Serological differentiation between Echinococcus granulosus and E. multilocularis infections in man.

An enzyme-linked immunosorbent assay (ELISA) was adapted for the serological differential diagnosis of cystic or alveolar echinococcosis in man caused by Echinococcus granulosus or E. multilocularis respectively. By affinity chromatography using rabbit anti hydatid fluid IgG coupled covalently to CNBr-Sepharose 4B a protein fraction (Em 1) containing shared antigens of both parasites could be isolated from an extract of E. multilocularis metacestode tissue. From the same source another antigen fraction (Em 2) with a high degree of specificity for E. multilocularis was prepared by immunosorption. Antigen Em 1 was equally sensitive for the detection of antibodies against E. granulosus and E. multilocularis, whereas antigen fraction Em 2 appeared to be more specific for E. multilocularis. A correct serological differential diagnosis was achieved in 95% of 57 confirmed cases of human cystic or alveolar echinococcosis by the simultaneous use of both antigen fractions in the ELISA and by comparison of their reactivities.

Retinal differentiation of human bone marrow-derived stem cells by co-culture with retinal pigment epithelium in vitro.

The goal of this study was to assess the in vitro differentiation capacity of human bone marrow-derived stem cells (hBMSCs) along retinal lineages. Mononuclear cells (MNC) were isolated from bone marrow (BM) and mobilized peripheral blood (mPB) using Ficoll-Paque density gradient centrifugation, and were sorted by magnetic-activated cell sorting (MACS) for specific stem cell subsets (CD34(+)CD38(+)/CD34(+)CD38(-)). These cells were then co-cultured on human retinal pigment epithelial cells (hRPE) for 7 days. The expression of stem cell, neural and retina-specific markers was examined by immunostaining, and the gene expression profiles were assessed after FACS separation of the co-cultured hBMSCs by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, in vitro functionality of the differentiated cells was analyzed by quantifying phagocytosis of CY5-labeled photoreceptor outer segments (POS). After 7 days of co-culture, hBMSCs adopted an elongated epithelial-like morphology and expressed RPE-specific markers, such as RPE65 and bestrophin. In addition, these differentiated cells were able to phagocytose OS, one of the main characteristics of native RPE cells. Our data demonstrated that human CD34(+)CD38(-) hBMSC may differentiate towards an RPE-like cell type in vitro and could become a new type of autologous donor cell for regenerative therapy in retinal degenerative diseases.

Mechanical Loading Promoted Discogenic Differentiation of Human Mesenchymal Stem Cells Incorporated in 3D-PEG Scaffolds with RhGDF5 and RGD

Hydrogels have been described as ideal scaffolds for cells of 3D tissue constructs and hold strong promises with respect to in vitro 3D-cell-culture, where cells are isolated from native extracellular matrix (ECM). Synthesized polyethyleneglycol (PEG) hydrogels are appealing with regard to potential for cell therapy or as vehicles for drug delivery or even to regenerate tissue with similar hydrogel-like properties such as the nucleus pulposus of the intervertebral disc (IVD). Here, we tested whether incorporation of RGD motive would hinder discogenic differentiation of primary bone marrow-derived human mesenchymal stem cells (hMSCs) but favor proliferation of undifferentiated hMSCs. HMSCs were embedded in +RGD containing or without RGD PEG hydrogel and pre-conditioned with or without growth and differentiation factor-5 (rhGDF-5) for 13 days. Afterwards, all hMSCs-PEG gels were subsequently cyclically loaded (15% strain, 1Hz) for 5 consecutive days in a bioreactor to generate an IVD-like phenotype. Higher metabolic activity (resazurin assay) was found in groups with rhGDF5 in both gel types with and without RGD. Cell viability and morphology measured by confocal laser microscopy and DNA content showed decreased values (~60%) after 18 days of culture. Real-time RT-PCR of an array of 15 key genes suspected to be distinctive for IVD cells revealed moderate response to rhGDF5 and mechanical loading as also shown by histology staining. Preconditioning and mechanical loading showed relatively moderate responses revealed from both RT-PCR and histology although hMSCs were demonstrated to be potent to differentiate into chondrocyte-progenitor cells in micro- mass and 3D alginate bead culture.

