation in hepatocytes and this reduction was prevented by palmitate. Herein, we show that the detrimental metabolic alteration seen with high doses of palmitate manifests itself early on even with non-obesogenic levels. This is achieved by modulating BMAL1 at several levels abrogating its activity and expression.

Establishment and maintenance of blood–lymph separation Abstract Hippocratic Corpus, a collection of Greek medical literature, described the functional anatomy of the lymphatic system in the fifth century B.C. Subsequent studies in cadavers and surgical patients firmly established that lymphatic vessels drain extravasated interstitial fluid, also known as lymph, into the venous system at the bilateral lymphovenous junctions. Recent advances revealed that lymphovenous valves and platelet-mediated hemostasis at the lymphovenous junctions maintain life-long separation of the blood and lymphatic vascular systems. Here, we review murine models that exhibit failure of blood–lymph separation to highlight the novel mechanisms and molecular targets for the modulation of lymphatic disorders. Specifically, we focus on the transcription factors, cofactors, and signaling pathways that regulate lymphovenous valve development and platelet-mediated lymphovenous hemostasis, which cooperate to maintain blood–lymph separation.

Therapeutic potential of menstrual blood-derived endometrial stem cells in cardiac diseases Abstract Despite significant developments in medical and surgical strategies, cardiac diseases remain the leading causes of morbidity and mortality worldwide. Numerous studies involving preclinical and clinical trials have confirmed that stem cell transplantation can help improve cardiac function and regenerate damaged cardiac tissue, and stem cells isolated from bone marrow, heart tissue, adipose tissue and umbilical cord are the primary candidates for transplantation. During the past decade, menstrual blood-derived endometrial stem cells (MenSCs) have gradually become a promising alternative for stem cell-based therapy due to their comprehensive advantages, which include their ability to be periodically and non-invasively collected, their abundant source material, their ability to be regularly donated, their superior proliferative capacity and their ability to be used for autologous transplantation. MenSCs have shown positive therapeutic potential for the treatment of various diseases. Therefore, aside from a brief introduction of the biological characteristics of MenSCs, this review focuses on the progress being made in evaluating the functional improvement of damaged cardiac tissue after MenSC transplantation through preclinical and clinical studies. Based on published reports, we conclude that the paracrine effect, transdifferentiation and immunomodulation by MenSC promote both regeneration of damaged myocardium and improvement of cardiac function.

Presynaptic NMDA receptors control nociceptive transmission at the spinal cord level in neuropathic pain Abstract Chronic neuropathic pain is a debilitating condition that remains challenging to treat. Glutamate N-methyl-d-aspartate receptor (NMDAR) antagonists have been used to treat neuropathic pain, but the exact sites of their actions have been unclear until recently. Although conventionally postsynaptic, NMDARs are also expressed presynaptically, particularly at the central terminals of primary sensory neurons, in the spinal dorsal horn. However, presynaptic NMDARs in the spinal cord are normally quiescent and are not actively involved in physiological nociceptive transmission. In this review, we describe the emerging role of presynaptic NMDARs at the spinal cord level in chronic neuropathic pain and the implications of molecular mechanisms for more effective treatment. Recent studies indicate that presynaptic NMDAR activity at the spinal cord level is increased in several neuropathic pain conditions but not in chronic inflammatory pain. Increased presynaptic NMDAR activity can potentiate glutamate release from primary afferent terminals to spinal dorsal horn neurons, which is crucial for the synaptic plasticity associated with neuropathic pain caused by traumatic nerve injury and chemotherapy-induced peripheral neuropathy. Furthermore, α2δ-1, previously considered a calcium channel subunit, can directly interact with NMDARs through its C-terminus to increase presynaptic NMDAR activity by facilitating synaptic trafficking of α2δ-1–NMDAR complexes in neuropathic pain caused by chemotherapeutic agents and peripheral nerve injury. Targeting α2δ-1–bound NMDARs with gabapentinoids or α2δ-1 C-terminus peptides can attenuate nociceptive drive form primary sensory nerves to dorsal horn neurons in neuropathic pain.

