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.     

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.     

Characterization of <Emphasis Type="Italic">Penicillium</Emphasis> Species by Ribosomal DNA Sequencing and BOX,ERIC and REP-PCR Analysis

The genus Penicillium is one of the largest and widely distributed fungal genera described to date. As a result, its taxonomic classification and species discrimination within this genus has become complicated. In this study, 52 isolates that belonged to the Penicillum genus and other related genera were characterized using two DNA-based methods: (i) analysis of the nucleotide sequences of internal transcribed spacers in ribosomal DNA and (ii) analysis of DNA fingerprints that were generated by polymerase chain reactions with specific primers for enterobacterial repetitive intergenic consensus (ERIC) and repetitive extragenic palindromic (REP) sequences, and BOX elements. Using both methods, Penicillium species were discriminated from other fungal genera. Furthermore, Penicillium species that include strains which are used as biocontrol agents, such as P. glabrum, P. purpurogenum, and P. oxalicum, could be distinguished from other Penicillium species using these techniques. Based on our findings, we propose that a polyphasic approach that includes analysis of the nucleotide sequences of ribosomal DNA and detecting the presence of highly conserved, repeated nucleotide sequences can be used to determine the genetic relationships between different Penicillium species. Furthermore, we propose that our results can be used as a start point to develop a strategy to monitor the environmental presence of particular strains of Penicillium species when they are used as biocontrol agents.     

Differential expression and regulation of expansin gene family members during fruit growth and development of ‘Shijia’ longan fruit

In this work, five expansin cDNAs (DlExp1–5) from ‘Shijia’ longan fruit were isolated and characterized. Moreover, the expression profiles of five expansin genes and the effect of naphthalene acetic acid (NAA) and thidiazuron (TDZ) on their expressions were investigated. The results showed that five expansins exhibited different expression patterns during fruit growth and development. DlExp1 was constitutively expressed in the pericarp while the levels of DlExp1 mRNA in the aril were very high at early stage of fruit development, and decreased gradually from 28 to 77 days after anthesis (DAA). DlExp2 and DlExp4 were related to the growth of pericarp, whereas the expression of DlExp2 and DlExp5 in the aril decreased from 28 to 77 DAA. In addition, NAA and TDZ applied at the stage of rapid pericarp (21 DAA) or aril growth (56 DAA) increased the accumulations of DlExp1 and DlExp2 mRNA in the pericarp and aril, while NAA and TDZ had no or little effect on the accumulations of DlExp3, DlExp4 and DlExp5. DlExp1 and DlExp2 also accumulated highly in rapidly growing tissues, such as young stems and leaves. These findings indicated that Exp genes played a different role in longan fruit growth and showed different response to plant growth substances.     

The effects of diet and caloric restriction on adipose tissue fatty acid signatures of tufted puffin (<Emphasis Type="Italic">Fratercula cirrhata</Emphasis>) nestlings

Fatty acid (FA) signature analysis is a powerful tool to investigate foraging ecology and food web dynamics in marine ecosystems. However, use of FA signatures to qualitatively or quantitatively infer diets is potentially complicated by effects of nutritional state on lipid metabolism. Estimation of diets using the quantitative fatty acid signature analysis (QFASA) model requires the use of calibration coefficients to account for predator metabolism of individual FAs. We conducted a captive feeding experiment to determine the effects of a 50% reduction in food intake on growth rate and adipose tissue FA signatures of tufted puffin (Fratercula cirrhata) nestlings, a species that routinely experiences food restriction during growth. FA signatures of chicks fed low- and high-calorie diets both exhibited a change in composition in response to the dietary shift with the direction of change in the composition of individual FAs matching the direction of change in the dietary FAs. Despite a growth rate in the restricted nestlings that was 38% of those in the well-fed group, rates of FA turnover were not different between high and low-calorie treatments, and turnover was close to, but not entirely complete, after 27 days on both high-calorie and restricted diets. FA signatures of tufted puffin nestlings were significantly affected by caloric restriction, but these effects were much less pronounced than those of dietary turnover, and calibration coefficients of puffins fed low and high-calorie diets were highly correlated. Our results demonstrate that changes in physiological state can affect FA metabolism, but future research is required to better understand whether the size of these effects is sufficient to substantially alter diet estimation using the QFASA model.