Urinary PGE2 metabolite levels in hospitalised infants with infections compared to age-matched controls.

AIM: To determine the urinary tetranor-prostaglandin E2 metabolite in healthy infants and in hospitalised infants with upper and lower respiratory tract as well as gastrointestinal infections. METHODS: A prospective cross-sectional study to determine baseline concentrations of urinary tetranor-prostaglandin E2 metabolite was conducted in 81 healthy infants aged one week to one year and in 142 hospitalised infants with infections. Prostaglandin metabolite levels were measured by liquid chromatography tandem mass spectrometry. RESULTS: In healthy infants, urinary prostaglandin E2 metabolite levels decreased with age and did not differ between girls and boys. Infections of the lower respiratory (n = 78) and gastrointestinal tract (n = 12) correlated with increased levels of the prostaglandin E2 metabolite. In contrast, infants hospitalised with upper respiratory tract infections (n = 23) exhibited similar levels as healthy, age-matched controls. Lower prostaglandin E2 levels were found after treatment with acetaminophen in hospitalised children. Prostaglandin E2 metabolite levels did not correlate with length of hospitalisation or need for respiratory support. CONCLUSION: This study first provides normal levels of urinary prostaglandin E2 metabolite in infants and secondly demonstrates elevated levels in hospitalised children with lower respiratory tract and gastrointestinal infections.

Muscle contractures in patients with cerebral palsy and acquired brain injury are associated with extracellular matrix expansion, pro-inflammatory gene expression, and reduced rRNA synthesis.

INTRODUCTION: Children with cerebral palsy (CP) and acquired brain injury (ABI) commonly develop muscle contractures with advancing age. An underlying growth defect contributing to skeletal muscle contracture formation in CP/ABI has been suggested. METHODS: The biceps muscles of children and adolescents with CP/ABI (n = 20) and typically developing controls (n = 10) were investigated. We used immunohistochemistry, quantitative real-time polymerase chain reaction, and Western blotting to assess gene expression relevant to growth and size homeostasis. RESULTS: Classical pro-inflammatory cytokines and genes involved in extracellular matrix (ECM) production were elevated in skeletal muscle of children with CP/ABI. Intramuscular collagen content was increased and satellite cell number decreased and this was associated with reduced levels of RNA polymerase I transcription factors, 45s pre-rRNA and 28S rRNA. DISCUSSION: The present study provides novel data suggesting a role for pro-inflammatory cytokines and reduced ribosomal production in the development/maintenance of muscle contractures, possibly underlying stunted growth and perimysial ECM expansion. Muscle Nerve 58: 277-285, 2018.

A subcellular map of the human proteome.

Resolving the spatial distribution of the human proteome at a subcellular level can greatly increase our understanding of human biology and disease. Here we present a comprehensive image-based map of subcellular protein distribution, the Cell Atlas, built by integrating transcriptomics and antibody-based immunofluorescence microscopy with validation by mass spectrometry. Mapping the in situ localization of 12,003 human proteins at a single-cell level to 30 subcellular structures enabled the definition of the proteomes of 13 major organelles. Exploration of the proteomes revealed single-cell variations in abundance or spatial distribution and localization of about half of the proteins to multiple compartments. This subcellular map can be used to refine existing protein-protein interaction networks and provides an important resource to deconvolute the highly complex architecture of the human cell.

Antibody Validation in Bioimaging Applications Based on Endogenous Expression of Tagged Proteins.

Antibodies are indispensible research tools, yet the scientific community has not adopted standardized procedures to validate their specificity. Here we present a strategy to systematically validate antibodies for immunofluorescence (IF) applications using gene tagging. We have assessed the on- and off-target binding capabilities of 197 antibodies using 108 cell lines expressing EGFP-tagged target proteins at endogenous levels. Furthermore, we assessed batch-to-batch effects for 35 target proteins, showing that both the on- and off-target binding patterns vary significantly between antibody batches and that the proposed strategy serves as a reliable procedure for ensuring reproducibility upon production of new antibody batches. In summary, we present a systematic scheme for antibody validation in IF applications using endogenous expression of tagged proteins. This is an important step toward a reproducible approach for context- and application-specific antibody validation and improved reliability of antibody-based experiments and research data.

