Ameboid cell motility: a model and inverse problem, with an application to live cell imaging data

In this article a mathematical model for ameboid cell movement is developed using a spring-dashpot system with Newtonian dynamics. The model is based on the facts that the cytoskeleton plays a primary role for cell motility and that the cytoplasm is viscoelastic. Based on the model, the inverse problem can be posed: if a structure like a spring-dashpot system is embedded into the living cell, what kind of characteristic properties must the structure have in order to reproduce a given movement of the cell? This inverse problem is the primary topic of this paper. On one side the model mimics some features of the movement, and on the other side, the solution to the inverse problem provides model parameters that give some insight, principally into the mechanical aspect, but also, through qualitative reasoning, into chemical and biophysical aspects of the cell. Moreover, this analysis can be done locally or globally and in different media by using the simplest possible information: positions of the cell and nuclear membranes. It is shown that the model and solution to the inverse problem for simulated data sets are highly accurate. An application to a set of live cell imaging data obtained from random movements of a human brain tumor cell (U87-MG human glioblastoma cell line) then provides an example of the efficiency of the model, through the solution of its inverse problem, as a way of understanding experimental data.     

Structural Framework for Covalent Inhibition of Clostridium botulinum Neurotoxin A by Targeting Cys165

Clostridium botulinum neurotoxin type A (BoNT/A) is one of the most potent toxins for humans and a major biothreat agent. Despite intense chemical efforts over the past 10 years to develop inhibitors of its catalytic domain (catBoNT/A), highly potent and selective inhibitors are still lacking. Recently, small inhibitors were reported to covalently modify catBoNT/A by targeting Cys¹⁶⁵, a residue located in the enzyme active site just above the catalytic zinc ion. However, no direct proof of Cys¹⁶⁵ modification was reported, and the poor accessibility of this residue in the x-ray structure of catBoNT/A raises concerns about this proposal. To clarify this issue, the functional role of Cys¹⁶⁵ was first assessed through a combination of site-directed mutagenesis and structural studies. These data suggested that Cys¹⁶⁵ is more involved in enzyme catalysis rather than in structural property. Then by peptide mass fingerprinting and x-ray crystallography, we demonstrated that a small compound containing a sulfonyl group acts as inhibitor of catBoNT/A through covalent modification of Cys¹⁶⁵. The crystal structure of this covalent complex offers a structural framework for developing more potent covalent inhibitors catBoNT/A. Other zinc metalloproteases can be founded in the protein database with a cysteine at a similar location, some expressed by major human pathogens; thus this work should find broader applications for developing covalent inhibitors.     

Survey of insect growth regulator (IGR) resistance in house flies (Musca domestica L.) from southwestern Turkey

Insect growth regulators (IGRs) are currently the fastest‐growing class of insecticides, and in Turkey these products represent a new approach to pest control. In recent years, several IGRs were also registered for the control of the house fly, Musca domestica L. (Diptera: Muscidae), in Turkey. A field survey was conducted in the summers of 2006 and 2007 to evaluate resistance to some agriculturally and medically used IGRs on house flies from livestock farms and garbage dumps in the greenhouse production areas (Merkez, Kumluca, Manavgat, and Serik) of Antalya province (Southwestern Turkey). The results of larval feeding assay with technical diflubenzuron, methoprene, novaluron, pyripoxyfen, and triflumuron indicate that low levels (RF<10‐fold) of resistance to the IGRs exist in the house fly populations from Antalya province. Exceptions, however, were two populations, Guzoren and Toptas, from the Kumluca area which showed moderate resistance to diflubenzuron with 11.8‐fold in 2006 and 13.2‐fold in 2007, respectively. We found substantial variation in susceptibility of field‐collected house fly populations from year to year and from product to product. We generally observed an increase in resistance at many localities sampled from 2006 to 2007. The implications of these results to the future use of IGRs for house fly control are discussed. It will be critically important to continue monitoring efforts so that appropriate steps can be taken if resistance levels start to increase.     

