Development of a High-Content Orthopoxvirus Infectivity and Neutralization Assays

Currently, a number of assays measure Orthopoxvirus neutralization with serum from individuals, vaccinated against smallpox. In addition to the traditional plaque reduction neutralization test (PRNT), newer higher throughput assays are based on neutralization of recombinant vaccinia virus, expressing reporter genes such as β-galactosidase or green fluorescent protein. These methods could not be used to evaluate neutralization of variola virus, since genetic manipulations of this virus are prohibited by international agreements. Currently, PRNT is the assay of choice to measure neutralization of variola virus. However, PRNT assays are time consuming, labor intensive, and require considerable volume of serum sample for testing. Here, we describe the development of a high-throughput, cell-based imaging assay that can be used to measure neutralization, and characterize replication kinetics of various Orthopoxviruses, including variola, vaccinia, monkeypox, and cowpox.     

Adverse events post smallpox-vaccination: insights from tail scarification infection in mice with Vaccinia virus

Adverse events upon smallpox vaccination with fully-replicative strains of Vaccinia virus (VACV) comprise an array of clinical manifestations that occur primarily in immunocompromised patients leading to significant host morbidity/mortality. The expansion of immune-suppressed populations and the possible release of Variola virus as a bioterrorist act have given rise to concerns over vaccination complications should more widespread vaccination be reinitiated. Our goal was to evaluate the components of the host immune system that are sufficient to prevent morbidity/mortality in a murine model of tail scarification, which mimics immunological and clinical features of smallpox vaccination in humans. Infection of C57BL/6 wild-type mice led to a strictly localized infection, with complete viral clearance by day 28 p.i. On the other hand, infection of T and B-cell deficient mice (Rag1(-/-)) produced a severe disease, with uncontrolled viral replication at the inoculation site and dissemination to internal organs. Infection of B-cell deficient animals (μMT) produced no mortality. However, viral clearance in μMT animals was delayed compared to WT animals, with detectable viral titers in tail and internal organs late in infection. Treatment of Rag1(-/-) with rabbit hyperimmune anti-vaccinia serum had a subtle effect on the morbidity/mortality of this strain, but it was effective in reduce viral titers in ovaries. Finally, NUDE athymic mice showed a similar outcome of infection as Rag1(-/-), and passive transfer of WT T cells to Rag1(-/-) animals proved fully effective in preventing morbidity/mortality. These results strongly suggest that both T and B cells are important in the immune response to primary VACV infection in mice, and that T-cells are required to control the infection at the inoculation site and providing help for B-cells to produce antibodies, which help to prevent viral dissemination. These insights might prove helpful to better identify individuals with higher risk of complications after infection with poxvirus.     

Bartonella henselae, B. quintana, and B. bacilliformis: historical pathogens of emerging significance

Bartonella species were virtually unrecognized as modern pathogens of humans until the last decade. However, identification of Bartonella species as the agents of cat-scratch disease, bacillary angiomatosis, urban trench fever, and possible novel presentations of Carrion's disease has left little doubt of the emerging medical importance of this genus of organisms. The three primary human pathogenic bartonellae, Bartonella bacilliformis (Carrion's disease), B. henselae (cat-scratch disease), and B. quintana (trench fever), present noteworthy comparisons in the epidemiology, natural history, pathology, and host-microbe interaction that this review will briefly explore.     

Challenges Faced by Behavioral Genetic Studies: Researchers Perspective from the MENA Region

BackgroundBehavioral genetic studies are important for the understanding of the contribution of genetic variations to human behavior. However, such studies might be associated with some ethical concerns.MethodsIn the current study, ethical challenges related to studies of genetic variations contributing to human behavior were examined among researchers. To achieve the study purpose, the Middle East and North Africa (MENA) region researchers were taken as an example, where the after- mentioned ethical challenges were discussed among a group of researchers, who were the participants of an online forum. Discussions and responses of the participants were monitored and were later qualitatively analyzed.ResultsDiscussions revealed that several ethical challenges, including subjects’ recruitment, the difficulty of obtaining informed consents, and issues of privacy and confidentiality of obtained data as information leakage, in this case, will lead to social stigma and isolation of the participants and their immediate family members. Jordanian social and cultural norms, faith, and the tribal nature of the population were raised as a major challenge that might face conducting behavioral genetic studies in the Arab populations of the MENA. The lack of regulation related to the conduction of genetic studies, misunderstanding, and misuse of genetic information are other challenges. A full explanation of genetic research and the current and future possible benefits/risks of such research could be potential solutions.ConclusionIn conclusion, the MENA populations are tackled with major challenges in relation to conducting research studies in genetics/antisocial behavior field/s. Establishment of guidelines related to genetic studies, capacity building, increasing public awareness about the importance of genetic testing, and enhancing responsible conduct of research will facilitate the conduct of such sensitive studies in the future in the region.     