Evaluation of urinary prostaglandin E2 metabolite as a biomarker in infants with fever due to viral infection.

We have investigated the clinical feasibility of the major urinary metabolite of prostaglandin (PG) E2, tetranor-PGEM, as a biomarker of inflammation in infants with fever. We tested two different and clinically relevant sampling methods, using self-adhesive urinary bags or gauze pads, with respect to stability of tetranor-PGEM and ease of sampling from infants. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was used to quantify tetranor-PGEM in urine, and different normalization parameters, i.e., urinary creatinine and body surface area, were investigated. To study inflammation, infants (1 month-1 year) that were hospitalized with fever of unknown origin at admittance (n=14) were compared to age-matched healthy controls (n=14). Levels of urinary tetranor-PGEM in infants with viral induced fever were increased compared to controls (102.4±56.2 vs. 37.0±21.6pmol/ml/m(2) body surface area, p<0.001). We conclude that urinary tetranor-PGEM is a potential non-invasive biomarker of inflammation in infants.

Experimental teratoma: at the crossroad of fetal- and onco-development.

Xenografting is the so far only available in vivo model for assessing pluripotency of human stem cells. This review describes known biological features of experimental teratoma from human pluripotent stem cells. We focus on the dual nature mimicking both normal and abnormal development, and propose this model system to be particularly interesting for investigations of the relationship between developmentally controlled differentiation and neoplasia of embryonic origin. In resemblance to the wide range of clinical teratomas, pluripotent stem cell (PSC) induced teratoma (PSCT) typically shows a mixture of developing tissues in randomly distributed compartments. The combined literature suggests that for teratomas derived from human diploid bona fide PSC the embryonic development in the separate tissue-niches can show a controlled differentiation into organoid patterns closely mimicking early development. In the experimental situation such PSCT human homologous in vivo tissue-niches have been shown to provide also matching microenvironment for a micrometastatic colonization and outgrowth of embryonic tumors transplanted directly from patients. Single or small clusters of normal and neoplastic cells can easily be visualized together in microscope-based imaging systems, enabling multi-parameter detection of in the scans of tissue slides/specimens.

PGE-metabolite levels in CSF correlate to HIE score and outcome after perinatal asphyxia.

AIM: Acute anoxic exposure rapidly increases prostaglandin E2 (PGE2 ) production and release in neonatal mice brains. We hypothesize that PGE2 is released in human cerebrospinal fluid (CSF) during perinatal asphyxia and that it might be used as a biomarker for perinatal asphyxia. METHODS: In full-term infants with lumbar puncture performed within 72 h of birth (n = 35), CSF was analysed for prostaglandin E2 metabolite (PGEM) using an enzyme immunoassay. Term infants with suspected but unverified infections were used as controls (n = 11). Hypoxic-ischaemic encephalopathy (HIE) was classified as mild, moderate or severe (HIE I-III). Neurological assessment of surviving patients was performed at 18 months of age. RESULTS: Prostaglandin E2 metabolite levels correlated to a low Apgar score at 5 min (p < 0.01) and 10 min (p < 0.01), a low pH (p < 0.001) and HIE score (p < 0.05). The HIE-III cases (n = 7) had significantly higher PGEM levels compared with both controls and the HIE-I group (n = 8). Irrespective of HIE grade, patients with adverse or fatal outcome had higher PGEM values compared with controls and asphyxiated infants with normal outcome (p < 0.05). CONCLUSIONS: PGE2 is released during anoxic events in newborn infants, and PGEM may be useful as a biomarker for estimating degree of insult and predicting long-term outcome after perinatal asphyxia.