Sodium-Dependent Phosphate Transporters in Osteoclast Differentiation and Function

Osteoclasts are multinucleated bone degrading cells. Phosphate is an important constituent of mineralized bone and released in significant quantities during bone resorption. Molecular contributors to phosphate transport during the resorptive activity of osteoclasts have been controversially discussed. This study aimed at deciphering the role of sodium-dependent phosphate transporters during osteoclast differentiation and bone resorption. Our studies reveal RANKL-induced differential expression of sodium-dependent phosphate transport protein IIa (NaPi-IIa) transcript and protein during osteoclast development, but no expression of the closely related NaPi-IIb and NaPi-IIc SLC34 family isoforms. In vitro studies employing NaPi-IIa-deficient osteoclast precursors and mature osteoclasts reveal that NaPi-IIa is dispensable for bone resorption and osteoclast differentiation. These results are supported by the analysis of structural bone parameters by high-resolution microcomputed tomography that yielded no differences between adult NaPi-IIa WT and KO mice. By contrast, both type III sodium-dependent phosphate transporters Pit-1 and Pit-2 were abundantly expressed throughout osteoclast differentiation, indicating that they are the relevant sodium-dependent phosphate transporters in osteoclasts and osteoclast precursors. We conclude that phosphate transporters of the SLC34 family have no role in osteoclast differentiation and function and propose that Pit-dependent phosphate transport could be pivotal for bone resorption and should be addressed in further studies.

Differentiation between Staphylococcus aureus and coagulase-negative Staphylococcus species by real-time PCR including detection of methicillin resistants in comparison to conventional microbiology testing.

BACKGROUND Staphylococcus aureus has long been recognized as a major pathogen. Methicillin-resistant strains of S. aureus (MRSA) and methicillin-resistant strains of S. epidermidis (MRSE) are among the most prevalent multiresistant pathogens worldwide, frequently causing nosocomial and community-acquired infections. METHODS In the present pilot study, we tested a polymerase chain reaction (PCR) method to quickly differentiate Staphylococci and identify the mecA gene in a clinical setting. RESULTS Compared to the conventional microbiology testing the real-time PCR assay had a higher detection rate for both S. aureus and coagulase-negative Staphylococci (CoNS; 55 vs. 32 for S. aureus and 63 vs. 24 for CoNS). Hands-on time preparing DNA, carrying out the PCR, and evaluating results was less than 5 h. CONCLUSIONS The assay is largely automated, easy to adapt, and has been shown to be rapid and reliable. Fast detection and differentiation of S. aureus, CoNS, and the mecA gene by means of this real-time PCR protocol may help expedite therapeutic decision-making and enable earlier adequate antibiotic treatment.

Within-plant distribution of 1,4-benzoxazin-3-ones contributes to herbivore niche differentiation in maize

Plant defences vary in space and time, which may translate into specific herbivore-foraging patterns and feeding niche differentiation. To date, little is known about the effect of secondary metabolite patterning on within-plant herbivore foraging. We investigated how variation in the major maize secondary metabolites, 1,4-benzoxazin-3-one derivatives (BXDs), affects the foraging behaviour of two leaf-chewing herbivores. BXD levels varied substantially within plants. Older leaves had higher levels of constitutive BXDs while younger leaves were consistently more inducible. These differences were observed independently of plant age, even though the concentrations of most BXDs declined markedly in older plants. Larvae of the well-adapted maize pest Spodoptera frugiperda preferred and grew better on young inducible leaves irrespective of plant age, while larvae of the generalist Spodoptera littoralis preferred and tended to grow better on old leaves. In BXD-free mutants, the differences in herbivore weight gain between old and young leaves were absent for both species, and leaf preferences of S. frugiperda were attenuated. In contrast, S. littoralis foraging patterns were not affected. In summary, our study shows that plant secondary metabolites differentially affect performance and foraging of adapted and non-adapted herbivores and thereby likely contribute to feeding niche differentiation

Linking the SUMO protease SENP5 to neutrophil differentiation of AML cells

In an mRNA profiling screen performed to unveil novel mechanisms of leukemogenesis, we found that the sentrin-specific protease 5 (SENP5) was significantly repressed in clinical acute myeloid leukemia when compared to healthy neutrophil samples. SENP5 is an enzyme that targets and cleaves small ubiquitin-like modifier (SUMO) residues from SUMOylated proteins. Further investigation with AML neutrophil differentiation cell models showed increased SENP5 expression upon induction of differentiation; in contrast, knocking down SENP5 resulted in significantly attenuated neutrophil differentiation. Our results support a new role of SENP5 in AML pathology, and in particular in the neutrophil differentiation of myeloid leukemic cells.