Tight junction proteins at the blood–brain barrier: far more than claudin-5 Abstract At the blood–brain barrier (BBB), claudin (Cldn)-5 is thought to be the dominant tight junction (TJ) protein, with minor contributions from Cldn3 and -12, and occludin. However, the BBB appears ultrastructurally normal in Cldn5 knock-out mice, suggesting that further Cldns and/or TJ-associated marvel proteins (TAMPs) are involved. Microdissected human and murine brain capillaries, quickly frozen to recapitulate the in vivo situation, showed high transcript expression of Cldn5, -11, -12, and -25, and occludin, but also abundant levels of Cldn1 and -27 in man. Protein levels were quantified by a novel epitope dilution assay and confirmed the respective mRNA data. In contrast to the in vivo situation, Cldn5 dominates BBB expression in vitro, since all other TJ proteins are at comparably low levels or are not expressed. Cldn11 was highly abundant in vivo and contributed to paracellular tightness by homophilic oligomerization, but almost disappeared in vitro. Cldn25, also found at high levels, neither tightened the paracellular barrier nor interconnected opposing cells, but contributed to proper TJ strand morphology. Pathological conditions (in vivo ischemia and in vitro hypoxia) down-regulated Cldn1, -3, and -12, and occludin in cerebral capillaries, which was paralleled by up-regulation of Cldn5 after middle cerebral artery occlusion in rats. Cldn1 expression increased after Cldn5 knock-down. In conclusion, this complete Cldn/TAMP profile demonstrates the presence of up to a dozen TJ proteins in brain capillaries. Mouse and human share a similar and complex TJ profile in vivo, but this complexity is widely lost under in vitro conditions.

Parasitology

Apparent kleptoparasitism in fish—parasitic gnathiid isopods" 10.1007/s00436-018-6152-8

Correction to: Epidemiology of Sulcascaris sulcata (Nematoda: Anisakidae) ulcerous gastritis in the Mediterranean loggerhead sea turtle ( Caretta caretta ) The original version of this article contained a mistake in the value of Bar in figure 3 caption. It should be Bar = 200 μm instead of Bar = 500 μm.

Correction to: In vitro schistosomicidal activity of tamoxifen and its effectiveness in a murine model of schistosomiasis at a single dose The original published version of this article contains error in Tables 1 and 2. Correct tables are presented here.

First report of sparganosis manifested by pleuritis and decreased peripheral blood eosinophils in Jiangsu province, China Abstract Sparganosis is a parasitic infection caused by the metacestode stage of Spirometra mansoni and some other related diphyllobothriidean cestodes. Although various internal organs were involved in sparganum infection, pulmonary and pleural involvement is rarely reported. We herein report an uncommon form of sparganosis manifested by pleuritis and decreased peripheral blood eosinophils. Sparganum worms were found in the pleural effusion accidentally and confirmed by pathological diagnosis. After being treated with praziquantel for 10 days, the patient's symptoms, laboratory examinations, and imaging findings were improved gradually.

Distribution of Cryptosporidium parvum gp60 subtypes in calf herds of Saxony, Germany Abstract Cryptosporidiosis is a common protozoan parasitic infection that causes diarrhoea in neonatal calves. The high shedding of environmentally resistant oocysts facilitates outbreaks of cryptosporidiosis in humans. In total, 58 farms (512 calves) in Germany (Saxony and Brandenburg) were visited three times each. Faecal samples of pre-weaned calves were microscopically examined for oocysts of Cryptosporidium spp. using Heine staining and were scored with regard to their consistency. Overall, 88.9% of calves tested microscopically positive for Cryptosporidium spp. in at least one sample, and the excretion of oocysts was significantly (P < 0.01) associated with a higher faecal score (diarrhoea). After DNA extraction from pooled farm isolates, 47 samples were successfully subtyped by sequence analysis of the 60 kDa glycoprotein gene (gp60). All isolates belonged to subtype family IIa. IIaA15G2R1 was the most common subtype (present on 66% of the farms), followed by IIaA16G3R1 (13%). Subtypes IIaA14G1R1, IIaA14G2R1, IIaA1612R1, IIaA16G2R1, IIaA17G1R1, IIaA17G2R1, IIaA17G4R1 and IIaA19G2R1 were found sporadically. This is the first description of gp60 subtype IIaA17G4R1 in cattle in Germany.

Combined morphology and DNA-barcoding to identify Plagiorhynchus cylindraceus cystacanths in Atelerix algirus Abstract The acanthocephalan parasite Plagiorhynchus cylindraceus has a global distribution and utilizes isopods and birds as intermediate and definitive hosts, respectively. Occasionally, mammals of various orders can act as paratenic hosts. In hedgehogs, severe cases have been reported in juvenile specimens due to secondary infections, as a consequence of complete penetrations of the intestinal wall by cystacanths. In a 66-month study period, we found seven larvae of this parasite encysted in both, the peritoneal cavity and intestine of the Algerian hedgehog, Atelerix algirus in Majorca. Morphology alone was insufficient to identify the species, due to the lack of previous reports and taxonomy-informative characters. In the present report, we combined the use of morphology and the DNA-barcoding approach to confirm to identify cystacanths as P. cylindraceus. This is the first report of this parasite in this hedgehog species. The epidemiological implications will be discussed, including the risk of zoonosis and the importance of using modern approaches to identify immature acanthocephalan larvae in wildlife hosts.

