Cloning and sequencing of M. tb gene Rv0378 for making subunit based DNA vaccine.

Mycobacterium tuberculosis has infected one third of the world's population and 1.5 million people die each year due to tuberculosis. The research was conducted to make clones of M. tb gene Rv0378 during January-November, 2015. Gene Rv0378 has a gene length 222bp and was amplified using proper sequence specific primers. The size and quality of DNA fragments were determined by agarose gel electrophoresis. PCR product was then ligated in Vector PtZ57R/T (T/A vector) in order to be transformed into competent alpha-DH5 (E. coli) cells. Ampicillin positive clones were selected from the plates, introduced in the autoclaved test tubes with 2 to 3 ml. SOB broth was then placed in a shaker incubator for overnight at 37ºC. Next day turbidity was clearly observed in the test tubes indicating the culture was ready to use for plasmid extraction. DNA was extracted by using Genejet Plasmid extraction miniprep Kit by Thermo scientific. Size of the extracted construct was about >3000bp (other impurities of proteins and salts are washed away in EZ-Spin columns). In next step it will be cloned into pND14 (A mammalian expression vector) to make candidate vaccine and will be tested for its efficacy against M. tb.

Differences in myelination between spinal cord and corticular tissue transplanted intraocularly in rats.

This paper compares the myelination in rat cortex and spinal cord transplanted on the embryonic day (E) 12, 14, 16, 18, 20 and right after birth (P0) into the anterior eye chamber of adult rats. Myelinated fibers were not observed in either cortical or spinal cord transplants of E12. When transplanted on E14, abundant myelinated fibers developed in the spinal cord and gathered at the periphery of the transplants as a "white matter". In the grafted cortex myelinated fibers were found when transplantation occurred on E16 or later. The myelinated fibers, however, remained scarce or formed only narrow bundles. The number and distribution of myelinated fibers did not depend on the donor's age between E16 and P0: even in the latter case transplanted cortex were found without myelination. The differences could be attributed to the different development of the cortical and spinal cord oligodendrocytes and to the different intrinsic organization of the grafted samples.

The early phase of vascularization in intraocular telencephalic transplants.

The present study focused on the early events of vascularization of intraocular cerebral transplants. Telencephalic pieces of rat embryos (E15) were transplanted into the anterior eye chamber of adult rats in deep ketamine-xylazine narcosis. At 3-, 4-, 5-, 6-, or 7-day postoperative survival periods, the rats were perfused and the transplants, with their iridic beds, were processed into serial, semi-thin sections. In 3- and 4-day transplants, neither dilated (perfused) nor collapsed blood vessels were found, but tissue defects, without proper wall and filled by non-nucleated (mature, host) erythrocytes, were seen. On post-operative day 5, large sinusoids were seen lines by endothelium and free of blood cells (as a consequence of perfusion). On days 6 and 7, the usual, although large, blood vessels were found. Our results suggest that the critical period of transplant vascularization is between postoperative days 4 and 5, and that the original vessels of donor tissue degenerate and disappear during the first postoperative days and thus, do not participate directly in transplant vascularization. Our hypothesis is that vascular invasion begins with the opening of host blood vessels into clefts formed by degeneration of graft tissue. For a period, a hemostasis occurs in these blood-filled lacunae, and then endothelium invasion from host vessels forms the proper wall. The transplant vasculature develops from these large sinusoids. The results challenge the role of the pre-existing donor vessels in transplant vascularization. A possible explanation of such paradoxical results is that the donor tissue must reach a stage of maturation to receive the ingrowing vessels, either host vessels, and the presence of vessels in the donor brain is the sign of this stage of maturation but has no direct role in transplant vascularization.

A rapid replacement of vimentin-containing radial glia by glial fibrillary acidic protein-containing astrocytes in transplanted telencephalon.

