Combined Fluorescent-Chromogenic In Situ Hybridization for Identification and Laser Microdissection of Interphase Chromosomes

Chromosome territories constitute the most conspicuous feature of nuclear architecture, and they exhibit non-random distribution patterns in the interphase nucleus. We observed that in cell nuclei from humans with Down Syndrome two chromosomes 21 frequently localize proximal to one another and distant from the third chromosome. To systematically investigate whether the proximally positioned chromosomes were always the same in all cells, we developed an approach consisting of sequential FISH and CISH combined with laser-microdissection of chromosomes from the interphase nucleus and followed by subsequent chromosome identification by microsatellite allele genotyping. This approach identified proximally positioned chromosomes from cultured cells, and the analysis showed that the identity of the chromosomes proximally positioned varies. However, the data suggest that there may be a tendency of the same chromosomes to be positioned close to each other in the interphase nucleus of trisomic cells. The protocol described here represents a powerful new method for genome analysis.     

Repair of long-bone pseudoarthrosis with autologous bone marrow mononuclear cells combined with allogenic bone graft

Background aimsLong-bone pseudoarthrosis is a major orthopedic concern because of numerous factors such as difficulty of the treatment, high recurrence, high costs and the devastating effects on the patients' quality of life, which sometimes ends in amputation. Although the “gold standard” for the treatment of this pathology is autologous bone grafting, which has high osteogenic, osteoconductive and osteoinductive properties, this treatment presents some restrictions such as the limited amount of bone that can be taken from the patient and donor site morbidity. Bone marrow mononuclear cells (BM-MNCs) comprise progenitor and stem cells with pro-angiogenic and pro-osteogenic properties. Allogenic cancellous bone graft is a natural and biodegradable osteoconductive and osteoinductive scaffold. Combination of these two components could mimic the advantages of autologous bone grafting while avoiding its main limitations.MethodsLong-bone pseudoarthrosis was treated in seven patients with autologous BM-MNCs from iliac crest combined with frozen allogenic cancellous bone graft obtained from the tissue bank.ResultsAll patients showed complete bone consolidation 5.3 ± 0.9 months (range, 2–9 months) after cell transplantation. Moreover, limb pain disappeared in all of them. The mean follow-up was 35.8 ± 4.6 months after transplantation (range, 24–51 months) without pseudoarthrosis recurrence or pain reappearing.ConclusionsCombination of autologous BM-MNCs and allogenic bone graft could constitute an easy, safe, inexpensive and efficacious attempt to treat long-bone pseudoarthrosis and non-union by reproducing the beneficial properties of autologous bone grafting while restricting its disadvantages.     

Identification of the Triticoid-type grains (Poaceae) from archaeobotanical assemblages in southwest Asia as Heteranthelium piliferum (Banks & Sol.) Hochst.

Vegetation History and Archaeobotany - The so-called Triticoid-type grains are known from several prehistoric sites in southwest Asia and their identification has long been unclear. They resemble...     

Osteopontin Deletion Prevents the Development of Obesity and Hepatic Steatosis via Impaired Adipose Tissue Matrix Remodeling and Reduced Inflammation and Fibrosis in Adipose Tissue and Liver in Mice

Osteopontin (OPN) is a multifunctional extracellular matrix (ECM) protein involved in multiple physiological processes. OPN expression is dramatically increased in visceral adipose tissue in obesity and the lack of OPN protects against the development of insulin resistance and inflammation in mice. We sought to unravel the potential mechanisms involved in the beneficial effects of the absence of OPN. We analyzed the effect of the lack of OPN in the development of obesity and hepatic steatosis induced by a high-fat diet (HFD) using OPN-KO mice. OPN expression was upregulated in epididymal white adipose tissue (EWAT) and liver in wild type (WT) mice with HFD. OPN-KO mice had higher insulin sensitivity, lower body weight and fat mass with reduced adipose tissue ECM remodeling and reduced adipocyte size than WT mice under a HFD. Reduced MMP2 and MMP9 activity was involved in the decreased ECM remodeling. Crown-like structure number in EWAT as well as F4/80-positive cells and Emr1 expression in EWAT and liver increased with HFD, while OPN-deficiency blunted the increase. Moreover, our data show for the first time that OPN-KO under a HFD mice display reduced fibrosis in adipose tissue and liver, as well as reduced oxidative stress in adipose tissue. Gene expression of collagens Col1a1, Col6a1 and Col6a3 in EWAT and liver, as well as the profibrotic cytokine Tgfb1 in EWAT were increased with HFD, while OPN-deficiency prevented this increase. OPN deficiency prevented hepatic steatosis via reduction in the expression of molecules involved in the onset of fat accumulation such as Pparg, Srebf1, Fasn, Mogat1, Dgat2 and Cidec. Furthermore, OPN-KO mice exhibited higher body temperature and improved BAT function. The present data reveal novel mechanisms of OPN in the development of obesity, pointing out the inhibition of OPN as a promising target for the treatment of obesity and fatty liver.     