Novel crown ethers on glucose based glycolipids

A series of crown ethers involving lauryl glucoside were synthesized and their assembly behavior in water was studied. The synthesis applied a simple protection scheme based on benzylidenation for the glycolipid, and cation templating for the macrocycle. A sequential build-up of the crown ether by bis-hydroxylethylation of the glucoside followed by reaction with di-, tri-, or tetraethylene glycol ditosylate provided better yields of the macrocycle compared to a single step cyclization with tetraethylene glycole ditosylate. The macrocycles containing up to six oxygens showed significantly higher affinity for sodium than for potassium, while more effective potassium complexation was found for the 21-crown-7 compound. The ion binding affinity leads to a slight but significant increase of the CMC of the crown ether containing surfactant in water upon the addition of sodium electrolyte.     

<Emphasis Type="Italic">Bifidobacterium adolescentis</Emphasis> (DSM 20083) and <Emphasis Type="Italic">Lactobacillus casei</Emphasis> (Lafti L26-DSL): Probiotics Able to Block the In Vitro Adherence of Rotavirus in MA104 Cells

Rotavirus is the leading worldwide cause of gastroenteritis in children under five years of age. Even though there are some available vaccines to prevent the disease, there are limited strategies for challenging diarrhea induced by rotavirus infection. For this reason, researchers are constantly searching for other approaches to control diarrhea by means of probiotics. In order to demonstrate the ability of some probiotic bacteria to interfere with the in vitro rotavirus infection in MA104 cells, strains of Lactobacillus sp. and Bifidobacterium sp. were tested in MA104 cells before the viral infection. As a preliminary assay, a blocking effect treatment was performed with viable bacteria. In this screening assay, four of initial ten bacteria showed a slight reduction of the viral infection (measured by percentage of infection). L. casei (Lafti L26-DSL), L. fermentum(ATCC 9338), B. adolescentis (DSM 20083), and B. bifidum (ATCC 11863) were used in further experiments. Three different treatments were tested in order to evaluate protein-based metabolites obtained from mentioned bacteria: (i) cell exposure to the protein-based metabolites before viral infection, (ii) exposure to protein-based metabolites after viral infection, and (iii) co-incubation of the virus and protein-based metabolites before viral infection to the cell culture. The best effect performed by protein-based metabolites was observed during the co-incubation assay of the virus and protein-based metabolites before adding them into the cell culture. The results showed 25 and 37% of infection in the presence of L. casei and B. adolescentis respectively. These results suggest that the antiviral effect may be occurring directly with the viral particle instead of making a blocking effect of the cellular receptors that are needed for the viral entrance.     

<Emphasis Type="Italic">Arbutus andrachne</Emphasis> L. Reverses Sleep Deprivation-Induced Memory Impairments in Rats

Sleep deprivation (SD) is associated with cognitive deficits. It was found to affect the hippocampus region of the brain by impairing memory formation. This impairment is suggested to be caused by elevation in oxidative stress in the body, including the brain during SD. It was hypothesized that the methanolic extract of the fruits of Arbutus andrachne L. (Ericaceae) will prevent chronic SD-induced impairment of hippocampal memory via its antioxidative properties. The methanolic extract of the fruits of A. andrachne was evaluated for its beneficial properties to reverse SD-induced cognitive impairment in rats. Animals were sleep deprived for 8 weeks using a multiple platform model. The extract was administered i.p. at three doses (50, 200, and 500 mg/kg). Behavioral studies were conducted to test the spatial learning and memory using radial arm water maze (RAWM). In addition, the hippocampus was dissected to analyze the following oxidative stress markers: glutathione (GSH), oxidized glutathione (GSSG), GSH/GSSG, glutathione peroxidase (GPx), and catalase. Chronic SD impaired short- and long-term memories (P < 0.05). Treatment of animals with A. andrachne fruit extract at all doses prevented long-term memory impairment induced by SD while such treatment prevented short-term memory impairment only at 200 and 500 mg/kg dose levels. Moreover, A. andrachne fruit extract normalized the reduction in the hippocampus GSH/GSSG ratio and activity of GPx, and catalase (P < 0.05) induced by chronic sleep deprivation. Chronic sleep deprivation impaired both short- and long-term memory formation, while methanolic extract of A. andrachne fruits reversed this impairment, probably through normalizing oxidative stress in the hippocampus.     

In vitro deposition of hydroxyapatite on cortical bone collagen stimulated by deformation-induced piezoelectricity

In the present work, we have studied the effect of the piezoelectricity of elastically deformed cortical bone collagen on surface using a biomimetic approach. The mineralization process induced as a consequence of the piezoelectricity effect was evaluated using scanning electron microscopy (SEM), thermally stimulated depolarization current (TSDC), and differential scanning calorimetry (DSC). SEM micrographs showed that mineralization occurred predominantly over the compressed side of bone collagen, due to the effect of piezoelectricity, when the sample was immersed in the simulated body fluid (SBF) in a cell-free system. The TSDC method was used to examine the complex collagen dielectric response. The dielectric spectra of deformed and undeformed collagen samples with different hydration levels were compared and correlated with the mineralization process followed by SEM. The dielectric measurements showed that the mineralization induced significant changes in the dielectric spectra of the deformed sample. DSC and TSDC results demonstrated a reduction of the collagen glass transition as the mineralization process advanced. The combined use of SEM, TSDC, and DSC showed that, even without osteoblasts present, the piezoelectric dipoles produced by deformed collagen can produce the precipitation of hydroxyapatite by electrochemical means, without a catalytic converter as occurs in classical biomimetic deposition.