Intestinal injury caused by Eimeria spp. impairs the phosphotransfer network and gain weight in experimentally infected chicken chicks Abstract Parasitic infections caused by protozoan belonging to genus Eimeria are considered important for the poultry industry, due to their severe intestinal lesions and high mortality rates, causing significant economic losses. Although several mechanisms of coccidiosis pathogenesis are known, the effects of this infection on intestinal enzymes linked to adenosine triphosphate (ATP) metabolism, as creatine kinase (CK), adenylate kinase (AK), and pyruvate kinase (PK), remain unknown. Thus, the aim of this study was to evaluate whether coccidiosis impairs enzymes linked ATP metabolism in the intestine of chicken chicks. For this, 42 animals that were 2 days old were divided into two groups: uninfected (the negative control group) and experimentally infected on second day of life (the positive control group). On days 5, 10, and 15 post-infection (PI), fecal samples were collected for oocyst counts; intestinal tissue was collected in order to evaluate CK, AK, and PK activities, as well as parameters of the oxidative stress and histopathology. On days 10 and 15 PI, infected animals showed high counts of oocysts in fecal samples and intestinal lesions compared to the control group. Cytosolic CK activity was higher in infected animals on days 10 and 15 PI compared to the control group, while mitochondrial CK activity was lower on days 5, 10, and 15 PI. Also, AK activity was lower in infected animals on days 10 and 15 PI compared to control group, while no differences were observed between groups regarding PK activity. In relation to parameters of oxidative stress, intestinal lipid peroxidation and reactive oxygen species levels were higher in infected animals on days 10 and 15 PI compared to the control group, while non-protein thiol levels were lower on day 10 PI. On the 15th day, infected animals had lower body weight (P < 0.05). Based on this evidence, inhibition of mitochondrial CK activity causes an impairment of intestinal energetic homeostasis possibly through depletion on ATP levels, although the cytosolic CK activity acted as an attempt to restore the mitochondrial ATP levels through a feedback mechanism. Moreover, the impairment on energy metabolism appears to be mediated by excessive production of intestinal ROS, as well as oxidation of lipids and thiol groups.

Oral infection of mice and host cell invasion by Trypanosoma cruzi strains from Mexico Abstract Oral infection by Trypanosoma cruzi has been responsible for frequent outbreaks of acute Chagas disease in the north of South America and in the Amazon region, where T. cruzi genetic group TcI predominates. TcI strains from different geographical regions have been used in oral infection in mice, but there is no information about strains from Mexico where TcI is prevalent. Here, we analyzed four Mexican strains as concerns the course of oral infection, the ability to invade host cells in vitro, and the profile of metacyclic trypomastigote surface molecules gp82 and gp90 that are implicated in parasite internalization. Oral infection of mice with metacyclic forms of all strains resulted in reduced blood and tissue parasitism, and mild to moderate inflammatory process in the heart/skeletal muscle. They expressed pepsin-resistant gp82 and gp90 molecules at high levels and invaded host cells poorly in full nutrient medium and efficiently under nutrient-deprived condition. The properties exhibited by Mexican strains were similar to those displayed by TcI strains from other geographical regions, reinforcing the notion that these features are common to the genetic group TcI as a whole.

Antarctophthirus microchir infestation in synanthropic South American sea lion ( Otaria flavescens ) males diagnosed by a novel non-invasive method Abstract Antarctophthirus microchir is a sucking louse species belonging to the family Echinophthiriidae and has been reported to parasitize all species of the subfamily Otariinae, the sea lions. Former studies on this ectoparasite mainly required fixation, immobilization, or death of host species and especially examinations of adult male sea lions are still very rare. Between March and May 2018, adult individuals of a unique "urban" bachelor group of South American sea lions (Otaria flavescens) living directly in the city of Valdivia, Chile, were studied regarding their ectoparasite infestation status. For first time, a non-invasive method in the form of a lice comb screwed on a telescopic rod and grounded with adhesive tape was used for sample taking process. Overall, during combing different stages of A. microchir were detected in 4/5 O. flavescens individuals, especially at the junction between the back and hind flippers. Our findings represent the first report of A. microchir infesting individuals of this synanthropic colony and fulfilling complete life cycle in a sea lion group despite inhabiting freshwater and in absence of females/pups. Our "telescopic lice comb apparatus" offers a new strategy to collect different stages of ectoparasites and a range of epidermal material, such as fur coat hair and superficial skin tissue for a broad spectrum of research fields in wildlife sciences in an unmolested and stress reduced manner.

