In Vivo Bioluminescence Imaging for Prolonged Survival of Transplanted Human Neural Stem Cells Using 3D Biocompatible Scaffold in Corticectomized Rat Model

Stem cell-based treatment of traumatic brain injury has been limited in its capacity to bring about complete functional recovery, because of the poor survival rate of the implanted stem cells. It is known that biocompatible biomaterials play a critical role in enhancing survival and proliferation of transplanted stem cells via provision of mechanical support. In this study, we noninvasively monitored in vivo behavior of implanted neural stem cells embedded within poly-l-lactic acid (PLLA) scaffold, and showed that they survived over prolonged periods in corticectomized rat model. Corticectomized rat models were established by motor-cortex ablation of the rat. F3 cells expressing enhanced firefly luciferase (F3-effLuc) were established through retroviral infection. The F3-effLuc within PLLA was monitored using IVIS-100 imaging system 7 days after corticectomized surgery. F3-effLuc within PLLA robustly adhered, and gradually increased luciferase signals of F3-effLuc within PLLA were detected in a day dependent manner. The implantation of F3-effLuc cells/PLLA complex into corticectomized rats showed longer-lasting luciferase activity than F3-effLuc cells alone. The bioluminescence signals from the PLLA-encapsulated cells were maintained for 14 days, compared with 8 days for the non-encapsulated cells. Immunostaining results revealed expression of the early neuronal marker, Tuj-1, in PLLA-F3-effLuc cells in the motor-cortex-ablated area. We observed noninvasively that the mechanical support by PLLA scaffold increased the survival of implanted neural stem cells in the corticectomized rat. The image-guided approach easily proved that scaffolds could provide supportive effect to implanted cells, increasing their viability in terms of enhancing therapeutic efficacy of stem-cell therapy.     

Hematological Parameters,and Hematopoietic Growth Factors: Epo and IL-3 in Response to Whole-Body Cryostimulation (WBC) in Military Academy Students

The effects of extreme cold on the human body are not fully understood, there are also no reports on the effect of cryogenic temperatures on the levels of erythropoietin (EPO) and interleukin 3 (IL-3), two important factors that regulate hematopoiesis. Aim: determination of changes in peripheral blood cell counts and EPO and IL-3 levels induced by a series of 10, 20 and 30 standard whole-body cryostimulation (WBC) treatments. The study involved 45 men, experimental group (EXP, n = 30) subjected to 30 WBC treatments (−130°C, treatment duration: 3 minutes) and a control group (CON, n = 15). Blood samples were collected before the series of treatments and after 10, 20 and 30 treatments. After 10 and 20 treatments we observed lower red blood cell counts and hematocrit and hemoglobin levels compared to baseline (p<0.05) and the control group (p<0.05). Additionally we observed an increase in hemoglobin concentration in plasma (p<0.05), and bilirubin after 10 and 20 treatments, and a decrease in plasma concentration of haptoglobin after 10, 20 and 30 treatments (p<0.05). The number of leukocytes was higher after 10 and 20 WBC treatments compared to baseline and the CON group. EPO concentration in plasma was elevated and the concentration of IL-3 was lower after 10, 20 and 30 WBC treatments. The decrease in indices of the erythrocytic system, plasma hemoglobin and bilirubin, with a simultaneous decrease in haptoglobin concentrations after 10 and 20 WBC treatments, may be due to increased intravascular hemolysis. At the same time there was a small, but statistically significant increase in the concentration of EPO stimulated erythropoiesis which could facilitate a return of erythrocytic system indices to initial levels after 30 WBC treatments. Changes in the white blood cell system showed transient mobilization of this system under the influence of WBC.     

Design of novel N-(2,4-dioxo-1,2,3,4-tetrahydro-thieno[3,2-d]pyrimidin-7-yl)-guanidines as thymidine phosphorylase inhibitors,and flexible docking to a homology model

A novel class of thymidine phosphorylase (TP) inhibitors has been designed based on analogy to the enzyme substrate as well as known inhibitors. Flexible docking studies, using a homology model of human TP, of the designed N-(2,4-dioxo-1,2,3,4-tetrahydro-thieno[3,2-d]pyrimidin-7-yl)-guanidines as well as their synthetic precursors provide insight into the observed experimental trends in binding affinity.     

