A simple method of DNA extraction from coffee seeds suitable for PCR analysis

High quality genomic DNA was successfully extracted from coffee seeds using a simple protocol devoid of liquid nitrogen or freeze-drying and proteinase K. The isolated DNA was quantified using spectrophotometer and using agarose gel electrophoresis. The DNA was free from polysaccharides, polyphenols, RNA and other contaminants. The quantity of DNA ranged from 180 to 630 g/g of seed powder. Quality of DNA was confirmed by digestion using EcoRI, HindIII and PstI restrictionendonucleases and complete digestion was observed. PCR with random decamer primers and consensus primers of mitochondria and chloroplast DNA and PCR-RFLP revealed the suitability of the DNA for PCR based marker techniques including diagnostics

Serum butyrylcholinesterase in type 2 diabetes mellitus: a biochemical and bioinformatics approach

BACKGROUND: Butyrylcholinesterase is an enzyme that may serve as a marker of metabolic syndrome. We (a) measured its level in persons with diabetes mellitus, (b) constructed a family tree of the enzyme using nucleotide sequences downloaded from NCBI. Butyrylcholinesterase was estimated colorimetrically using a commercially available kit (Randox Lab, UK). Phylogenetic trees were constructed by distance method (Fitch and Margoliash method) and by maximum parsimony method. RESULTS: There was a negative correlation between serum total cholesterol and butyrylcholinesterase (-0.407; p < 0.05) and between serum LDL cholesterol and butyrylcholinesterase (-0.435; p < 0.05). There was no statistically significant correlation among the other biochemical parameters. In the evolutionary tree construction both methods gave similar trees, except for an inversion in the position of Sus scrofa (M62778) and Oryctolagus cuniculus (M62779) between Fitch and Margoliash, and maximum parsimony methods. CONCLUSION: The level of butyrylcholinesterase enzyme was inversely related to serum cholesterol; dendrogram showed that the structures from evolutionarily close species were placed near each other

Hippobosca longipennis - a potential intermediate host of a species of Acanthocheilonema in dogs in northern India

Background Hippobosca longipennis (the 'dog louse fly') is a blood sucking ectoparasite found on wild carnivores such as cheetahs and lions and domesticated and feral dogs in Africa, the Middle East and Asia, including China. Known as an intermediate host for Acanthocheilonema dracunculoides and a transport host for Cheyletiella yasguri, it has also been suggested that H. longipennis may be a vector for other pathogens, including Acanthocheilonema sp.? nov., which was recently reported to infect up to 48% of dogs in northern India where this species of fly is known to commonly infest dogs. To test this hypothesis, hippoboscid flies feeding on dogs in Ladakh in northern India were collected and subjected to microscopic dissection. Results A total of 12 infective larvae were found in 10 out of 65 flies dissected; 9 from the head, 2 from the thorax and 1 from the abdomen. The larvae averaged 2, 900 (± 60) μm in length and 34 (± 5) μm in width and possessed morphological features characteristic of the family Onchocercidae. Genetic analysis and comparison of the 18S, ITS-2, 12S and cox-1 genes confirmed the identity of the larvae as the Acanthocheilonema sp.? nov. reported in dogs in Ladakh. Conclusion This study provides evidence for a potential intermediate host-parasite relationship between H. longipennis and the canine Acanthocheilonema sp.? nov. in northern India

Identification of chemokine receptors as potential modulators of endocrine resistance in oestrogen receptor–positive breast cancers