On the occurrence and molecular identification of Contracaecum larvae (Nematoda: Anisakidae) in Mugil cephalus from Turkish waters Abstract Anisakis and Contracaecum species are fish borne zoonotic nematodes. In our previous studies, other larval anisakid and raphidascarid nematodes, Anisakis and Hysterothylacium species, were genetically identified in marine fish from Turkish waters. However, there is no information on molecular identification of larval Contracaecum species in marine fish from Turkey. Therefore, the aim of this study was only to investigate the presence and molecular identification of Contracaecum species in commonly commercialized marine fish from Turkish waters. A total of 475 marine fish, which belong to 21 different species, were sampled from the Aegean (FAO 37.3.1), Mediterranean (FAO 37.3.2), and Black Sea (FAO 37.4.2). The prevalence of Contracaecum L3 larvae in the Aegean Sea was identified as 10% in Mugil cephalus. All Contracaecum L3 larvae were molecularly characterized with RFLP targeting the ITS region and rrnS gene. Moreover, all larvae were analyzed by sequencing of ITS region, rrnS and cox2 gene. All Contracaecum larvae were identified as C. overstreeti based on the cox2 sequence analysis. This is the first report of C. overstreeti larvae in M. cephalus as paratenic and intermediate hosts. Furthermore, the analysis reveals novel information on ITS region. Additionally, the rrnS gene of C. overstreeti was also achieved and deposited in Genbank for the first time. The PCR-RFLP patterns of the ITS region and rrnS gene from C. overstreeti were presented in the present study. Consequently, the presence of C. overstreeti larvae in M. cephalus from the Aegean Sea may also potentially capable of inducing allergic sensitization in humans.

NeuroOncology

Neuro-radiological characteristics of adult diffuse grade II and III insular gliomas classified according to WHO 2016 In the initial, online publication, the authors' given names were captured as family names and vice versa. The names are correctly shown here. The original article has been corrected.

Correction to: Monitoring of intracerebellarly-administered natural killer cells with fluorine-19 MRI There was a typo in author Andrew Wahba's name in the initial online publication. The original article has been corrected.

Comment on: Effects of surgery on neurocognitive function in patients with glioma: a meta-analysis of immediate post-operative and long-term follow-up neurocognitive outcomes

Incidence and clinicopathologic features of H3 K27M mutations in adults with radiographically-determined midline gliomas Abstract Purpose H3 K27 mutations, most commonly in H3F3A, are common in diffuse midline glioma. The exact frequency of these mutations in adults with gliomas in the midline location is unknown. This study was conducted to define the incidence of H3 K27M mutations in this location and to compare clinicopathological features with those of patients who do not harbor this mutation. Methods Consecutive glioma cases from 2007 to 2017 were screened for gliomas in the midline location. Immunohistochemistry was performed on all available tissue for mutations of H3 K27M, IDH1, and ARTX. Results Of 850 gliomas screened, 163 cases had midline glioma on MRI. Sufficient FFPE tissue was available for 123 cases (75%). H3 K27M mutation was identified in 18 of 123 cases (15%). All except one H3 K27M-mutant tumors were WHO grade III or IV on histology, while non-mutant tumors encompassed all four grades. The most common midline locations for H3 K27M-mutated tumors were midbrain (2/3; 67%), pons (4/11; 36%), and cerebellum (6/24; 25%). As compared to H3 K27M-wildtype tumors, there were no differences in age at diagnosis, sex, tumor grade, contrast enhancement on MRI, extent of resection, or treatment received. In this cohort, median survival was longer for patients with H3 K27M-mutated tumors (n = 18; 17.6 months) compared with high-grade wildtype tumors (n = 74; 7.7 months, p = 0.03). Conclusions H3 K27M mutations are common in midline gliomas in adults and can present in all midline locations. Survival comparison between H3 K27M-mutant and wildtype midline gliomas suggests that survival may be similar or possibly improved if the mutation is present.

Targeted radioimmunotherapy for embryonal tumor with multilayered rosettes Abstract Purpose We explored the use of intraventricular 131I-Omburtamab targeting B7-H3 in patients with ETMR. Methods Patients were enrolled in an IRB approved, phase 1, 3 + 3 dose escalation trial. Patients with CNS disease expressing the antibody target antigen B7-H3 were eligible. We report on a cohort of three patients with ETMR who were enrolled on the study. Three symptomatic children (ages 14 months, 3 and 3.5 years) had large parietal masses confirmed to be B7-H3-reactive ETMR. Patients received 2 mCi 131I-Omburtamab as a tracer followed by one or two therapeutic 131I-Omburtamab injections. Dosimetry was based on serial CSF, blood samplings and region of interest (ROI) on nuclear scans. Brain and spine MRIs and CSF cytology were done at baseline, 5 weeks after 131I-Omburtamab, and approximately every 3 months thereafter. Acute toxicities and survival were noted. Results Patients received surgery, focal radiation, and high dose chemotherapy. Patients 1 and 2 received 131I-Omburtamab (80 and 53 mCi, respectively). Patient 3 had a local recurrence prior to 131I-Omburtamab treated with surgery, external beam radiation, chemotherapy, then 131I-Omburtamab (36 mCi). 131I-Omburtamab was well-tolerated. Mean dose delivered by 131I-Omburtamab was 68.4 cGy/mCi to CSF and 1.95 cGy/mCi to blood. Mean ROI doses were 230.4 (ventricular) and 58.2 (spinal) cGy/mCi. Patients 1 and 2 remain in remission 6.8 years and 2.3 years after diagnosis, respectively; patient 3 died of progressive disease 7 months after therapy (2 years after diagnosis). Conclusions 131I-Omburtamab appears safe with favorable dosimetry therapeutic index. When used as consolidation following surgery and chemoradiation therapy, 131I-Omburtamab may have therapeutic benefit for patients with ETMR.

