Gill lesions and mortality in common carp (Cyprinus carpio) with a dense bloom of Heterosigma-like algae in Khuzestan province.

BACKGROUND: Widespread common carp (Cyprinus carpio) mortalities have been recorded in Khuzestan province fish farms in recent years. In summer of 2017 two cases of harmful algal bloom were encountered that led to massive mortality in common carp in Khuzestan, Iran. AIMS: The aim of this study was to identify the possible etiologic agent of two mortalities with characteristic symptoms of gill lesions due to harmful algae. METHODS: Water samples were collected and 5 moribund fishes were examined by histophatologic, scanning electron microscopic and PCR examination. RESULTS: In wet smear preparations, a lot of algal cells and fragments, and sloughed, necrotic epithelial cells were observed between the lamellae. In histopathologic examination of gills, hyperplasia, necrosis and algal cells surrounded by hyperplastic cells were seen in tissue sections. No inflammatory cell aggregation was noticed. In scanning electron microscopic examination the algae was found attached to the gill surface (cell diameter: 8.5 ± 4.2 µm) with 2 equal flagella. CONCLUSION: Phytoplankton analysis using direct microscopy and electron microscopy, morphologically resembling Heterosigma was identified, however, in PCR tests, Heterosigma analysis showed negative results, therefore the causative agent was called "Heterosigma-like" algae.

The effects of respiratory muscle training on improvement of the internal and external thoraco-pulmonary respiratory mechanism in COPD patients.

INTRODUCTION: Chronic obstructive pulmonary disease (CPOD) is a severe, incapacitating pathology. Inspiratory and/or expiratory muscle training may favorably impact the indicators of both specific and general improvement with regard to this disease. We are hypothesizing that when combined with bronchial decluttering, this training will have a beneficial effect on lung function and quality of life in these patients. METHOD: Fourty COPD subjects classified Gold I and Gold II and aged 60.38±8.02years were divided into four groups of 10. Three of the groups were trained with the help of Threshold(®) tools used for (1) inspiratory, (2) expiratory and (3) inspiratory and expiratory purposes; their training supplemented the decluttering and lower limb muscle exercise that the 4th group concurrently received. The patients underwent 16 rehabilitation sessions over an 8-week period. The variables consisted in: (1) forced expiratory volume in 1s (FEV1) and spirometrically measured peak expiratory and inspiratory flow rates (PEFR and PIFR); (2) fatigability, dyspnea, heart rate and walking distance evaluated during the 6-minute walk test; (3) maximum inspiratory pressure and (4) maximum expiratory pressure as assessed by the Threshold(®) tools and (5) the signs of quality of life in terms of the Saint-George's respiratory questionnaire (SGRQ) score. RESULTS: Only in group 1, there was significant improvement with regard to FEV1 and PEFR. There was no PIFR modification in any of the groups. On the other hand, signs of quality of life scores along with dyspnea, fatigability and heart rate showed significant improvement in the three experimental groups, and significant improvement in maximum inspiratory pressure was observed in groups 1 and 3. DISCUSSION: When associated with decluttering techniques, diaphragmatic rehabilitation and lower limb muscle exercise along with psychological support and educational efforts, respiratory muscle training is beneficial when compared with the usual protocols in rehabilitation of COPD patients.

Determination of the electrophoretic pattern of somatic and excretory-secretory proteins of Ligula intestinalis parasite in spirlin (Alburnoides bipunctatus).

Ligula intestinalis parasite is a cestodes that causes remarkable damages to fish. It is also of prime importance in economic and hygienic aspects. SDS-PAGE and western blotting are the methods that can be used to determine the electerophoretic pattern of somatic and excretory-secretory proteins of parasites. In this study, after obtaining the plerocercoidal stage of this parasite from the spirlin (Alburnoides bipunctatus), its somatic proteins were prepared using ultrasonicae, and excretory-secretory proteins were prepared using the PBS solution. After protein assay, which included using the Bradford method and then SDS-PAGE on these two antigenic solutions, 5 protein bands of 26, 33, 38, 58, 70kDa in somatic antigens, and 7 bands of 25, 28, 33, 43, 49, 60, 70kDa in excretory-secretory antigens were observed. After western blotting on both antigens and adding the primary antibody (the sera of infected fish) and then the secondary antibody (Rabbit Anti-fish Polyclonal Antibody Conjugated from Abnova Corporation) no band was seen in excretory-secretory antigen. And only in the 55kDa band of somatic antigen, a positive response, in comparison of fish positive serum was observed.