Introduction Endocrine therapies target oestrogenic stimulation of breast cancer (BC) growth, but resistance remains problematic. Our aims in this study were (1) to identify genes most strongly associated with resistance to endocrine therapy by intersecting global gene transcription data from patients treated presurgically with the aromatase inhibitor anastrazole with those from MCF7 cells adapted to long-term oestrogen deprivation (LTED) (2) to assess the clinical value of selected genes in public clinical data sets and (3) to determine the impact of targeting these genes with novel agents. Methods Gene expression and Ki67 data were available from 69 postmenopausal women with oestrogen receptor–positive (ER+) early BC, at baseline and 2 weeks after anastrazole treatment, and from cell lines adapted to LTED. The functional consequences of target genes on proliferation, ER-mediated transcription and downstream cell signalling were assessed. Results By intersecting genes predictive of a poor change in Ki67 with those upregulated in LTED cells, we identified 32 genes strongly correlated with poor antiproliferative response that were associated with inflammation and/or immunity. In a panel of LTED cell lines, C-X-C chemokine receptor type 7 (CXCR7) and CXCR4 were upregulated compared to their wild types (wt), and CXCR7, but not CXCR4, was associated with reduced relapse-free survival in patients with ER+ BC. The CXCR4 small interfering RNA variant (siCXCR4) had no specific effect on the proliferation of wt-SUM44, wt-MCF7 and their LTED derivatives. In contrast, siCXCR7, as well as CCX733, a CXCR7 antagonist, specifically suppressed the proliferation of MCF7-LTED cells. siCXCR7 suppressed proteins associated with G1/S transition and inhibited ER transactivation in MCF7-LTED, but not wt-MCF7, by impeding association between ER and proline-, glutamic acid– and leucine-rich protein 1, an ER coactivator. Conclusions These data highlight CXCR7 as a potential therapeutic target warranting clinical investigation in endocrine-resistant BC

Hidden attractors in fundamental problems and engineering models

Recently a concept of self-excited and hidden attractors was suggested: an attractor is called a self-excited attractor if its basin of attraction overlaps with neighborhood of an equilibrium, otherwise it is called a hidden attractor. For example, hidden attractors are attractors in systems with no equilibria or with only one stable equilibrium (a special case of multistability and coexistence of attractors). While coexisting self-excited attractors can be found using the standard computational procedure, there is no standard way of predicting the existence or coexistence of hidden attractors in a system. In this plenary survey lecture the concept of self-excited and hidden attractors is discussed, and various corresponding examples of self-excited and hidden attractors are considered

A survey of canine tick-borne diseases in India

Background: There are few published reports on canine Babesia, Ehrlichia, Anaplasma, Hepatozoon and haemotropic Mycoplasma infections in India and most describe clinical disease in individual dogs, diagnosed by morphological observation of the microorganisms in stained blood smears. This study investigated the occurrence and distribution of canine tick-borne disease (TBD) pathogens using a combination of conventional and molecular diagnostic techniques in four cities in India. Results: On microscopy examination, only Hepatozoon gamonts were observed in twelve out of 525 (2.3%; 95% CI: 1.2, 4) blood smears. Using polymerase chain reaction (PCR), a total of 261 from 525 dogs (49.7%; 95% CI: 45.4, 54.1) in this study were infected with one or more canine tick-borne pathogen. Hepatozoon canis (30%; 95% CI: 26.0, 34.0) was the most common TBD pathogen found infecting dogs in India followed by Ehrlichia canis (20.6%; 95% CI: 17.2, 24.3), Mycoplasma haemocanis (12.2%; 95% CI: 9.5, 15.3), Anaplasma platys (6.5%; 95% CI: 4.5, 8.9), Babesia vogeli (5.5%, 95% CI: 3.7, 7.8) and Babesia gibsoni (0.2%, 95% CI: 0.01, 1.06). Concurrent infection with more than one TBD pathogen occurred in 39% of cases. Potential tick vectors, Rhipicephalus (most commonly) and/or Haemaphysalis ticks were found on 278 (53%) of dogs examined. Conclusions: At least 6 species of canine tick-borne pathogens are present in India. Hepatozoon canis was the most common pathogen and ticks belonging to the genus Rhipicephalus were encountered most frequently. Polymerase chain reaction was more sensitive in detecting circulating pathogens compared with peripheral blood smear examination. As co-infections with canine TBD pathogens were common, Indian veterinary practitioners should be cognisant that the discovery of one such pathogen raises the potential for multiple infections which may warrant different clinical management strategies