Impact of overall corticosteroid exposure during chemoradiotherapy on lymphopenia and survival of glioblastoma patients Abstract Purpose Corticosteroids are commonly used to alleviate symptoms from cerebral vasogenic edema in glioblastoma (GBM) patients. This study evaluated the impact of overall corticosteroid exposure during chemoradiotherapy (CRT) on acute severe lymphopenia (ASL) and survival outcomes of GBM patients. Methods GBM patients treated with CRT from 2007 to 2016 were retrospectively analyzed. Overall corticosteroid exposure was estimated as the average daily dexamethasone dose during 6 weeks of CRT. ASL was defined as grade 3 or higher lymphopenia within 3 months of starting CRT. ASL rates, overall survival (OS), and progression-free survival (PFS) were analyzed using Kaplan–Meier method. Multivariable analysis (MVA) was performed using logistic and Cox regression to identify independent predictors of ASL and survival outcomes, respectively. Results Of the 319 eligible patients, the median daily dexamethasone use was 2 mg/day. The high-dose dexamethasone cohort (> 2 mg/day) had significantly higher ASL and worse OS than the low-dose dexamethasone cohort: 3-month ASL of 43.7% versus 19.8% (p < 0.003) and median OS of 12.6 months versus 17.9 months (p < 0.001), respectively. On MVA, higher dexamethasone use was independently associated with higher ASL and worse OS, but not worse PFS. A subset analysis of patients with gross-total resection found that higher dexamethasone use was significantly associated with ASL, but not OS. Conclusion Increased corticosteroid use among GBM patients during CRT appears to be an independent risk factor for developing subsequent ASL. Its apparent association with worse OS may be influenced by other confounding factors and would need to be validated through prospective investigations.

Gamma Knife Stereotactic Radiosurgery favorably changes the clinical course of hemangioblastoma growth in von Hippel-Lindau and sporadic patients Abstract Purpose This is the first single-institution study of its size to characterize the treatment impact and to address the question of whether hemangioblastoma treatment with Gamma Knife Stereotactic Radiosurgery (GKRS) in both sporadic and VHL patients changes the characteristic saltatory hemangioblastoma growth pattern. Methods The authors reviewed a single-institution tumor registry to identify patients who had received GKRS for hemangioblastomas between January 1st, 1999, and December 31st, 2017. Results 15 patients with 101 lesions met search criteria with a median age of first GKRS of 39.2 years (interquartile range [IQR] of 25.7–57.4 years), including 96 VHL and 5 sporadic lesions. The median time from GKRS to last follow-up was 5.4 years (IQR 2.3–11.5 years). 4 lesions (4%) and 3 patients (20%) experienced a local failure. The 1-year, 3-year, and 5-year freedom from new hemangioblastoma formation rates were 97%, 80%, and 46% respectively. Multivariate analysis revealed a reduction in tumor volume after GKRS. Several variables associated with a greater percent reduction in volume from GKRS to last follow-up: non-cystic status (p = .01), no prior craniotomy (p = .04), and follow-up time from GKRS (p < .0001). Conclusions GKRS is a successful long-term treatment option for hemangioblastomas changing the clinical course from saltatory growth to reduction in tumor volume. Non-cystic tumors and those without prior craniotomy were associated with a greater percent reduction in volume from GKRS at last follow-up.