Mosquito (Diptera: Culicidae) repellency field tests of essential oils from plants traditionally used in Laos.

Essential oils of Hyptis suaveolens (Lamiaceae), Croton roxburghii (Euphorbiaceae), and Litsea cubeba (Lauraceae) were tested in the field near Vientiane city, Lao PDR, on humans for repellent activity against mosquitoes. Landing mosquitoes were collected and later identified. The most abundant mosquitoes captured belonged to the genera Armigeres, Culex, and Aedes. All the plant oils tested at concentrations of 1.7 microg/cm(2), 3.3 microg/cm(2), and 6.3 microg/cm(2) were significantly more mosquito repellent than the negative control. Croton oil was significantly repellent against mosquitoes of the three genera at the highest (6.3 microg/cm(2)) concentration tested. Litsea oil was significantly repellent against Armigeres at all (1.7 microg/cm(2), 3.3 microg/cm(2), and 6.3 microg/cm(2)) concentrations tested. Hyptis oil was significantly repellent against Armigeres at 3.3 microg/cm(2) and 6.3 microg/cm(2) and against Culex at 1.7 microg/cm(2) and 6.3 microg/cm(2). The oils were analyzed for chemical content of volatiles, mainly terpenes. Main constituents were beta-pinene, sabinene, and 1,8-cineol from oils of the green parts of H. suaveolens; alpha-pinene, beta-pinene, and alpha-phellandrene from fresh bark of C. roxburghii; and alpha-pinene, beta-phellandrene, sabinene, and 1,8-cineol from fresh fruits of L. cubeba.

Functional stem cell integration assessed by organotypic slice cultures.

Re-formation or preservation of functional, electrically active neural networks has been proffered as one of the goals of stem cell-mediated neural therapeutics. A primary issue for a cell therapy approach is the formation of functional contacts between the implanted cells and the host tissue. Therefore, it is of fundamental interest to establish protocols that allow us to delineate a detailed time course of grafted stem cell survival, migration, differentiation, integration, and functional interaction with the host. One option for in vitro studies is to examine the integration of exogenous stem cells into an existing active neuronal network in ex vivo organotypic cultures. Organotypic cultures leave the structural integrity essentially intact while still allowing the microenvironment to be carefully controlled. This allows detailed studies over time of cellular responses and cell-cell interactions, which are not readily performed in vivo. This unit describes procedures for using organotypic slice cultures as ex vivo model systems for studying neural stem cell and embryonic stem cell engraftment and communication with CNS host tissue.

Molecular identification of commercialized medicinal plants in southern Morocco.

BACKGROUND: Medicinal plant trade is important for local livelihoods. However, many medicinal plants are difficult to identify when they are sold as roots, powders or bark. DNA barcoding involves using a short, agreed-upon region of a genome as a unique identifier for species- ideally, as a global standard. RESEARCH QUESTION: What is the functionality, efficacy and accuracy of the use of barcoding for identifying root material, using medicinal plant roots sold by herbalists in Marrakech, Morocco, as a test dataset. METHODOLOGY: In total, 111 root samples were sequenced for four proposed barcode regions rpoC1, psbA-trnH, matK and ITS. Sequences were searched against a tailored reference database of Moroccan medicinal plants and their closest relatives using BLAST and Blastclust, and through inference of RAxML phylograms of the aligned market and reference samples. PRINCIPAL FINDINGS: Sequencing success was high for rpoC1, psbA-trnH, and ITS, but low for matK. Searches using rpoC1 alone resulted in a number of ambiguous identifications, indicating insufficient DNA variation for accurate species-level identification. Combining rpoC1, psbA-trnH and ITS allowed the majority of the market samples to be identified to genus level. For a minority of the market samples, the barcoding identification differed significantly from previous hypotheses based on the vernacular names. CONCLUSIONS/SIGNIFICANCE: Endemic plant species are commercialized in Marrakech. Adulteration is common and this may indicate that the products are becoming locally endangered. Nevertheless the majority of the traded roots belong to species that are common and not known to be endangered. A significant conclusion from our results is that unknown samples are more difficult to identify than earlier suggested, especially if the reference sequences were obtained from different populations. A global barcoding database should therefore contain sequences from different populations of the same species to assure the reference sequences characterize the species throughout its distributional range.