Assembly and Development of the Pseudomonas aeruginosa Biofilm Matrix

Virtually all cells living in multicellular structures such as tissues and organs are encased in an extracellular matrix. One of the most important features of a biofilm is the extracellular polymeric substance that functions as a matrix, holding bacterial cells together. Yet very little is known about how the matrix forms or how matrix components encase bacteria during biofilm development. Pseudomonas aeruginosa forms environmentally and clinically relevant biofilms and is a paradigm organism for the study of biofilms. The extracellular polymeric substance of P. aeruginosa biofilms is an ill-defined mix of polysaccharides, nucleic acids, and proteins. Here, we directly visualize the product of the polysaccharide synthesis locus (Psl exopolysaccharide) at different stages of biofilm development. During attachment, Psl is anchored on the cell surface in a helical pattern. This promotes cell–cell interactions and assembly of a matrix, which holds bacteria in the biofilm and on the surface. Chemical dissociation of Psl from the bacterial surface disrupted the Psl matrix as well as the biofilm structure. During biofilm maturation, Psl accumulates on the periphery of 3-D-structured microcolonies, resulting in a Psl matrix-free cavity in the microcolony center. At the dispersion stage, swimming cells appear in this matrix cavity. Dead cells and extracellular DNA (eDNA) are also concentrated in the Psl matrix-free area. Deletion of genes that control cell death and autolysis affects the formation of the matrix cavity and microcolony dispersion. These data provide a mechanism for how P. aeruginosa builds a matrix and subsequently a cavity to free a portion of cells for seeding dispersal. Direct visualization reveals that Psl is a key scaffolding matrix component and opens up avenues for therapeutics of biofilm-related complications

Histopathological alterations in Senegal sole, Solea Senegalensis, from a polluted Huelva estuary (SW, Spain)

As a component of a large research project to evaluate the effects of contaminants on fish health in the field, histopathological studies have been conducted to help establish causal relationship between pollutants (heavy metals and aromatic polycyclic hydrocarbons—PAHs) and histopathological responses in Senegal sole, Solea senegalensis, from an estuary of SW Spain. Heavy metals (As, Zn, Cd, Pb, Cu and Fe) and 16 PAHs (proprietary USEPA) concentrations in water, sediment and tissues (liver and gills) and histopathological alterations in S. senegalensis from three sampling sites of Ria de Huelva estuary during 2004–2006 years have been analysed. The histopathological studies revealed seasonal and spatial differences in the lesion grade of alterations observing the highest lesion grades in fish from Odiel River and autumn season. No significant differences were observed in the alterations prevalence between sampling sites, but significant differences were observed between seasons observing the highest prevalence in autumn season. However, calculated IPAT demonstrated a low–moderate impact of pollutants on health fish. Correlations between histopathological alterations and pollutants analysed were observed being heavy metals the group that presented a major number of correlations with alterations in several organs of S. senegalensis. In evaluating the general health of fish, the use of histopathological studies in recommended for making more reliable assessment of biochemical responses in fish exposed to a variety of environmental stressors. Statistical analysis using semiquantitative data on pathological lesions can help to establish correlation between cause (stressor) and effect (biomarker)

First case of Anaplasma platys infection in a dog from Croatia

<p>Abstract</p> <p>Background</p> <p>It is known that <it>Anaplasma (A.) platys</it>, the causative agent of infectious canine cyclic thrombocytopenia, is endemic in countries of the Mediterranean basin. However, few reports are available from the Balkans. This case report describes a dog, which was imported from Croatia to Germany in May 2010. One month later the dog was presented to a local veterinarian in Germany due to intermittent/recurrent diarrhoea. Diagnostic tests were performed to identify infections caused by <it>Anaplasma </it>spp., <it>Ehrlichia </it>spp., <it>Hepatozoon canis, Babesia </it>spp., <it>Leishmania </it>spp., <it>Borrelia burgdorferi </it>and/or <it>Dirofilaria immitis</it>.</p> <p>Findings</p> <p>Haematological examination of a blood smear revealed basophilic inclusions in thrombocytes, which were confirmed as <it>A. platys </it>with a species-specific real-time PCR. Additionally, an infection with <it>Babesia (B.) vogeli </it>was also detected (PCR and serology). No specific antibodies against <it>Anaplasma </it>antigen were detectable. Although the dog showed no specific clinical signs, thrombocytopenia, anaemia and elevated C-reactive protein (CRP) were observed. Sequencing of a 1,348-bp partial ribosomal RNA gene revealed highest homology to <it>A. platys </it>sequences from Thailand, Japan and France.</p> <p>Conclusions</p> <p><it>A. platys </it>was detected for first time in a dog imported from Croatia. As the dog was also co-infected by <it>B. vogeli</it>, unique serological and haematological findings were recorded. Thrombocytopenia, anaemia and elevated values of C-reactive protein were the laboratory test abnormalities observed in this case. <it>A. platys </it>infections should be considered in dogs coming from Croatia and adjacent regions.</p