Impact on survival of early tumor growth between surgery and radiotherapy in patients with de novo glioblastoma Abstract Purpose Systematic pre-radiotherapy MRI in patients with newly resected glioblastoma (OMS 2016) sometimes reveals tumor growth in the period between surgery and radiotherapy. We evaluated the relation between early tumor growth and overall survival (OS) with the aim of finding predictors of regrowth. Methods Seventy-five patients from 25 to 84 years old (Median age 62 years) with preoperative, immediate postoperative, and preradiotherapy MRI were included. Volumetric measurements were made on each of the three MRI scans and clinical and molecular parameters were collected for each case. Results Fifty-four patients (72%) had an early regrowth with a median contrast enhancement volume of 3.61 cm3—range 0.12–71.93 cm3. The median OS was 24 months in patients with no early tumor growth and 17.1 months in those with early tumor regrowth (p = 0.0024). In the population with initial complete resection (27 patients), the median OS was 25.3 months (19 patients) in those with no early tumor growth between surgery and radiotherapy compared to 16.3 months (8 patients) in those with tumor regrowth. In multivariate analysis, the initial extent of resection (p < 0.001) and the delay between postoperative MRI and preradiotherapy MRI (p < 0.001) were significant independent prognostic factors of regrowth and of poorer outcome. Conclusions We demonstrated that, in addition to the well known issue of incomplete resection, longer delays between surgery and adjuvant treatment is an independent factors of tumor regrowth and a risk factor of poorer outcomes for the patients. To overcome the delay factor, we suggest shortening the usual time between surgery and radiotherapy.

Tumor control and survival in patients with ten or more brain metastases treated with stereotactic radiosurgery: a retrospective analysis Abstract Introduction To assess tumor control and survival in patients treated with stereotactic radiosurgery (SRS) for 10 or more metastatic brain tumors. Methods Patients were retrospectively identified. Clinical records were reviewed for follow-up data, and post-treatment MRI studies were used to assess tumor control. For tumor control studies, patients were separated based on synchronous or metachronous treatment, and control was assessed at 3-month intervals. The Kaplan–Meier method was employed to create survival curves, and regression analyses were employed to study the effects of several variables. Results Fifty-five patients were treated for an average of 17 total metastases. Forty patients received synchronous treatment, while 15 received metachronous treatment. Univariate analysis revealed an association between larger brain volumes irradiated with 12 Gy and decreased overall survival (p = 0.0406); however, significance was lost on multivariate analysis. Among patients who received synchronous treatment, the median percentage of tumors controlled was 100%, 91%, and 82% at 3, 6, and 9 months, respectively. Among patients who received metachronous treatment, the median percentage of tumors controlled after each SRS encounter was 100% at all three time points. Conclusions SRS can be used to treat patients with 10 or more total brain metastases with an expectation of tumor control and overall survival that is equivalent to that reported for patients with four or fewer tumors. Development of new metastases leading to repeat SRS is not associated with worsened tumor control or survival. Survival may be adversely affected in patients having a higher volume of normal brain irradiated.

A multicenter phase II study of temozolomide plus disulfiram and copper for recurrent temozolomide-resistant glioblastoma Abstract Purpose Preclinical studies have suggested promising activity for the combination of disulfiram and copper (DSF/Cu) against glioblastoma (GBM) including re-sensitization to temozolomide (TMZ). A previous phase I study demonstrated the safety of combining DSF/Cu with adjuvant TMZ for newly diagnosed GBM. This phase II study aimed to estimate the potential effectiveness of DSF/Cu to re-sensitize recurrent GBM to TMZ. Methods This open-label, single-arm phase II study treated recurrent TMZ-resistant GBM patients with standard monthly TMZ plus concurrent daily DSF 80 mg PO TID and Cu 1.5 mg PO TID. Eligible patients must have progressed after standard chemoradiotherapy and within 3 months of the last dose of TMZ. Known isocitrate dehydrogenase (IDH) mutant or secondary GBMs were excluded. The primary endpoint was objective response rate (ORR), and the secondary endpoints included progression-free survival (PFS), overall survival (OS), clinical benefit (response or stable disease for at least 6 months), and safety. Results From March 2017 to January 2018, 23 recurrent TMZ-resistant GBM patients were enrolled across seven centers, and 21 patients were evaluable for response. The median duration of DSF/Cu was 1.6 cycles (range: 0.1–12.0). The ORR was 0%, but 14% had clinical benefit. Median PFS was 1.7 months, and median OS was 7.1 months. Only one patient (4%) had dose-limiting toxicity (grade three elevated alanine transaminase). Conclusions Addition of DSF/Cu to TMZ for TMZ-resistant IDH-wild type GBM appears well tolerated but has limited activity for unselected population.

Correction to: Cognitive assessment in multiple sclerosis—an Italian consensus In the original article, Maria Pia Amato's second affiliation was not included. The second affiliation is IRCCS Fondazione Don Carlo Gnocchi, Florence, Italy. The correct affiliation is presented here.

Correction to: Normative values of the Rao's Brief Repeatable Battery in an Italian young adolescent population: the influence of age, gender, and education The published version of this article unfortunately contained a mistake in Table 2.

