Multispectral imaging flow cytometry reveals distinct frequencies of γ-H2AX foci induction in DNA double strand break repair defective human cell lines

The measurement of γ-H2AX foci induction in cells provides a sensitive and reliable method for the quantitation of DNA damage responses in a variety of cell types. Accurate and rapid methods to conduct such observations are desirable. In this study, we have employed the novel technique of multispectral imaging flow cytometry to compare the induction and repair of γ-H2AX foci in three human cell types with different capacities for the repair of DNA double strand breaks (DSB). A repair normal fibroblast cell line MRC5-SV1, a DSB repair defective ataxia telangiectasia (AT5BIVA) cell line, and a DNA-PKcs deficient cell line XP14BRneo17 were exposed to 2 Gy gamma radiation from a (60)Cobalt source. Thirty minutes following exposure, we observed a dramatic induction of foci in the nuclei of these cells. After 24 hrs, there was a predictable reduction on the number of foci in the MRC5-SV1 cells, consistent with the repair of DNA DSB. In the AT5BIVA cells, persistence of the foci over a 24-hr period was due to the failure in the repair of DNA DSB. However, in the DNA-PKcs defective cells (XP14BRneo17), we observed an intermediate retention of foci in the nuclei indicative of partial repair of DNA DSB. In summary, the application of imaging flow cytometry has permitted an evaluation of foci in a large number of cells (20,000) for each cell line at each time point. This provides a novel method to determine differences in repair kinetics between different cell types. We propose that imaging flow cytometry provides an alternative platform for accurate automated high through-put analysis of foci induction in a variety of cell types.     

Acetylcholinesterase associates differently with its anchoring proteins ColQ and PRiMA

Acetylcholinesterase tetramers are inserted in the basal lamina of neuromuscular junctions or anchored in cell membranes through the interaction of four C-terminal t peptides with proline-rich attachment domains (PRADs) of cholinesterase-associated collagen Q (ColQ) or of the transmembrane protein PRiMA (proline-rich membrane anchor). ColQ and PRiMA differ in the length of their proline-rich motifs (10 and 15 residues, respectively). ColQ has two cysteines upstream of the PRAD, which are disulfide-linked to two AChE(T) subunits ("heavy" dimer), and the other two subunits are disulfide-linked together ("light" dimer). In contrast, PRiMA has four cysteines upstream of the PRAD. We examined whether these cysteines could be linked to AChE(T) subunits in complexes formed with PRiMA in transfected COS cells and in the mammalian brain. For comparison, we studied complexes formed with N-terminal fragments of ColQ, N-terminal fragments of PRiMA, and chimeras in which the upstream regions containing the cysteines were exchanged. We also compared the effect of mutations in the t peptides on their association with the two PRADs. We report that the two PRADs differ in their interaction with AChE(T) subunits; in complexes formed with the PRAD of PRiMA, we observed light dimers, but very few heavy dimers, even though such dimers were formed with the PQ chimera in which the N-terminal region of PRiMA was associated with the PRAD of ColQ. Complexes with PQ or with PRiMA contained heavy components, which migrated abnormally in SDS-PAGE but probably resulted from disulfide bonding of four AChE(T) subunits with the four upstream cysteines of the associated protein.     

Bioenergetic theory predicts infection dynamics of human schistosomes in intermediate host snails across ecological gradients

Epidemiological dynamics depend on the traits of hosts and parasites, but hosts and parasites are heterogeneous entities that exist in dynamic environments. Resource availability is a particularly dynamic and potent environmental driver of within‐host infection dynamics (temporal patterns of growth, reproduction, parasite production and survival). We developed, parameterised and validated a model for resource‐explicit infection dynamics by incorporating a parasitism module into dynamic energy budget theory. The model mechanistically explained the dynamic multivariate responses of the human parasite Schistosoma mansoni and its intermediate host snail to variation in resources and host density. At the population level, feedbacks mediated by resource competition could create a unimodal relationship between snail density and human risk of exposure to schistosomes. Consequently, weak snail control could backfire if reductions in snail density release remaining hosts from resource competition. If resource competition is strong and relevant to schistosome production in nature, it could inform control strategies.     

