Pathobiology Research
Year:2014 | Volume:16 | Issue:4|Papers Count:10

DNA sequence determination is a tremendous human achievement. DNA sequencing includes several methods and technologies in use for determining the order of the nucleotide bases (adenine, guanine, cytosine, and thymine) in a molecule of DNA.Knowledge of DNA sequences has become indispensable for basic biological research, other research branches utilizing DNA sequencing, and in numerous applied fields such as diagnostic, biotechnology, forensic biology and biological systematics. The advent of DNA sequencing has significantly accelerated biological research and discovery. Rapid sequencing, the result of modern DNA sequencing technology, is instrumental in the sequencing of the human genome for the Human Genome Project. Related projects, often by scientific collaboration across continents, have generated complete DNA sequences of humans as well as numerous animals, plants and microbial genomes.DNA sequencing methods currently under development include labeling DNA polymerase and reading the sequence as a DNA strand transits through nanopores. Additional methods include microscopy-based techniques such as atomic force microscopy or transmission electron microscopy that are used to identify the positions of individual nucleotides within long DNA fragments (>5000 bp) by nucleotide labeling with heavier elements (e.g., halogens) for visual detection and recording. Third generation technologies aim to increase throughput and decrease the time to result and cost by eliminating the need for excessive reagents and harnessing the processivity of DNA polymerase.Researchers in the field of genetics in Iran use this technology in their studies, but unfortunately our literature lack proper Persian language resources. The authors intend to write a series of review articles in this field. The present paper is an introduction to the summary of important techniques in DNA sequencing.

Objective: Acinetobacter baumannii (A. baumannii) is a major hospital pathogen with a high capacity to resist most common anti-microbial agents. A. baumannii is the etiologic agent for various illnesses including pneumonia, meningitis, and bloodstream infections. Biofilm associated proteins (Bap) are specific cell surface proteins essential for the formation of biofilm and play a main role in its pathogenicity. Previously, we have studied various regions of this protein. Considering different criteria, some regions were introduced as conserved and immunogenic. The immunogenicity of one of those regions pertaining to amino acids 706-1076 previously examined has shown that its expression triggers high antibody levels when injected to mice thereby protecting the animals against the bacterium. The present study examines region 4 of the Bap protein in order to validate the previous bioinformatics studies and its immunogenicity.Methods: In order to obtain immunity against this pathogen, a 1620 bp gene from Bap was amplified and cloned in pET32a. This region from Bap was cloned, expressed and verified by monoclonal antibodies. BALB/c mice were immunized by subcutaneous injection of the pure recombinant protein. Mice immune response was determined by ELISA.Results: High titer of raised antibodies implied that the recombinant protein was a strong antigen and immunogen.Conclusion: The results indicate that this protein can be a suitable choice for developing a new recombinant vaccine against A. baumannii.

Objective: The study of physiological changes in recombinant cell lines provides useful information to improve production performance. In this study, we investigate the effects of an anti-CD33 chimeric IgG4 expression on Sp2.0 cell growth.Methods: Variable region genes of light and heavy chains of monoclonal antibody produced by M195 were cloned in pFUSE-CLIg-hk and pFUSE-CHIg-hG4 expression vectors, respectively. Transfection of recombinant plasmids into Sp2.0 cell lines was performed using lipofectamine in two steps. Positive transformant cells were isolated and subjected to PCR, RT-PCR and Western blot analysis to confirm the integration of gene cassettes and the expression of recombinant IgG4.To assess the growth parameters, recombinant and parent Sp2.0 cell lines were seeded at a density of 1´105 cells/ml in duplicate into 12-well plates. For nine days, culture plates were sampled daily and viable cell count and viability determined.Results: The results of PCR, RT-PCR and Western blot analyses confirmed the generation of stable producer cell lines. In recombinant cells, the maximum cell density decreased by 46%. However, it was observed that IgG4 expression had no effect on cell viability of these transfectants.Conclusion: Our results showed that the expression of recombinant IgG4 can change growth parameters in Sp2.0 cell lines that express the pFUSE-CHIg-hG4-pFUSE-CLIghk construct.

Objective: A recent field of research in epigenetics is DNA methylation which involves the CpG island in the genome that subsequently controls transcription and translation of targeted genes. In the hepatitis B virus (HBV) genome, there are three CpG islands which tend to be methylated. The aim of the current study is to determine the methylation pattern of the HBV X gene in chronically infected HBV patients.Methods: Study participants comprised 45 chronically infected HBV patients. According to the presence of the HBeAg, patients were divided into two groups, HBeAg positive (n=24) and HBeAg negative (n=21). Initially, viral DNA was treated with natrium bisulfate. Then, analysis was performed with two sets of methylated and non-methylated primers by the MSP method.Results: The overall methylation rate in serum samples of hepatitis B infected patients was 35.5%; the rate in the HBeAg positive patients was 20.8%, whereas it was 52.3% in HBeAg negative patients. There was a significantly higher rate of methylation in serum samples of HBeAg negative patients compared to HBeAg positive patients (student's ttest; P=0.02).Conclusion: Methylation of HBV can be used as a new mechanism to control the progression of viral infection. This methodology can be useful for determining the characteristics of clinical stages of this infection.

