Quantifying the internal deformation of the rodent spinal cord during acute spinal cord injury – the validation of a method

Visualization and analysis of the rodent spinal cord subject to experimental spinal cord injury (SCI) has almost completely been limited to naked-eye observations, and a single measure of gross spinal cord motion due to injury. This study introduces a novel method which utilizes MRI to quantify the deformation of the rodent spinal cord due to imposed, clinically-relevant injuries – specifically, cervical contusion and dislocation mechanisms. The image registration methods were developed using the Advanced Normalization Tools package, which incorporate rigid, affine and deformable registration steps. The proposed method is validated against a fiducial-based, ‘gold-standard’ measure of spinal cord tissue motion. The validation analysis yielded accuracy (and precision) values of 62 μm (49 μm), 73 μm (79 μm) and 112 μm (110 μm), for the medio-lateral, dorso-ventral and cranio-caudal directions, respectively. The internal morphological change of the spinal cord has never before been quantified, experimentally. This study demonstrates the capability of this method and its potential for future application to in vivo rodent models of SCI.     

Studies on the viability of metacercariae of Fasciola gigantica.

The viability of metacercariae of Fasciola gigantica was tested by in vitro and in vivo methods. In vitro testing was based upon the motility of juvenile flukes within the inner cyst as examined under the light microscope. In vivo testing was undertaken through experimental infections of rabbits (two groups) and natural definitive hosts, lambs (one group). In the first group, out of six rabbits each given 25 metacercariae, worm establishment only took place in one rabbit with a single fluke recovery on 60 days post infection. In the second group of six rabbits each given 200 metacercariae, five were infected, with two or three flukes per host. All the lambs given 250 metacercariae became infected showing prevalences of 7.2-40% in comparison with rabbits in which low prevalences (0-4%) were recorded. The results indicated that even viable metacercariae which were already tested in vitro could not readily establish in rabbits. Such variability in worm establishment suggests that immunological and chemotherapeutic studies in rabbits infected with F. gigantica are likely to be unreliable.     

The ptsH gene from Bacillus thuringiensis israelensis. Characterization of a new phosphorylation site on the protein HPr.

The ptsH gene from Bacillus thuringiensis israelensis (Bti), coding for the phosphocarrier protein HPr of the phosphotransferase system has been cloned and overexpressed in Escherichia coli. Comparison of its primary sequence with other HPr sequences revealed that the conserved His15 and Ser46 residues were shifted by one amino acid and located at positions 14 and 45, respectively. The biological activity of the protein was not affected by this change. When expressed in a Bacillus subtilis ptsH deletion strain, Bti HPr was able to complement the functions of HPr in sugar uptake and glucose catabolite repression of the gnt and iol operons. A modified form of HPr was detected in Bti cells, and also when Bti ptsH was expressed in E. coli or B. subtilis. This modification was identified as phosphorylation, because alkaline phosphatase treatment converted the modified form to unmodified HPr. The phosphoryl bond in the new form of in vivo phosphorylated HPr was resistant to alkali treatment but sensitive to acid treatment, suggesting phosphorylation at a histidine residue. Replacement of His14 with alanine in Bti HPr prevented formation of the new form of phosphorylated HPr. The phosphorylated HPr was stable at 60 degrees C, in contrast with HPr phosphorylated at the N delta 1 position of His14 with phosphoenolpyruvate and enzyme I. (31)P-NMR spectroscopy was used to show that the new form of P-HPr carried the phosphoryl group bound to the N epsilon 2 position of His14 of Bti HPr. Phosphorylation of HPr at the novel site did not occur when Bti HPr was expressed in an enzyme I-deficient B. subtilis strain. In addition, P-(N epsilon 2)His-HPr did not transfer its phosphoryl group to the purified glucose-specific enzyme IIA domain of B. subtilis.     

A Single-Dose PLGA Encapsulated Protective Antigen Domain 4 Nanoformulation Protects Mice against Bacillus anthracis Spore Challenge

Bacillus anthracis, the etiological agent of anthrax, is a major bioterror agent. Vaccination is the most effective prophylactic measure available against anthrax. Currently available anthrax vaccines have issues of the multiple booster dose requirement, adjuvant-associated side effects and stability. Use of biocompatible and biodegradable nanoparticles to deliver the antigens to immune cells could solve the issues associated with anthrax vaccines. We hypothesized that the delivery of a stable immunogenic domain 4 of protective antigen (PAD4) of Bacillus anthracis encapsulated in a poly (lactide-co-glycolide) (PLGA) - an FDA approved biocompatible and biodegradable material, may alleviate the problems of booster dose, adjuvant toxicity and stability associated with anthrax vaccines. We made a PLGA based protective antigen domain 4 nanoparticle (PAD4-NP) formulation using water/oil/water solvent evaporation method. Nanoparticles were characterized for antigen content, morphology, size, polydispersity and zeta potential. The immune correlates and protective efficacy of the nanoparticle formulation was evaluated in Swiss Webster outbred mice. Mice were immunized with single dose of PAD4-NP or recombinant PAD4. The PAD4-NP elicited a robust IgG response with mixed IgG1 and IgG2a subtypes, whereas the control PAD4 immunized mice elicited low IgG response with predominant IgG1 subtype. The PAD4-NP generated mixed Th1/Th2 response, whereas PAD4 elicited predominantly Th2 response. When we compared the efficacy of this single-dose vaccine nanoformulation PAD4-NP with that of the recombinant PAD4 in providing protective immunity against a lethal challenge with Bacillus anthracis spores, the median survival of PAD4-NP immunized mice was 6 days as compared to 1 day for PAD4 immunized mice (p<0.001). Thus, we demonstrate, for the first time, the possibility of the development of a single-dose and adjuvant-free protective antigen based anthrax vaccine in the form of PAD4-NP. Further work in this direction may produce a better and safer candidate anthrax vaccine.     