Rapid and simple identification of human pathogenic heterophyid intestinal fluke metacercariae by PCR-RFLP

Six species of heterophyid intestinal flukes (HIFs) constitute the major endemic zoonotic fish-borne pathogens in Asia: Haplorchis taichui, H. pumilio, H. yokogawai, Procerovum varium, Stellantchasmus falcatus, and Centrocestus formosanus. Several different species of these parasites are often found co-infecting the same second intermediate fish host. Because of their morphological similarities, differentiating between species of HIF metacercariae is difficult, time-consuming, and frequently results in misidentification. In this study, we aimed to develop an efficient and accurate method of identifying metacercariae of these 6 HIFs. Metacercariae were roughly grouped together, based on morphological characteristics seen under a stereomicroscope. Then, PCR-restriction fragment length polymorphism (PCR-RFLP) was used to identify the exact species of each metacercaria, using the 28S ribosomal RNA gene as the genetic marker and MboII as the restriction enzyme. Using a combination of morphological and molecular methods eliminates the need for DNA sequencing and infecting animal subjects to obtain adult worms, increases accuracy, and decreases the need for laborious morphological identification. Because the method is simple, rapid, and relatively cheap compared with PCR-sequencing, it may be an effective tool for epidemiological studies of HIFs in endemic areas.     

Salmonella exploits Arl8B-directed kinesin activity to promote endosome tubulation and cell-to-cell transfer

The facultative intracellular pathogen Salmonella enterica serovar Typhimurium establishes a replicative niche, the Salmonella-containing vacuole (SCV), in host cells. Here we demonstrate that these bacteria exploit the function of Arl8B, an Arf family GTPase, during infection. Following infection, Arl8B localized to SCVs and to tubulated endosomes that extended along microtubules in the host cell cytoplasm. Arl8B(+) tubules partially colocalized with LAMP1 and SCAMP3. Formation of LAMP1(+) tubules (the Salmonella-induced filaments phenotype; SIFs) required Arl8B expression. SIFs formation is known to require the activity of kinesin-1. Here we find that Arl8B is required for kinesin-1 recruitment to SCVs. We have previously shown that SCVs undergo centrifugal movement to the cell periphery at 24 h post infection and undergo cell-to-cell transfer to infect neighbouring cells, and that both phenotypes require kinesin-1 activity. Here we demonstrate that Arl8B is required for migration of the SCV to the cell periphery 24 h after infection and for cell-to-cell transfer of bacteria to neighbouring cells. These results reveal a novel host factor co-opted by S. Typhimurium to manipulate the host endocytic pathway and to promote the spread of infection within a host.     

(1)H NMR-based metabolomic profiling in mice infected with Mycobacterium tuberculosis

Tuberculosis (TB) is one of three major infectious diseases, and the control of TB is becoming more difficult because of the emergence of multidrug-resistant and extensively drug-resistant strains. In this study, we explored the (1)H NMR-based metabolomics of TB using an aerobic TB infection model. Global profiling was applied to characterize the responses of C57Bl/6 mice to an aerobic infection with virulent Mycobacterium tuberculosis (MTB). The metabolic changes in organs (i.e., the lung, the target organ of TB, and the spleen and liver, remote systemic organs) and in serum from control and MTB-infected rats were investigated to clarify the host-pathogen interactions in MTB-infected host systems. Principal components analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) score plots showed distinct separation between control and MTB-infected rats for all tissue and serum samples. Several tissue and serum metabolites were changed in MTB-infected rats, as compared to control rats. The precursors of membrane phospholipids, phosphocholine, and phosphoethanolamine, as well as glycolysis, amino acid metabolism, nucleotide metabolism, and the antioxidative stress response were altered based on the presence of MTB infection. This study suggests that NMR-based global metabolite profiling of organ tissues and serum could provide insight into the metabolic changes in host infected aerobically with virulent Mycobacterium tuberculosis.