Quantitative detection of BCR–ABL fusion gene and its application in monitoring chronic myeloid leukemia treatment

The BCR–ABL fusion gene in chromosome translocation, t (9; 22), and its product, p210BCR/ABL oncogenic tyrosine kinase, is the underlying molecular mechanism that leads to the development of CML. Quantitative detection of BCR–ABL fusion gene has become a reliable approach to diagnose and monitor CML. The aim of this study was to evaluate a Roche t (9; 22) kit in CML diagnosis, monitoring treatment responses, and identification of relapse. Using BCR–ABL fusion gene-expressing K562 cells, a series of standard samples were prepared and used to establish a curve for the calculation of BCR–ABL fusion gene expression in patient samples. Our results indicate that PCR detection system with aforementioned kit has good reproducibility. In addition, the relative concentration of BCR–ABL measured by PCR was in agreement with the patient’s response to the Imatinib treatment and bone marrow morphology remission. Furthermore, we found that the relative concentration of BCR–ABL fusion gene increased 1–3 months before CML relapse was clinically and cytogenetically diagnosed, suggesting that the PCR-based BCR–ABL fusion gene detection with t (9; 22) kit is able to diagnose the recurrence of CML at least 1 month earlier than the classic cytogenetic analysis. In conclusion, detection of BCR–ABL fusion gene expression in CML using Roche t (9; 22) kit has great clinical value in the primary diagnosis, monitoring treatment responses, and identification of relapse in CML patients.     

Evaluating osteochondral defect repair potential of autologous rabbit bone marrow cells on type II collagen scaffold

The feasibility of using genipin cross-linked type II collagen scaffold with rabbit bone marrow mesenchymal stem cells (RBMSCs) to repair cartilage defect was herein studied. Induction of RBMSCs into chondrocytic phenotype on type II collagen scaffold in vitro was conducted using TGF-β 3 containing medium. After 3-weeks of induction, chondrocytic behavior, including marker genes expression and specific extracellular matrix (ECM) secretion, was observed. In the in vivo evaluation experiment, the scaffolds containing RBMSCs without prior induction were autologous implanted into the articular cartilage defects made by subchondral drilling. The repairing ability was evaluated. After 2 months, chondrocyte-like cells with lacuna structure and corresponding ECM were found in the repaired sites without apparent inflammation. After 24 weeks, we could easily find cartilage structure the same with normal cartilage in the repair site. In conclusion, it was shown that the scaffolds in combination of in vivo conditions can induce RBMSCs into chondrocytes in repaired area and would be a possible method for articular cartilage repair in clinic and cartilage tissue engineering.     

Kinetic analysis of protein aggregation monitored by real-time 2D solid-state NMR spectroscopy

It is shown that real-time 2D solid-state NMR can be used to obtain kinetic and structural information about the process of protein aggregation. In addition to the incorporation of kinetic information involving intermediate states, this approach can offer atom-specific resolution for all detectable species. The analysis was carried out using experimental data obtained during aggregation of the 10.4 kDa Crh protein, which has been shown to involve a partially unfolded intermediate state prior to aggregation. Based on a single real-time 2D ¹³C–¹³C transition spectrum, kinetic information about the refolding and aggregation step could be extracted. In addition, structural rearrangements associated with refolding are estimated and several different aggregation scenarios were compared to the experimental data.     

Reorganization of the nuclear lamina and cytoskeleton in adipogenesis

A thorough understanding of fat cell biology is necessary to counter the epidemic of obesity. Although molecular pathways governing adipogenesis are well delineated, the structure of the nuclear lamina and nuclear-cytoskeleton junction in this process are not. The identification of the ‘linker of nucleus and cytoskeleton’ (LINC) complex made us consider a role for the nuclear lamina in adipose conversion. We herein focused on the structure of the nuclear lamina and its coupling to the vimentin network, which forms a cage-like structure surrounding individual lipid droplets in mature adipocytes. Analysis of a mouse and human model system for fat cell differentiation showed fragmentation of the nuclear lamina and subsequent loss of lamins A, C, B1 and emerin at the nuclear rim, which coincides with reorganization of the nesprin-3/plectin/vimentin complex into a network lining lipid droplets. Upon 18 days of fat cell differentiation, the fraction of adipocytes expressing lamins A, C and B1 at the nuclear rim increased, though overall lamin A/C protein levels were low. Lamin B2 remained at the nuclear rim throughout fat cell differentiation. Light and electron microscopy of a subcutaneous adipose tissue specimen showed striking indentations of the nucleus by lipid droplets, suggestive for an increased plasticity of the nucleus due to profound reorganization of the cellular infrastructure. This dynamic reorganization of the nuclear lamina in adipogenesis is an important finding that may open up new venues for research in and treatment of obesity and nuclear lamina-associated lipodystrophy.     