Induction of autophagy is a key component of all-trans-retinoic acid-induced differentiation in leukemia cells and a potential target for pharmacologic modulation

Acute myeloid leukemia (AML) is characterized by the accumulation of immature blood cell precursors in the bone marrow. Pharmacologically overcoming the differentiation block in this condition is an attractive therapeutic avenue, which has achieved success only in a subtype of AML, acute promyelocytic leukemia (APL). Attempts to emulate this success in other AML subtypes have thus far been unsuccessful. Autophagy is a conserved protein degradation pathway with important roles in mammalian cell differentiation, particularly within the hematopoietic system. In the study described here, we investigated the functional importance of autophagy in APL cell differentiation. We found that autophagy is increased during all-trans-retinoic acid (ATRA)-induced granulocytic differentiation of the APL cell line NB4 and that this is associated with increased expression of LC3II and GATE-16 proteins involved in autophagosome formation. Autophagy inhibition, using either drugs (chloroquine/3-methyladenine) or short-hairpin RNA targeting the essential autophagy gene ATG7, attenuates myeloid differentiation. Importantly, we found that enhancing autophagy promotes ATRA-induced granulocytic differentiation of an ATRA-resistant derivative of the non-APL AML HL60 cell line (HL60-Diff-R). These data support the development of strategies to stimulate autophagy as a novel approach to promote differentiation in AML.

Low DICER1 expression is associated with attenuated neutrophil differentiation and autophagy of NB4 APL cells

Successful myeloid differentiation depends on the expression of a series of miRNAs. Thus, it is hardly surprising that miRNAs are globally repressed in AML, a disease mainly characterized by a block in cellular myeloid differentiation. Studies investigating the mechanisms for low miRNA expression in AML has mostly focused on altered transcriptional regulation or deletions, whereas defective miRNA processing has received less attention. In this study, we report that the expression of the key miRNA processing enzyme DICER1 is down-regulated in primary AML patient samples and healthy CD34(+) progenitor cells as compared with granulocytes. In line with these findings, Dicer1 expression was induced significantly in AML cell lines upon neutrophil differentiation. The knocking down of DICER1 in AML cells significantly attenuated neutrophil differentiation, which was paralleled by decreased expression of miRNAs involved in this process. Moreover, we found that inhibiting DICER1 attenuated the activation of autophagy, a cellular recycling process that is needed for proper neutrophil differentiation of AML cells. Our results clearly indicate that DICER1 plays a novel role in neutrophil differentiation as well as in myeloid autophagy of AML cells.

Genomics of Divergence along a Continuum of Parapatric Population Differentiation

The patterns of genomic divergence during ecological speciation are shaped by a combination of evolutionary forces. Processes such as genetic drift, local reduction of gene flow around genes causing reproductive isolation, hitchhiking around selected variants, variation in recombination and mutation rates are all factors that can contribute to the heterogeneity of genomic divergence. On the basis of 60 fully sequenced three-spined stickleback genomes, we explore these different mechanisms explaining the heterogeneity of genomic divergence across five parapatric lake and river population pairs varying in their degree of genetic differentiation. We find that divergent regions of the genome are mostly specific for each population pair, while their size and abundance are not correlated with the extent of genome-wide population differentiation. In each pair-wise comparison, an analysis of allele frequency spectra reveals that 25–55% of the divergent regions are consistent with a local restriction of gene flow. Another large proportion of divergent regions (38–75%) appears to be mainly shaped by hitchhiking effects around positively selected variants. We provide empirical evidence that alternative mechanisms determining the evolution of genomic patterns of divergence are not mutually exclusive, but rather act in concert to shape the genome during population differentiation, a first necessary step towards ecological speciation.

Disentangling the role of phenotypic plasticity and genetic divergence in contemporary ecotype formation during a biological invasion

The occurrence of contemporary ecotype formation through adaptive divergence of populations within the range of an invasive species typically requires standing genetic variation but can be facilitated by phenotypic plasticity. The relative contributions of both of these to adaptive trait differentiation have rarely been simultaneously quantified in recently diverging vertebrate populations. Here we study a case of intraspecific divergence into distinct lake and stream ecotypes of threespine stickleback that evolved in the past 140 years within the invasive range in Switzerland. Using a controlled laboratory experiment with full-sib crosses and treatments mimicking a key feature of ecotypic niche divergence, we test if the phenotypic divergence that we observe in the wild results from phenotypic plasticity or divergent genetic predisposition. Our experimental groups show qualitatively similar phenotypic divergence as those observed among wild adults. The relative contribution of plasticity and divergent genetic predisposition differs among the traits studied, with traits related to the biomechanics of feeding showing a stronger genetic predisposition, whereas traits related to locomotion are mainly plastic. These results implicate that phenotypic plasticity and standing genetic variation interacted during contemporary ecotype formation in this case.