Latent Dirichlet Allocation reveals spatial and taxonomic structure in a DNA‐based census of soil biodiversity from a tropical forest

High‐throughput sequencing of amplicons from environmental DNA samples permits rapid, standardized and comprehensive biodiversity assessments. However, retrieving and interpreting the structure of such data sets requires efficient methods for dimensionality reduction. Latent Dirichlet Allocation (LDA) can be used to decompose environmental DNA samples into overlapping assemblages of co‐occurring taxa. It is a flexible model‐based method adapted to uneven sample sizes and to large and sparse data sets. Here, we compare LDA performance on abundance and occurrence data, and we quantify the robustness of the LDA decomposition by measuring its stability with respect to the algorithm's initialization. We then apply LDA to a survey of 1,131 soil DNA samples that were collected in a 12‐ha plot of primary tropical forest and amplified using standard primers for bacteria, protists, fungi and metazoans. The analysis reveals that bacteria, protists and fungi exhibit a strong spatial structure, which matches the topographical features of the plot, while metazoans do not, confirming that microbial diversity is primarily controlled by environmental variation at the studied scale. We conclude that LDA is a sensitive, robust and computationally efficient method to detect and interpret the structure of large DNA‐based biodiversity data sets. We finally discuss the possible future applications of this approach for the study of biodiversity.     

Phytotoxic and Metabolic Effects of Exogenous Quinate on Pisum sativum L.

Quinate (1,3,4,5-tetrahydroxycyclohexanecarboxylate) is a compound synthesized in plants through a side branch of the shikimate biosynthesis pathway. Plants treated with herbicides that inhibit amino acid biosynthesis (branched-chain and aromatic) accumulate quinate in their leaves. The objective of this study was to evaluate whether quinate mimics the effects of herbicides in plants. In pea plants, exogenous application of quinate through the nutrient solution was compared with leaf spraying at a concentration of 4 and 400 mM, respectively, and evaluated in parallel to the effects of herbicides. The analysis facilitated an assessment of the phytotoxicity and potential use of quinate as a natural herbicide. The application of quinate through the nutrient solution, but not the spray, was lethal, although both treatments affected plant growth. Quinate was absorbed and translocated to other plant organs remote from the application site, and an increase in the levels of aromatic amino acids and caffeic acid (that is, compounds located after quinate in the shikimate biosynthesis pathway) was detected, which indicates that quinate was metabolized and incorporated into the shikimate pathway. Exogenous application of quinate affected the carbohydrate content in the leaves and roots in a way similar to the toxic effects of herbicides. The phytotoxic effects of quinate reported in this study suggest that this compound deregulates the shikimate pathway and mimics some physiological effects described in the mode of action of herbicides inhibiting amino acid biosynthesis.     

A highly sensitive DNA bead-based suspension array for the detection and species identification of bovine piroplasms

Piroplasms are among the most harmful tick-borne pathogens for livestock and sensitive and specific diagnostic methods for rapid detection and identification of the different species are needed for effective control. Reverse Line Blot has been the molecular technique of choice but it is laborious, time-consuming and highly susceptible to subjective variation in the interpretation of the hybridisation signal. Here, an oligonucleotide multiplex suspension microarray (Luminex® microsphere system) was developed for bovine piroplasms. Probes previously used in Reverse Line Blot for Babesia divergens, Babesia bovis, Babesia occultans, Babesia bigemina and Theileria buffeli, and a catch-all Theileria and Babesia control probe, were included in the Luminex assay together with newly designed probes for Theileria annulata and Babesia major. An internal amplification control that was detected with a Luminex probe was included to monitor for inhibition. Serially diluted linearised recombinant plasmids of the different species were used to assess the analytical sensitivity and specificity, and the detection limit of the Luminex assay was determined using serial dilutions of infected blood from an animal with a known level of T. annulata parasitaemia. The assay was then validated on 214 bovine blood samples analysed in parallel by Reverse Line Blot and Luminex. The Luminex assay proved to be highly specific and more sensitive than Reverse Line Blot, detecting 0.05 parasites/μl of blood. Technically, the Luminex procedure was rapid, provided high throughput screening, transformed the subjective interpretation of Reverse Line Blot results into numerical objective values, and allowed more flexibility in array preparation than Reverse Line Blot. The method described herein can substantially improve the detection of piroplasm carriers and thus better protect livestock trade and facilitate preventive control programs.