Objective: This study aimed to evaluate the incidence of apoptosis in vitrified and non-vitrified human ovarian tissue by the use of morphological analysis and apoptosis assay techniques.Methods: We obtained human ovarian tissue biopsies from 30 women who underwent elective caesarean sections. Tissues were transported to the laboratory in pre-warmed, equilibrated Leibovitz L-15 medium within 2 hours. The tissues were cut into small pieces and divided into two groups, vitrified and non-vitrified (control). Apoptosis incidence was assessed by light microscope and the TUNEL assay and DNA laddering. Evaluation of caspase 3.7 protein levels was performed by the luminescent assay.Results: We observed no morphological signs of apoptosis in the vitrified samples. There were no apoptosis signals as evidenced by TUNEL staining and no DNA laddering pattern observed in the vitrified group. Caspase 3.7 activity was 2294±169.19 RLU/mg protein in the non-vitrified control group and 2231±89.271 RLU/mg protein in the vitrified group, which was not significantly different.Conclusion: The structure of human vitrified ovarian tissue was well preserved. Vitrification could not increase apoptosis and caspase 3.7 activity in human ovarian tissue.

Objective: Trichomonas species usually reside in the mouth and occasionally in the respiratory tract. These species can be found in the lungs of humans. Although the pathogenicity of the parasite in the respiratory system has not been proven, it is more prevalent in people who lack good oral health or suffer from asthma or chronic pulmonary diseases. In the present descriptive study, we have identified Trichomonas by direct microscopic observation of stained smears and by PCR on the lung sputum of patients with asthma and chronic lung diseases that include lung cancer, bronchiectasis, COPD, and malignant pulmonary disease who were admitted to Masih Daneshvari Hospital, Tehran, Iran.Methods: For direct examination of 133 sputum samples, we stained the smears with giemsa. In addition a total of 60 samples were used for DNA extraction by an extraction kit (Cinnagen). Nested-PCR was used for amplifying the ITS1 target gene of the parasite. Finally the DNA sequence of the gene was determined.Results: According to the results, in direct examination of the sputum smears there were only 4 positive cases identified, whereas 22 (36.66%) of the samples were identified as Trichomonas by nested PCR. According to gender, 33.33% of the female samples and 38.46% of the male samples were found to be positive.Conclusion: Considering the high prevalence of this parasite in the study group, chronic pulmonary disease and asthmatic patients may be more susceptible to Trichomonas.

Objective: This study investigated tissue damages induced by chronic subcutaneous administration of nano- and microparticles of manganese dioxide (MnO2) on the liver, kidneys and testes of rats.Methods: Rats (n=210) were divided into three groups: control, MnO2 nanoparticle injected and MnO2 microparticle injected. The experimental groups received subcutaneous injections with either nano- or microparticles of a solution that contained MnO2 (100 mg/kg) once per two weeks for 14 weeks. Once every two weeks, we randomly selected five rats from each group for histological evaluations of the liver, kidneys, and testes. Tissue lesions were initially evaluated by hematoxylin and eosin staining, then kidney and liver tissue sections were stained by the Jones and Masson' s trichrome methods, respectively. The changes in diameter of basement membrane and cell numbers of the various parts of the nephrons in different groups were measured by Image Tools version 2 software.Results: The liver tissues of the nano- and microparticle groups exhibited severe damage histopathologically. Cloudy swelling was observed in the cytoplasm of hepatocytes. The liver tissue and its canaliculi structures were severely damaged. Inflammation and ductular reaction signs were seen in liver tissue. Deposition of particles in the basement membrane of the nephrons were observed in the nanoparticle-treated group. There was a significant reduction in glomerular and tubular cells in the nanoparticle-treated group compared to the control and microparticle-treated groups. Some of the structural and functional parameters of the testes in the nanoparticle-treated group had significant pathobiological variations.Conclusion: Administration of MnO2 nanoparticles when compared with the same dose of MnO2 microparticles caused more tissue damage in all examined tissues. Reduction in particle size from micrometer to nanometer appeared to exacerbate the damaging mechanisms of these particles in the examined tissues.

Objective: Inflammation and proinflammatory cytokines play an important role in the initiation and maintenance of central neuropathic pain. There are several reports that cytokine production is increased at the lesion site following spinal injury. A few studies have investigated the supraspinal levels of these cytokines. This study intended to determine TNF-a and IL-6 release in the ventroposterolateral nucleus of the thalamus in spinal cord injury-related neuropathic pain in rats.Methods: Male Sprague-Dawley rats that weighed 200-230 g were used. Following administration of anesthesia, spinothalamic tract injury was performed by a laminectomy at the T9-T10 level in male rats. Mechanical allodynia and motor performance were evaluated at 3, 7, 14, 21 and 28 days after spinal injury by Von Frey filament and the open field test, respectively, in the sham and lesion groups. Concentrations of TNF-a and IL-6 in the VPL microdialysate were detected by ELISA in both spinal cord injured and sham groups during four weeks after surgery.Results: Mechanical pain threshold reduced in both hind paws following lateral spinothalamic tract injury. Paw withdrawal threshold in the Spinothalamic tract-injured group was significantly (P<0.05) lower than in sham group at day 14 post-surgery. Motor performance did not show any significant change after surgery. In the microdialysate, TNF-a reduced significantly (P<0.05) at days 3 and 7 post-injury compared to the sham group which returned to a level close to the pre-surgery level. VPL concentration of IL-6 increased significantly (P<0.05) at day 21 post-injury compared to the sham group.Conclusion: Lesions in spinal pathways that contain afferent pain fibers appear to change the supraspinal levels of inflammatory mediators, including VPL, concentrations of TNF-a and IL-6 which are consistent with spinal injury related pain behavior. Cytokine production results in hyperexcitability of the thalamocortical neurons, a decrease in pain threshold, and persistent neuropathic pain after spinal injury.