Impact of dengue virus (serotype DENV-2) infection on liver of BALB/c mice: A histopathological analysis

In this research, we characterized the histopathological impact of dengue virus (serotype DENV-2) infection in livers of BALB/c mice. The mice were infected with different doses of DENV-2 via intraperitoneal injection and liver tissues were processed for histological analyses and variation was documented. In the BALB/c mouse model, typical liver tissues showed regular hepatocyte architecture, with normal endothelial cells surrounding sinusoid capillary. Based on histopathological observations, the liver sections of BALB/c mice infected by DENV-2 exhibited a loss of cell integrity, with a widening of the sinusoidal spaces. There were marked increases in the infiltration of mononuclear cells. The areas of hemorrhage and micro- and macrovesicular steatosis were noted. Necrosis and apoptosis were abundantly present. The hallmark of viral infection, i.e., cytopathic effects, included intracellular edema and vacuole formation, cumulatively led to sinusoidal and lobular collapse in the liver. The histopathological studies on autopsy specimens of fatal human DENV cases are important to shed light on tissue damage for preventive and treatment modalities, in order to manage future DENV infections. In this framework, the method present here on BALB/c mouse model may be used to study not only the effects of infections by other DENV serotypes, but also to investigate the effects of novel drugs, such as recently developed nano-formulations, and the relative recovery ability with intact immune functions of host.     

Molecular signals in the trafficking of Toxoplasma gondii protein MIC3 to the micronemes

The protozoan parasite Toxoplasma gondii is equipped with a sophisticated secretory apparatus, including three distinct exocytic organelles, named micronemes, rhoptries, and dense granules. We have dissected the requirements for targeting the microneme protein MIC3, a key component of T. gondii infection. We have shown that MIC3 is processed in a post-Golgi compartment and that the MIC3 propeptide and epidermal growth factor (EGF) modules contain microneme-targeting information. The minimal requirement for microneme delivery is defined by the propeptide plus any one of the three EGF domains. We have demonstrated that the cleavage of the propeptide, the dimerization of MIC3, and the chitin binding-like sequence, which are crucial for host cell binding and virulence, are dispensable for proper targeting. Finally, we have shown that part of MIC3 is withheld in the secretory pathway in a cell cycle-dependent manner.     

Radiobiological effects and medical applications of non-ionizing radiation

Radiation is used in medicine to diagnose and treat diseases but it can also cause harm to the body by burning or mutation. This depends on whether the radiation is ionizing or nonionizing. Despite its vast applications in surgery, dermatology and cosmetics, little is taught and thus known about non-ionizing radiation.This review article discusses the fundamentals of non-ionizing electromagnetic radiations. The main aim is to extensively explain the different types of non-ionizing radiation. This will equip students and medical personnel with knowledge on different medical applications and expose them to a variety of specializations in medicine that utilize non-ionizing radiation. The article discusses the physics, hazard, means of protection and medical application of each type of radiation: ultraviolet radiation, light (both visible light and LASER), infrared radiation, microwaves and extremely low frequency radiation separately. It presents these terms in a simple manner that avoids rigors mathematics and physics, which makes them comprehensible for medical students.The development of new diagnostic and therapeutic approaches could also lead to increased hazards to the body unless they are treated with precaution. If not adequately monitored, a significant health risk may be posed to potentially exposed employees. Hence proper dosage should be used for non-ionizing radiation. This is only possible through understanding of the risks/benefits of these radiations by studying the physics and radiobiological effects of each individual radiation.     

ROP18 is a rhoptry kinase controlling the intracellular proliferation of Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite for which the discharge of apical organelles named rhoptries is a key event in host cell invasion. Among rhoptry proteins, ROP2, which is the prototype of a large protein family, is translocated in the parasitophorous vacuole membrane during invasion. The ROP2 family members are related to protein-kinases, but only some of them are predicted to be catalytically active, and none of the latter has been characterized so far. We show here that ROP18, a member of the ROP2 family, is located in the rhoptries and re-localises at the parasitophorous vacuole membrane during invasion. We demonstrate that a recombinant ROP18 catalytic domain (amino acids 243-539) possesses a protein-kinase activity and phosphorylate parasitic substrates, especially a 70-kDa protein of tachyzoites. Furthermore, we show that overexpression of ROP18 in transgenic parasites causes a dramatic increase in intra-vacuolar parasite multiplication rate, which is correlated with kinase activity. Therefore, we demonstrate, to our knowledge for the first time, that rhoptries can discharge active protein-kinases upon host cell invasion, which can exert a long-lasting effect on intracellular parasite development and virulence.