Langerhans cell histiocytosis presenting as a rapidly evolving frontotemporal syndrome Abstract Langerhans cell histiocytosis (LCH) is a rare disorder in adults which usually manifests with involvement of multiple organ systems, including the central nervous system. We describe an unusual case of biopsy-proven LCH presenting with frontotemporal-dominant cognitive impairment with hypothalamic involvement, along with multisystem disease. We propose that the dementia was probably an immune-mediated process triggered by LCH which responded dramatically to high-dose steroids.

Assessing seasonal dynamics of Guillain-Barré syndrome with search engine query data Abstract Background and objective In previous studies, data deriving from Google Trends showed promising correlation with disease incidence trends assessed with public health control systems. The aim of this work is to use search engine query data to investigate seasonal dynamics in Guillain-Barré syndrome (GBS) in the USA. Methods Average Google monthly search volumes for GBS from 2008 to 2017 were analysed for the USA overall and on regional base with generalized estimating equation models. Association with monthly historical temperature variations was tested. Results Monthly search volume for GBS displayed the greatest positive anomaly for October, clustering with September and November. Region-wide analysis confirmed this pattern and showed secondary spring (Feb/Apr) subpeaks in Pacific and Midwest. Association of GBS search volume with month-to-month temperature variations showed J-shaped relationship, with the highest peak occurring in months with greatest temperature falls, and subpeak in months with sharpest temperature rises. Conclusions This study represents the first approach in investigating digital epidemiology of GBS and establishing possible links with traditional epidemiology. Cold season GBS peak has been observed by some traditional studies; hypothetical pathogenic relationship with infectious antecedents is supported from finding GBS peaks clustering with greatest temperature change. Further studies are needed to compare these findings to traditional public health approaches.

Highlights of the issue 5, 2019

Bilateral trigeminal root entry zone enhancement in MOG-IgG-associated brainstem encephalitis

Status and perspectives of acute stroke care in Europe

A new neurobehavioral phenotype of familial Creutzfeldt–Jakob disease: impaired theory of mind

An unusual case of PML in HIV patient presenting with diplopia

Serum orexin-A levels are associated with disease progression and motor impairment in multiple sclerosis Abstract Objective Diencephalon is frequently affected in multiple sclerosis (MS), and lesions of this region are associated with increased disability. Orexin-A and melatonin, two foremost mediators of diencephalon, modulate cognitive and motor functions through several pathways including the brain-derived neurotrophic factor (BDNF)-cAMP response element-binding protein (CREB) signaling pathway. In this pilot study, our aim was to investigate the prognostic value of these factors in progression of cognitive and physical disability. Methods Levels of BDNF, melatonin, CREB, and orexin-A were determined by ELISA in sera of 25 relapsing remitting MS (RRMS) patients, 15 secondary progressive MS (SPMS) patients, and 20 healthy controls. Cognitive and motor functions were assessed by a neuropsychological test battery, timed 25-ft walk (T25-FW) and 9-hole peg (9-HP) tests. Results MS patients had significantly lower serum levels of orexin-A and BDNF than healthy controls, and SPMS patients had significantly lower levels of melatonin and orexin-A than RRMS patients. Serum orexin-A levels were negatively correlated with 9-HP, T25-FW test scores, and progression index in RRMS patients. BDNF, CREB, and melatonin levels did not show any significant correlation with clinical features including EDSS and cognitive/motor performance of the patients. Conclusion Our results suggest that orexin-A levels are decreased in parallel to disease progression and motor system deterioration in the earlier stages of the disease. Thus, orexin-A might be used as a potential biomarker of physical disability.

Theoretical Medicine and Bioethics

Death, unity, and the brain Abstract The dead donor rule holds that removing organs from living human beings without their consent is wrongful killing. The rule still prevails in most countries, and I assume it without argument in order to pose the question: is it possible to have a metaphysically correct, clinically relevant analysis of human death that makes organ donation ethically permissible? I argue that the two dominant criteria of death—brain death and circulatory death—are both empirically and metaphysically inadequate as definitions of human death and therefore hold no epistemic value in themselves. I first set out a neo-Aristotelian theory of death as separation of soul (understood as organising principle) and body, which is then fleshed out as loss of organismic integrity. The brain and circulatory criteria are shown to have severe weaknesses as physiological manifestations of loss of integrity. Given the mismatch between what death is, metaphysically speaking, and the dominant criteria accepted by clinicians and philosophers, it turns out that only actual bodily decomposition is a sure sign of death. In this I differ from Alan Shewmon, whose important work I discuss in detail.