A new approach to chemotherapy: drug-induced differentiation kills African trypanosomes

Human African trypanosomiasis (sleeping sickness) is a neglected tropical disease caused by Trypanosoma brucei spp. The parasites are transmitted by tsetse flies and adapt to their different hosts and environments by undergoing a series of developmental changes. During differentiation, the trypanosome alters its protein coat. Bloodstream form trypanosomes in humans have a coat of variant surface glycoprotein (VSG) that shields them from the immune system. The procyclic form, the first life-cycle stage to develop in the tsetse fly, replaces the VSG coat by procyclins; these proteins do not protect the parasite from lysis by serum components. Our study exploits the parasite-specific process of differentiation from bloodstream to procyclic forms to screen for potential drug candidates. Using transgenic trypanosomes with a reporter gene in a procyclin locus, we established a whole-cell assay for differentiation in a medium-throughput format. We screened 7,495 drug-like compounds and identified 28 hits that induced expression of the reporter and loss of VSG at concentrations in the low micromolar range. Small molecules that induce differentiation to procyclic forms could facilitate studies on the regulation of differentiation as well as serving as scaffolds for medicinal chemistry for new treatments for sleeping sickness.

Postmortem quantitative 1.5-T MRI for the differentiation and characterization of serous fluids, blood, CSF, and putrefied CSF.

The purpose of the present study was to investigate whether serous fluids, blood, cerebrospinal fluid (CSF), and putrefied CSF can be characterized and differentiated in synthetically calculated magnetic resonance (MR) images based on their quantitative T 1, T 2, and proton density (PD) values. Images from 55 postmortem short axis cardiac and 31 axial brain 1.5-T MR examinations were quantified using a quantification sequence. Serous fluids, fluid blood, sedimented blood, blood clots, CSF, and putrefied CSF were analyzed for their mean T 1, T 2, and PD values. Body core temperature was measured during the MRI scans. The fluid-specific quantitative values were related to the body core temperature. Equations to correct for temperature differences were generated. In a 3D plot as well as in statistical analysis, the quantitative T 1, T 2 and PD values of serous fluids, fluid blood, sedimented blood, blood clots, CSF, and putrefied CSF could be well differentiated from each other. The quantitative T 1 and T 2 values were temperature-dependent. Correction of quantitative values to a temperature of 37 °C resulted in significantly better discrimination between all investigated fluid mediums. We conclude that postmortem 1.5-T MR quantification is feasible to discriminate between blood, serous fluids, CSF, and putrefied CSF. This finding provides a basis for the computer-aided diagnosis and detection of fluids and hemorrhages.

Morphological differentiation of Betula (birch) pollen in northwest North America and its palaeoecological application

Lake sediments from arcto-boreal regions commonly contain abundant Betula pollen. However, palaeoenvironmental interpretations of Betula pollen are often ambiguous because of the lack of reliable morphological features to distinguish among ecologically distinct Betula species in western North America. We measured the grain diameters and pore depths of pollen from three tree-birch species (B. papyrifera, B. kenaica and B. neoalaskana) and two shrub-birch species (B. glandulosa and B. nana), and calculated the ratio of grain diameter to pore depth (D/P ratio). No statistical difference exists in all three parameters between the shrub-birch species or between two of the tree-birch species (B. kenaica and B. papyrifera), and B. neoalaskana is intermediate between the shrub-birch and the other two tree-birch species. However, mean pore depth is significantly larger for the tree species than for the shrub species. In contrast, mean grain diameter cannot distinguish tree and shrub species. Mean D/P ratio separates tree and shrub species less clearly than pore depth, but this ratio can be used for verification. The threshold for distinguishing pollen of tree versus shrub birch lies at 2.55 μm and 8.30 for pore depth and D/P ratio, respectively. We'applied these thresholds to the analysis of Betula pollen in an Alaskan lake-sediment core spanning the past 800 years. Results show that shrub birch increased markedly at the expense of tree birch during the‘Little Ice Age’; this patten is not discernible in the profile of total birch pollen.

Assessment of bovine adipose-derived stem cells osteogenic and adipogenic differentiation in Normoxic and hypoxic conditions

Background: The differentiation of ADSC is regulated by many factors, including oxygen tensions. Evidences have suggested that low oxygen tension or hypoxia is involved in the osteogenic, adipogenic differentiations of MSCs. Expansion and induction of ADSCs under hypoxia generally result in enhanced osteogenic, differentiation. Therefore, we analyzed bovine ADSC differentiations in Normoxia and hypoxia conditions Methodology: Recently (<8h) cow tail from a slaughterhouse, take out some fat from the tail and fat cells was isolated by using for isolation of ADSC protocol, the expansion cells were put into osteogenic and adipogenic medium for 3 weeks in hypoxia and normoxia conditions separately and characterized by Von kossa, Alizarin red and Oil red O staining and further by using real-time PCR by using primers of osteocalcin, Collagen type1, cbfa1/runx2, ALP, ostepontin, osteonectin, BMP2, BMP24, BMP27, noggin, gremlin, Nestin and HIF1A,VEGFA,PPARG,Leptin. Results: Our experiment results show hypoxia promotes osteogenesis but suppresses adipogenesis.