Comparative study of mesenchymal stem cells from C57BL/10 and mdx mice

<p>Abstract</p> <p>Background</p> <p>Human mesenchymal stem cells (MSCs) have been studied and applied extensively because of their ability to self-renew and differentiate into various cell types. Since most human diseases models are murine, mouse MSCs should have been studied in detail. The mdx mouse – a Duchenne muscular dystrophy model – was produced by introducing a point mutation in the dystrophin gene. To understand the role of dystrophin in MSCs, we compared MSCs from mdx and C57BL/10 mice, focusing particularly on the aspects of light and electron microscopic morphology, immunophenotyping, and differentiation potential.</p> <p>Results</p> <p>Our study showed that at passage 10, mdx-MSCs exhibited increased heterochromatin, larger vacuoles, and more lysosomes under electron microscopy compared to C57BL/10-MSCs. C57BL/10-MSCs formed a few myotubes, while mdx-MSCs did not at the same passages. By passage 21, mdx-MSCs but not C57BL/10-MSCs had gradually lost their proliferative ability. In addition, a significant difference in the expression of CD34, not Sca-1 and CD11b, was observed between the MSCs from the 2 mice.</p> <p>Conclusion</p> <p>Our current study reveals that the MSCs from the 2 mice, namely, C57BL/10 and mdx, exhibit differences in proliferative and myogenic abilities. The results suggest that the changes in mouse MSC behavior may be influenced by lack of dystrophin protein in mdx mouse.</p

Development and efficacy evaluation of remodeled canine parvovirus-like particles displaying major antigenic epitopes of a giant panda derived canine distemper virus

Canine parvovirus (CPV) and Canine distemper virus (CDV) can cause fatal diseases in giant panda (Ailuropoda melanoleuca). The main capsid protein of CPV VP2 can be self-assembled to form virus-like particles (VLPs) in vitro, which is of great significance for potential vaccine development. In the present study, we remodeled the VP2 protein of a giant panda-derived CPV, where the major CDV F and N epitopes were incorporated in the N-terminal and loop2 region in two combinations to form chimeric VLPs. The reactivity ability and morphology of the recombinant proteins were confirmed by Western blot, hemagglutination (HA) test and electron microscopy. Subsequently, the immunogenicity of the VLPs was examined in vivo. Antigen-specific antibodies and neutralizing activity were measured by ELISA, hemagglutination inhibition (HI) test and serum neutralization test (SNT), respectively. In addition, antigen specific T cell activation were determined in splenic lymphocytes. The results indicated that the VLPs displayed good reaction with CDV/CPV antibodies, and the heterologous epitopes do not hamper solubility or activity. The VLPs showed decent HA activity, and resembled round-shaped particles with a diameter of 22–26 nm, which is identical to natural virions. VLPs could induce high levels of specific antibodies to CPV and CDV, shown by the indication of neutralizing antibodies in both VP2N and VP2L VLPs group. In addition, splenic lymphocytes of mice immunized with VLPs could proliferate rapidly after stimulation by specific antigen. Taken together, the CPV VP2 VLPs or chimeric VLPs are highly immunogenic, and henceforth could function as CPV/CDV vaccine candidates for giant pandas

Comprehensive genomic profiles of small cell lung cancer

We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Dex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Global patient outcomes after elective surgery: prospective cohort study in 27 low-, middle- and high-income countries.

BACKGROUND: As global initiatives increase patient access to surgical treatments, there remains a need to understand the adverse effects of surgery and define appropriate levels of perioperative care. METHODS: We designed a prospective international 7-day cohort study of outcomes following elective adult inpatient surgery in 27 countries. The primary outcome was in-hospital complications. Secondary outcomes were death following a complication (failure to rescue) and death in hospital. Process measures were admission to critical care immediately after surgery or to treat a complication and duration of hospital stay. A single definition of critical care was used for all countries. RESULTS: A total of 474 hospitals in 19 high-, 7 middle- and 1 low-income country were included in the primary analysis. Data included 44 814 patients with a median hospital stay of 4 (range 2-7) days. A total of 7508 patients (16.8%) developed one or more postoperative complication and 207 died (0.5%). The overall mortality among patients who developed complications was 2.8%. Mortality following complications ranged from 2.4% for pulmonary embolism to 43.9% for cardiac arrest. A total of 4360 (9.7%) patients were admitted to a critical care unit as routine immediately after surgery, of whom 2198 (50.4%) developed a complication, with 105 (2.4%) deaths. A total of 1233 patients (16.4%) were admitted to a critical care unit to treat complications, with 119 (9.7%) deaths. Despite lower baseline risk, outcomes were similar in low- and middle-income compared with high-income countries. CONCLUSIONS: Poor patient outcomes are common after inpatient surgery. Global initiatives to increase access to surgical treatments should also address the need for safe perioperative care. STUDY REGISTRATION: ISRCTN5181700

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Identification of an immunodominant IgE epitope of Der f 40, a novel allergen of Dermatophagoides farinae

Background: House dust mites (HDMs), including Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farinae (Der f) species, represent a major source of inhalant allergens that induce IgE-mediated anaphylactic reactions. HDM allergen identification is important to the diagnosis and treatment of allergic diseases. Here, we report the identification of a novel HDM allergen, which we suggest naming Der f 40, and its immunodominant IgE epitopes. Methods: The recombinant protein Der f 40 was expressed using a pET prokaryotic expression system and purified with Ni-NTA resins. IgE binding activity was evaluated by IgE-western blot, dot-blot, and ELISA. Mast cell activation testing was performed to assess the cellular effects of IgE binding in mouse bone marrow derived mast cells (BMMCs) expressing human FcεRI. IgE binding assays were performed with truncated and hybrid Der f 40 protein molecules to find immunodominant IgE epitopes. Results: A 106-amino acid (aa) recombinant Der f Group 40 protein (rDer f 40) was obtained (GenBank accession No. XP_046915420.1) as thiredoxin-like protein. Der f 40 was shown to bind IgE from HDM allergic serum in vitro (9.68%; 12/124 in IgE-ELISA), and shown to promote the release of β-hexosaminidase from BMMCs dose-dependently when administered with HDM allergic sera. The Der f Group 40 protein was named Der f 40 and listed in the World Health Organization and International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-committee. IgE binding assays with Der f 40-based truncated and hybrid proteins indicated that IgE binding epitopes are likely located in the C-terminal region and dependent on conformational structure. The 76–106-aa region of C-terminus was identified as an immunodominant IgE epitope of Der f 40. Conclusion: A novel HDM allergen with robust IgE binding activity was identified and named Der f 40. An immunodominant IgE epitope of Der f 40 with conformational dependency was identified in the C-terminus (aa 76–106). These findings provide new information that may be useful in the development of diagnostic and therapeutic agents for HDM allergy

Synthesis of L-Indospicine, [5,5,6-2H3 ]- L-Indospicine and L-Norindospicine

Indospicine is a non-proteogenic amino acid that accumulates as the free amino acid in livestock grazing Indigofera plant species and causes both reproductive losses and hepatotoxic effects. An efficient synthetic route to l-indospicine from l-homoserine lactone is described. The methodology is applicable for the synthesis of both deuterium labelled isotopomers and structural analogues for utilisation in biological studies. The key steps are a zinc mediated Barbier reaction with acrylonitrile and a Pinner reaction that together introduce the target amidine moiety