Akira Akabayashi (ed): The future of bioethics: international dialogues

Joo-Young Lee: A human rights framework for intellectual property, innovation and access to medicines

Outcome-adaptive randomization in clinical trials: issues of participant welfare and autonomy Abstract Outcome-adaptive randomization (OAR) has been proposed as a corrective to certain ethical difficulties inherent in the traditional randomized clinical trial (RCT) using fixed-ratio randomization. In particular, it has been suggested that OAR redresses the balance between individual and collective ethics in favour of the former. In this paper, I examine issues of welfare and autonomy arising in relation to OAR. A central issue in discussions of welfare in OAR is equipoise, and the moral status of OAR is crucially influenced by the way in which this concept is construed. If OAR is based on a model of equipoise that demands strict indifference between competing interventions throughout the trial, such equipoise is disturbed by accruing data favouring one treatment over another; OAR seeks to redress this by weighting randomization to the seemingly superior treatment. However, this is a partial response, as patients continue to be allocated to the inferior therapy. Moreover, it rests upon considerations of aggregate harms and benefits, and does not therefore uphold individual ethics. Issues of fairness also arise, as early and late enrollees are randomized on a different basis. Fixed-ratio randomization represents a fuller and more consistent response to a loss of equipoise, as so construed. With regard to consent, the complexity of OAR poses challenges to adequate disclosure and comprehension. Additionally, OAR does not offer a remedy to the therapeutic misconception—participants' tendency to attribute treatment allocation in an RCT to individual clinical judgments, rather than to scientific considerations—and, if anything, accentuates rather than alleviates this misconception. In relation to these issues, OAR fails to offer ethical advantages over fixed-ratio randomization. More broadly, the ethical basis of OAR can be seen to lie more in collective than in individual ethics, and overall it fares worse in this territory than fixed-ratio randomization.

Paula Gerber and Katie O'Byrne (eds): Surrogacy, law and human rights

Taking patient virtue seriously Abstract Virtue theory in philosophical bioethics has influenced clinical ethics with depictions of the virtuous doctor or nurse. Comparatively little has been done with the concept of the virtuous patient, however. Bioethicists should correct the asymmetry in virtue theory between physician virtues and patient virtues in a way that provides a practical theory for the new patient-centered medicine—something clinicians and administrators can take seriously.

Evidence for personalised medicine: mechanisms, correlation, and new kinds of black box Abstract Personalised medicine (PM) has been discussed as a medical paradigm shift that will improve health while reducing inefficiency and waste. At the same time, it raises new practical, regulatory, and ethical challenges. In this paper, we examine PM strategies epistemologically in order to develop capacities to address these challenges, focusing on a recently proposed strategy for developing patient-specific models from induced pluripotent stem cells (iPSCs) so as to make individualised treatment predictions. We compare this strategy to two main PM strategies—stratified medicine and computational models. Drawing on epistemological work in the philosophy of medicine, we explain why these two methods, while powerful, are neither truly personalised nor, epistemologically speaking, novel strategies. Both are forms of correlational black box. We then argue that the iPSC models would count as a new kind of black box. They would not rely entirely on mechanistic knowledge, and they would utilise correlational evidence in a different way from other strategies—a way that would enable personalised predictions. In arguing that the iPSC models would present a novel method of gaining evidence for clinical practice, we provide an epistemic analysis that can help to inform the practical, regulatory, and ethical challenges of developing an iPSC system.

Is "aid in dying" suicide? Abstract The practice whereby terminally ill patients choose to end their own lives painlessly by ingesting a drug prescribed by a physician has commonly been referred to as physician-assisted suicide. There is, however, a strong trend forming that seeks to deny that this act should properly be termed suicide. The purpose of this paper is to examine and reject the view that the term suicide should be abandoned in reference to what has been called physician-assisted suicide. I argue that there are no good conceptual or philosophical reasons to avoid the suicide label. I contend that intending one's death is essential to the nature of suicide, and this intention is normally required on the part of the terminally ill patient when she knowingly takes a life-ending drug. Additionally, the analysis shows that any plausible strategy that avoids the term suicide is counteracted by the way in which advocates of the practice want to make it legal.

Henk ten Have: Global bioethics: an introduction

The harm of medical disorder as harm in the damage sense Abstract Jerome Wakefield has argued that a disorder is a harmful dysfunction. This paper develops how Wakefield should construe harmful in his harmful dysfunction analysis (HDA). Recently, Neil Feit has argued that classic puzzles involved in analyzing harm render Wakefield's HDA better off without harm as a necessary condition. Whether or not one conceives of harm as comparative or non-comparative, the concern is that the HDA forces people to classify as mere dysfunction what they know to be a disorder. For instance, one can conceive of cases where simultaneous disorders prevent each other from being, in any traditional sense, actually harmful; in such cases, according to the HDA, neither would be a disorder. I argue that the sense of harm that Wakefield should employ in the HDA is dispositional, similar to the sense of harm used when describing a vile of poison: "Be careful! That's poison. It's harmful." I call this harm in the damage sense. Using this sense of harm enables the HDA to avoid Feit's arguments, and thus it should be preferred to other senses when analyzing harmful dysfunction.