Comparing medicinal plant knowledge using similarity indices: a case of the Brou, Saek and Kry in Lao PDR.

BACKGROUND: Medicinal plant traditional knowledge is one of the most widely known traditional ecosystem services, as it provides primary healthcare, contributes to subsistence livelihoods, and for its potential value as a source of novel pharmaceuticals. People living in close contact with their surroundings for many generations are hypothesized to have developed, through trial-and-error, in-depth knowledge of ecosystems, biodiversity, and their management and utility. In the case of medicinal plant knowledge it could lead to an asymptotic climax or a constantly evolving equilibrium of cures with proven efficacy and those under assessment. METHODS: An in-depth study of 97 plant species used in traditional medicine by the Brou, Saek and Kry ethnic groups in Lao PDR was made to test similarity in medicinal plant knowledge. RESULTS: Medicinal plants were used in 99 different ways in 510 species-use combinations. Medicinal uses could be generalized into 12 use categories with 747 species-category combinations. Similarity indices show Brou and Saek plant use appears to be most similar (QS(BS): 60.0; JI(BS): 75.1) followed by Kry and Saek (QS(KS): 51.6; JI(KS): 53.4), and then Kry and Brou (QS(BK): 46.9; JI(BK): 44.1). DISCUSSION: Intercultural similarities found are quite low, considering that all three groups share the same geographical and ecological area and have the same dependence on medicinal plants. Intercultural transmission is unimpeded but many treatments are likely to be ineffective. Comparison of the similarities found here with similarities computed from other data show that these results are homologous with other sympatric ethnic groups, and much higher than those for allopatrically living groups. CONCLUSION: Medicinal plant knowledge does not reach a stable climax, but appears to evolve continually by trial-and-error, as effective cures to many ailments are unavailable.

Steam sauna and mother roasting in Lao PDR: practices and chemical constituents of essential oils of plant species used in postpartum recovery.

BACKGROUND: Fundamental in traditional postpartum recovery in Lao PDR is the use of hotbeds, mother roasting, steam sauna and steam baths. During these treatments medicinal plants play a crucial role, but little has been published about how the treatments are carried out precisely, which species are used, the medicinal properties of these species, and the medicinal efficacy of their chemical constituents. METHODS: Sixty-five interviews, in 15 rural villages, with women of 4 different ethnic groups were conducted to survey confinement rituals, and postpartum plant use and salience. Essential oils from the main species used were extracted using steam distillation and the main chemical constituents characterized using gas chromatography-mass spectrometry (GC-MS). RESULTS: A total of 10 different species were used by three or more of the ethnic groups included in this study. All species were used in steam sauna and bath, but only 3 species were used in hotbed and mother roasting. Essential oils of Amomum villosum, Amomum microcarpum and Blumea balsamifera were found to contain significant amounts of the following terpenes: ß-pinene, camphor, bornyl acetate, borneol, linalool, D-limonene, fenchone, terpinen-4-ol and α-terpinene. CONCLUSIONS: Many of these terpenes have documented antimicrobial and analgesic properties, and some have also synergistic interactions with other terpenes. The mode of application in hotbed and mother roasting differs from the documented mechanisms of action of these terpenes. Plants in these two practices are likely to serve mainly hygienic purposes, by segregating the mother from infection sources such as beds, mats, stools, cloth and towels. Steam sauna medicinal plant use through inhalation of essential oils vapors can possibly have medicinal efficacy, but is unlikely to alleviate the ailments commonly encountered during postpartum convalescence. Steam sauna medicinal plant use through dermal condensation of essential oils, and steam bath cleansing of the perineal area is possibly a pragmatic use of the reported medicinal plants, as terpene constituents have documented antimicrobial, analgesic and anti-inflammatory properties.