The present study follows the early events in the development of astroglia in rat embryonic (E15) tissue grafted into the cortex of adult rats. Astroglial elements (radial glia and astrocytes) were studied by glial fibrillary acidic protein (GFAP) and vimentin immunohistochemistry on post-transplantation (PT) days 7, 11, 14, 17, and 21. At PT7, GFAP-immunopositive elements were only scarce fibers in the transplants. At PT11, a dense network of long, GFAP-immunopositive fibers enmeshed the entire transplant, and astrocytes were already recognized. The fibers also showed vimentin immunoreactivity. By PT14, astrocytes became the predominant GFAP-labeled elements, although a few long fibers persisted. When compared with in situ development, the grafts showed earlier GFAP-immunoreactivity and earlier appearance of astrocytes, as well as a more rapid transition from the immature to the mature form of the glial system.

Mature but not fetal or neonatal rat superior cervical ganglion transplants survive in the cortex of adult rats.

The transplantation of catecholaminergic tissues is a possible therapy for parkinsonism. Central nervous tissue is suitable for transplantation only in the immature stage, whereas peripheral nervous tissue can also be transplanted when mature. The present study compares the development of fetal (17-20 embryonic day, E17-20), neonatal (1-3 postnatal day, P1-3) and mature (5-6-week-old) rat superior cervical ganglia after transplantation into the cerebral cortex of adult rats. The mature transplants survived in greater proportion and preserved their structural characteristics, although a considerable proportion of the neurons died. The perinatal transplants only survived sporadically, decreased in size and the surviving remnants failed to display a structure comparable to the adult ganglion in situ. Thus, the use of adult donors is not only a possibility but a necessity when superior cervical ganglion (probably any ganglion) is transplanted. This principle is radically different from that seen in the case of central nervous tissues, and can be understood by the analysis of the time curves of cell proliferation and programmed cell death (apoptosis) observed during the perinatal development of sympathetic ganglia.

[The significance of the vascularization of embryonic donor brain for the viability of intraocular transplants in the rat]. / Znachenie vaskuliarizatsii donorskogo mozga émbrionov dlia zhiznesposobnosti intraokuliarnykh transplantatov u krysy.

Telencephalic regions of rat embryonic brain were transplanted into the adult rat eye anterior chamber on E12 and E14 stages of development. These stages fall on the onset of the vascularization and the presence of the well developed vascular network in the embryonic brain. After a month of cultivation E14 transplants were much more viable than E12 (appropriately 58% and 15%). Only few E12 grafts had no initial vascularization. These transplants displayed growth retardation, while differentiated neurons could be seen only on their periphery. The results of the investigation demonstrate the meaning of vascularization extent in donor embryonic nervous tissue in its survival in conditions of transplantation into the rat eye anterior chamber.

Development of ependyma in neural transplants.

Under in situ conditions, the innermost (juxtaventricular) neuroepithelial layer of the embryonic brain wall develops into ependyma. No development of ependyma was usually observed, however, in transplanted embryonic brain wall. In our telencephalic transplants, however, cysts lined by epithelium resembling ependyma were observed, although only sporadically. We supposed that occasional foldings of the transplanted telencephalic wall enclosed the aforementioned cysts and so induced the formation of ependyma. This hypothesis was supported by the observation that ependyma developed frequently in a model system in which the telencephalic wall was folded artificially prior to transplantation.

Survival of embryonic rat mesencephalic tissue transplanted into cerebral cortex in the presence of co-grafted embryonic striatum.

The transplantation of embryonic dopaminergic tissue is an approach to the therapy of parkinsonism. In this study we compared the development of the rat embryonic ventral mesencephalon transplanted together or without striatal tissue into the cerebral cortex of adult rats. After one month, the survival ratio was 17 of 44 when striatal tissue was co-grafted, while only 4 of 45 transplants survived when the co-graft was omitted. In the mesencephalic graft, the appearance of tyrosine hydroxylase-contaning cells was investigated by immunohistochemical method. Applying a fluorescent tracer dye, we demonstrated the growth of neural fibers from the mesencephalic tissue to the striatal co-graft. In situ, the embryonic striatal tissue is the natural target area for the developing nigrostriatal pathway. The possible role of trophic factors and postsynaptic target surfaces provided by the co-graft is discussed as basis of the adjuvant effect.