The generation of live offspring from vitrified oocytes

Oocyte cryopreservation is extremely beneficial for assisted reproductive technologies, the treatment of infertility and biotechnology and offers a viable alternative to embryo freezing and ovarian grafting approaches for the generation of embryonic stem cells and live offspring. It also offers the potential to store oocytes to rescue endangered species by somatic cell nuclear transfer and for the generation of embryonic stem cells to study development in these species. We vitrified mouse oocytes using a range of concentrations of trehalose (0 to 0.3 M) and demonstrated that 0.1 and 0.3 M trehalose had similar developmental rates, which were significantly different to the 0.2 M cohort (P<0.05). As mitochondria are important for fertilisation outcome, we observed that the clustering and distribution of mitochondria of the 0.2 M cohort were more affected by vitifrication than the other groups. Nevertheless, all 3 cohorts were able to develop to blastocyst, following in vitro fertilisation, although developmental rates were better for the 0.1 and 0.3 M cohorts than the 0.2 M cohort (P<0.05). Whilst blastocysts gave rise to embryonic stem-like cells, it was apparent from immunocytochemistry and RT-PCR that these cells did not demonstrate true pluripotency and exhibited abnormal karyotypes. However, they gave rise to teratomas following injection into SCID mice and differentiated into cells of each of the germinal layers following in vitro differentiation. The transfer of 2-cell embryos from the 0.1 and 0.3 M cohorts resulted in the birth of live offspring that had normal karyotypes (9/10). When 2-cell embryos from vitrified oocytes underwent vitrification, and were thawed and transferred, live offspring were obtained that exhibited normal karyotypes, with the exception of one offspring who was larger and died at 7 months. We conclude that these studies highlight the importance of the endometrial environment for the maintenance of genetic stability and thus the propagation of specific genetic traits.     

Detection of anti-leishmanial effect of the Lucilia sericata larval secretions in vitro and in vivo on Leishmania tropica: First work

It is known that some of the enzymes and substances secreted by 2nd and 3rd stages of the Lucilia sericata larvae to have bacteriostatic and bactericidal effects. From this point of view, we investigated the anti-leishmanial effect of larval secretions of the L. sericata on the Leishmania tropica both in vitro and in vivo conditions. In vitro: It was observed that promastigotes of L. tropica had undergone lyzis within 1min in the larval secretions of L. sericata. However, larval secretion was ineffective on the promastigotes within Novy-MacNeal-Nicolle (NNN) cultures and RPMI 1640 medium. In vivo: Seven groups of male Balb/C mice (6 study groups and 1 control group), each composed of eight weeks old 10 mice were formed. L. tropica promastigotes were injected subcutaneosly to the soles of the SG mice' feet. In study groups, cutaneous lesions were developed Limoncu et al., 1997 in 2 (20%) and 1 (10%) of the SG-1 and SG-2, respectively after 15days. There were L. tropica in the smears prepared from the lesions and L. tropica was observed in the cultures. Cutaneous lesions were not developed in 8 (80%), 9 (90%) and 10 (100%) of the SG-I, SG-II and SG-III, respectively. There were no cutaneous lesions developed in the soles of the feet. There were no L. tropica in the smears prepared from the infected soles of the feet neither L. tropica was observed in the cultures. Larval secretions were given into the cutaneous lesions to the feet soles of the SG-IV, V and VI mice after 6months. No healing was observed in the cutaneos lesions of 4 (40%), 5 (50%) and 1 (10%) of SG-IV, SG-V and SG-VI, after 6months, respectively. There were L. tropica in the smears prepared from the lesions and L. tropica was observed in the cultures. On the other hand, the lesions of 6 (60%), 5 (50%) and 9 (90%) of SG-IV, SG-V and SG-VI were diminished in size and disappeared completely after 6months. There were L. tropica observed in the smears prepared from the infected soles of the feet and no growth was observed in the cultures. In the smears prepared from the cutaneous lesions developed in the soles of the feet of the control group mice, L. tropica was visualized and observed in the cultures. A statistical significant difference was observed between study groups and control group (p<0.001). In our study we demonstrated for the first time that the secretions of the 2nd and 3rd stages sterile and pure larvae of L. sericata had effects on promastigotes of L. tropica in in vitro and very effective on amastigote forms in in vivo conditions.