Traditions and plant use during pregnancy, childbirth and postpartum recovery by the Kry ethnic group in Lao PDR.

BACKGROUND: Activities and diet during the postpartum period are culturally dictated in many Southeast Asian cultures, and a period of confinement is observed. Plants play an important role in recovery during the postpartum period in diet and traditional medicine. Little is known of the Kry, a small ethnic group whose language was recently described, concerning its traditions and use of plants during pregnancy, parturition, postpartum recovery and infant healthcare. This research aims to study those traditions and identify medicinal plant use. METHODS: Data were collected in the 3 different Kry villages in Khammouane province, Lao PDR, through group and individual interviews with women by female interviewers. RESULTS: A total of 49 different plant species are used in women's healthcare. Plant use is culturally different from the neighboring Brou and Saek ethnic groups. Menstruation, delivery and postpartum recovery take place in separate, purpose-built, huts and a complex system of spatial restrictions is observed. CONCLUSIONS: Traditions surrounding childbirth are diverse and have been strictly observed, but are undergoing a shift towards those from neighboring ethnic groups, the Brou and Saek. Medicinal plant use to facilitate childbirth, alleviate menstruation problems, assist recovery after miscarriage, mitigate postpartum haemorrhage, aid postpartum recovery, and for use in infant care, is more common than previously reported (49 species instead of 14). The wealth of novel insights into plant use and preparation will help to understand culturally important practices such as traditional delivery, spatial taboos, confinement and dietary restrictions, and their potential in modern healthcare.

Botanical repellents and pesticides traditionally used against hematophagous invertebrates in Lao People's Democratic Republic: a comparative study of plants used in 66 villages.

Hematophagous parasites such as leeches, ticks, mites, lice, bedbugs, mosquitoes, and myiasis-producing fly larvae are common health problems in Lao People's Democratic Republic. Several arthropod-borne infections, e.g., malaria, dengue fever, and Japanese encephalitis, are endemic there. Effective vector control methods including the use of pesticides, insecticide-treated bed nets, and synthetic and plant-based repellents are important means of control against such invertebrates and the pathogens they may transmit or directly cause. In this study, we documented traditional knowledge on plants that are used to repel or kill hematophagous arthropods, including mosquitoes, bedbugs, human lice, mites and ticks, fly larvae, and blood-sucking leeches. Structured interviews were carried out in 66 villages comprising 17 ethnic groups, covering a range of cultures, throughout Lao People's Democratic Republic. A total of 92 plant species was recorded as traditional repellents (including plants for pesticidal usages) in 123 different plant-ectoparasite combinations. The number and species of plants, and animal taxa repelled (or killed) per plant species differed per region, village, and ethnic group. Traditional use was confirmed in the scientific literature for 74 of these plant species, and for an additional 13 species using literature on closely related species. The use of botanical repellents and pesticides from many plant species is common and widespread in the Lao countryside. In the future, the identification of the active components in certain plants to develop more optimal, inexpensive repellents, insecticides, acaricides, or antileech compounds as alternatives to synthetic repellents/pesticides against blood-feeding insects, ticks, mites, and leeches is warranted.

Down-regulation of the strawberry Bet v 1-homologous allergen in concert with the flavonoid biosynthesis pathway in colorless strawberry mutant.

Proteomic screening of strawberry (Fragaria ananassa) yielded a 58% success rate in protein identification in spite of the fact that no genomic sequence is available for this species. This was achieved by a combination of MALDI-MS/MS de novo sequencing of double-derivatized peptides and indel-tolerant searching against local protein databases built on both EST and full-length nucleotide sequences. The amino acid sequence of a strawberry allergen, homologous to the well-known major birch pollen allergen Bet v 1, was partially determined. This strawberry allergen, named Fra a 1 according to the nomenclature for allergen proteins, showed sequence identity of 54 and 77%, respectively, with corresponding allergens from birch and apple. Differential expression, as evaluated by 2-D DIGE, occurred in 10% of protein spots when red strawberries were compared to a colorless (white) strawberry mutant. White strawberries, known to be tolerated by individuals affected by allergy, were found to be virtually free from the strawberry allergen. Also several enzymes in the pathway for biosynthesis of flavonoids, to which the red color pelargonidin belongs, were down-regulated. This approach to assess differential protein expression without access to genomic sequence information can also be applied to other crop plants and phenotypic traits.