Cytomorphological study of soft tissue neoplasms: role of fluorescent immunocytochemistry in diagnosis

OBJECTIVES: Exact categorization of soft tissue tumours (STTs) on smears requires application of various ancillary techniques. This study was aimed at evaluating the role of fluorescent immunocytochemistry (FICC) in cyto-diagnosis of 30 STT cases. METHODS: Thirty cases of soft tissue tumours were included in the present study. All cases were subjected to routine Giemsa and Papanicolaou stain. Extra smears were made and kept for fluorescent immunostaining. A panel of cytoskeletal antibodies, tagged with FITC (Fluorescein isothyocynate), was employed in all these cases. Fluorescent immunostained smears were examined under Zeiss Confocal Laser scanning microscope, using double immunofluorescence (red-green). Finally, all cases were subjected to biopsy and again immunoperoxidase staining. RESULTS: Among the 30 cases in the present study, unaided cytological diagnoses ranged from 'spindle cell' tumour in four (13.3%) cases, benign and malignant spindle cell tumour in 17 (56.6%) cases, to malignant mesenchymal tumour in nine (30%) cases. FICC helped in further correct categorization of 25/30 (83.3%) cases viz. leiomyoma (three), benign neurogenic tumour (six), schwannoma (one), dermatofibrosarcoma protuberans (three), synovial sarcoma (two), rhabdomyosarcoma (two), malignant fibrous histiocytoma (five) and malignant peripheral nerve sheath tumour (three). Aggressive fibromatosis was found to be a missed diagnosis in two cases. Overall concordance between cyto-diagnosis with FICC, and histopathology results was 83.3% (P < 0.05). CONCLUSION: Fluorescent immunocytochemistry is a significant ancillary technique for making a rapid and specific diagnosis of STT, as required for their timely management. Incorporation of a wide panel of antibody markers with clinico-cytological correlation is recommended in forming an exact diagnosis in these cases.     

Conformational states of Leu5- and Met5-enkephalins in solution

The molecular conformations of Leu5- and Met5-enkephalins in aqueous and DMSO solutions were investigated by FT-IR and laser Raman spectroscopic methods. The amide I, II, and III regions in the FT-IR spectra of Leu5- and Met5-enkephalins in aqueous solution were analyzed by performing Fourier self-deconvolution of the bands. Leu5-enkephalin in aqueous solution is found to exist in both type II beta-turn and beta-sheet structures, whereas Met5-enkephalin has a lesser tendency to form beta-turn structure in aqueous solution. It is likely that these different conformers of enkephalins might bind to different receptor types.     

Stimulation of poly(ADP-ribose) synthetase activity in the lungs of mice exposed to a low level of ozone

Toxic effects of O3 are mediated through the formation of free radicals, which can cause DNA strand breaks. Cellular DNA repair is dependent upon the formation of poly(ADP-ribose) (polyADPR) catalyzed by polyADPR synthetase. In order to evaluate whether O3 exposure inflicted DNA damage in lung tissue, we measured the activity of polyADPR synthetase (known to be activated in response to DNA damage) in mouse lungs after exposure to 0.45 ppm (882 micrograms/m3) O3 for up to 7 days. The enzyme activity was stimulated with O3 exposure relative to unexposed controls, showing a 20% (P less than 0.05) increase at Day 5 and 42% (P less than 0.001) at Day 7 of O3 exposure. In addition, the activity of superoxide dismutase (SOD), known to be stimulated in response to production of superoxide anion (.O2-), was measured as an indicator of free radical involvement. Relative to unexposed controls, the SOD activity in exposed animal lungs increased to the peak level at Day 5 (48%, P less than 0.001) and then declined at Day 7 of O3 exposure but was still higher than controls (17%, P less than 0.05). When animals, after 5 days of O3 exposure, were allowed to recover in filtered room air, the activities of both enzymes declined to their respective control values in 6 days. These results suggest a possible temporal relationship between O3 injury and the activities of polyADPR synthetase and a free radical scavenging enzyme, SOD. The stimulation of polyADPR synthetase activity with O3 exposure, reflecting a response to lung cellular DNA repair, may be a sensitive indicator for assessing DNA damage in oxidant injury.