Nematicidal activity of fervenulin isolated from a nematicidal actinomycete, <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CMU-MH021, on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis>

An isolate of the actinomycete, Streptomyces sp. CMU-MH021 produced secondary metabolites that inhibited egg hatch and increased juvenile mortality of the root-knot nematode Meloidogyne incognita in vitro. 16S rDNA gene sequencing showed that the isolate sequence was 99% identical to Streptomyces roseoverticillatus. The culture filtrates form different culture media were tested for nematocidal activity. The maximal activity against M. incognita was obtained by using modified basal (MB) medium. The nematicidal assay-directed fractionation of the culture broth delivered fervenulin (1) and isocoumarin (2). Fervenulin, a low molecular weight compound, shows a broad range of biological activities. However, nematicidal activity of fervenulin was not previously reported. The nematicidal activity of fervenulin (1) was assessed using the broth microdilution technique. The lowest minimum inhibitory concentrations (MICs) of the compound against egg hatch of M. incognita was 30 μg/ml and juvenile mortality of M. incognita increasing was observed at 120 μg/ml. Moreover, at the concentration of 250 μg/ml fervenulin (1) showed killing effect on second-stage nematode juveniles of M. incognita up to 100% after incubation for 96 h. Isocoumarin (2), another bioactive compound produced by Streptomyces sp. CMU-MH021, showed weak nematicidal activity with M. incognita.     

Rho kinase inhibitors stimulate the migration of human cultured osteoblastic cells by regulating actomyosin activity

We investigated the effects of Rho-associated kinase (ROCK) on migration and cytoskeletal organization in primary human osteoblasts and Saos-2 human osteosarcoma cells. Both cell types were exposed to two different ROCK inhibitors, Y-27632 and HA-1077. In the improved motility assay used in the present study, Y-27632 and HA-1077 significantly increased the migration of both osteoblasts and osteosarcoma cells on plastic in a dose-dependent and reversible manner. Fluorescent images showed that cells of both types cultured with Y-27632 or HA-1077 exhibited a stellate appearance, with poor assembly of stress fibers and focal contacts. Western blotting showed that ROCK inhibitors reduced myosin light chain (MLC) phosphorylation within 5 min without affecting overall myosin light-chain protein levels. Inhibition of ROCK activity is thought to enhance the migration of human osteoblasts through reorganization of the actin cytoskeleton and regulation of myosin activity. ROCK inhibitors may be potentially useful as anabolic agents to enhance the biocompatibility of bone and joint prostheses.     

Multiple strategies to improve sensitivity,speed and robustness of isothermal nucleic acid amplification for rapid pathogen detection

Background  

In the past decades the rapid growth of molecular diagnostics (based on either traditional PCR or isothermal amplification technologies) meet the demand for fast and accurate testing. Although isothermal amplification technologies have the advantages of low cost requirements for instruments, the further improvement on sensitivity, speed and robustness is a prerequisite for the applications in rapid pathogen detection, especially at point-of-care diagnostics. Here, we describe and explore several strategies to improve one of the isothermal technologies, helicase-dependent amplification (HDA).     

Sex-driven differences in Egyptian mongoose’s (<Emphasis Type="Italic">Herpestes ichneumon</Emphasis>) diet in its northwestern European range

Sexual dimorphism in carnivores can result from, or induce, variations in diet and foraging behaviour between individuals of different sexes. Sex-driven behavioural changes in feeding habits may also result in a reduction in intraspecific competition for resources, avoiding dietary overlap by concentrating on different prey sizes/types. We therefore evaluated the variation in feeding habits of both males and females of an invasive and range expanding southern European carnivore, the Egyptian mongoose (Herpestes ichneumon), through gut content analysis of road or predator-control killed animals. The analysis of 59 gut samples, collected in Portugal, revealed that northwestern populations of mongooses prey mostly upon mammals [especially lagomorphs, percentage of biomass (PB) = 44%], reptiles (PB = 28%) and arthropods (PB = 2% but percentage of occurrence = 35%). However, females seem to focus mainly on reptiles (PB = 51%) and mammals (PB = 38%), whilst males consume mostly mammals (PB = 75%). The results suggest that this variation maybe a result of sexual dimorphism (since some variation is documented in a few Egyptian mongoose’s skull measures, namely condylobasal length and canine diameter), with males shifting their diets to prey from which they can obtain more energy. Females seem to be focused on predating lighter animals, probably females or juveniles, a fact that has important game management implications.