Expanded substrate screenings of human and Drosophila type 10 17beta-hydroxysteroid dehydrogenases (HSDs) reveal multiple specificities in bile acid and steroid hormone metabolism: characterization of multifunctional 3alpha/7alpha/7beta/17beta/20beta/21-HSD.

17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyse the conversion of 17beta-OH (-hydroxy)/17-oxo groups of steroids, and are essential in mammalian hormone physiology. At present, eleven 17beta-HSD isoforms have been defined in mammals, with different tissue-expression and substrate-conversion patterns. We analysed 17beta-HSD type 10 (17beta-HSD10) from humans and Drosophila, the latter known to be essential in development. In addition to the known hydroxyacyl-CoA dehydrogenase, and 3alpha-OH and 17beta-OH activities with sex steroids, we here demonstrate novel activities of 17beta-HSD10. Both species variants oxidize the 20beta-OH and 21-OH groups in C21 steroids, and act as 7beta-OH dehydrogenases of ursodeoxycholic or isoursodeoxycholic acid (also known as 7beta-hydroxylithocholic acid or 7beta-hydroxyisolithocholic acid respectively). Additionally, the human orthologue oxidizes the 7alpha-OH of chenodeoxycholic acid (5beta-cholanic acid, 3alpha,7alpha-diol) and cholic acid (5beta-cholanic acid). These novel substrate specificities are explained by homology models based on the orthologous rat crystal structure, showing a wide hydrophobic cleft, capable of accommodating steroids in different orientations. These properties suggest that the human enzyme is involved in glucocorticoid and gestagen catabolism, and participates in bile acid isomerization. Confocal microscopy and electron microscopy studies reveal that the human form is localized to mitochondria, whereas Drosophila 17beta-HSD10 shows a cytosolic localization pattern, possibly due to an N-terminal sequence difference that in human 17beta-HSD10 constitutes a mitochondrial targeting signal, extending into the Rossmann-fold motif.

Abrogated lymphocyte infiltration and lowered CD14 in dextran sulfate induced colitis in mice treated with p65 antisense oligonucleotides.

BACKGROUND AND AIMS: Dextran sulfate sodium (DSS) induced colitis exhibits a predominantly NF-kappaB dependent proinflammatory cytokine profile and shares similarities with human inflammatory bowel disease (IBD). Lamina propria macrophages of IBD patients display elevated levels of NF-kappaB p65. Knowing the role of NF-kappaB in IBD, we investigated the beneficial cellular mechanisms underlying the lasting effect of a single p65 antisense treatment in DSS-colitis mice. METHODS: One local dose of p65 antisense oligonucleotides was administered in DSS colitis mice. Ten days later the mice were killed and examined at cellular and biochemical levels. The level of p65 in lamina propria cells was determined by electrophoretic mobility shift assay and by intracellular immunofluorescent staining of nuclear p65 levels, using laser scanning cytometer. RESULTS: FACS analysis demonstrated a considerable drop in infiltrating lymphocytes and a drastic reduction in CD14+ cells in mice treated with p65 antisense oligonucleotides. Moreover, abrogation of inflammation extended all the way to the cecum in treated mice. Treatment was correlated with decreased DNA binding activity of NF-kappaB. CONCLUSION: Our data strongly support a model in which p65 antisense treatment possesses the capacity to disrupt the pathogenic autocrine loop propagated by NF-kappaB at the chronic phase of IBD.