Determination of the electrophoretic pattern of somatic and excretory-secretory proteins of Ligula intestinalis parasite in spirlin (Alburnoides bipunctatus)

Ligula intestinalis parasite is a cestodes that causes remarkable damages to fish. It is also of prime importance in economic and hygienic aspects. SDS-PAGE and western blotting are the methods that can be used to determine the electerophoretic pattern of somatic and excretory-secretory proteins of parasites. In this study, after obtaining the plerocercoidal stage of this parasite from the spirlin (Alburnoides bipunctatus), its somatic proteins were prepared using ultrasonicae, and excretory-secretory proteins were prepared using the PBS solution. After protein assay, which included using the Bradford method and then SDS-PAGE on these two antigenic solutions, 5 protein bands of 26, 33, 38, 58, 70kDa in somatic antigens, and 7 bands of 25, 28, 33, 43, 49, 60, 70kDa in excretory-secretory antigens were observed. After western blotting on both antigens and adding the primary antibody (the sera of infected fish) and then the secondary antibody (Rabbit Anti-fish Polyclonal Antibody Conjugated from Abnova Corporation) no band was seen in excretory-secretory antigen. And only in the 55kDa band of somatic antigen, a positive response, in comparison of fish positive serum was observed.

Effect of ghrelin on protein kinase C-ε and protein kinase C-δ gene expression in the pulmonary arterial smooth muscles of chronic hypoxic rats.

INTRODUCTION: Protein kinase C (PKC), can be activated in pulmonary arterial smooth muscle cells during hypoxia, leading to hypoxic pulmonary vasoconstriction (HPV). Studies are going on to detect the strict PKC isoform involved in the phenomenon. It has been shown that ghrelin, a 28-amino-acid peptide, may protect lungs from HPV side effects, to some extent. The aim of study was to evaluate the effect of exogenous ghrelin on PKC-ε and PKC-δ gene expression during chronic hypoxia. MATERIAL AND METHODS: Twenty-four adult male Wistar rats were divided randomly in 3 groups. Hypoxic rats with saline or ghrelin treatment were placed in a normobaric hypoxic chamber for 2 weeks. Controls remained in room air. PKC-ε and PKC-δ gene expression was measured by real-time RT-PCR. RESULTS: Morphometric analysis showed that ghrelin reversed the hypoxia induced pulmonary artery wall thickness. In hypoxic animals, there was a 2- and 4-fold increment in PKC-ε and PKC- δ gene expression, respectively. Ghrelin treatment reduced the overexpression of PKC-ε and PKC-δ to control animals' value. CONCLUSION: Ghrelin by decreasing the expression of PKC-ε and PKC-δ in hypoxic animals reduces the HPV. Although more studies are needed, it could be an honest deduction that ghrelin affects HPV in a multifunctional manner and might be used as a therapeutic agent in the future.

Expression of epidermal growth factor receptor tyrosine kinase family in fine needle aspiration and permanent specimens of invasive lobular and ductal breast cancers.

Recently, the role of HER-2/Neu gene amplification has been enthusiastically investigated in breast cancer. Determining the HER-2/Neu status could be achieved by evaluating either histologic samples or cytologic specimens obtained by Fine Needle Aspiration (FNA). This study aimed at determining the concordance of HER-2/Neu expression in FNA and histologic sections. FNA samples, as well as their corresponding histologic sections of 90 cases with breast cancer were evaluated in Tabriz Sina Teaching Center in a 13-month period of time. The immunohistochemistry was employed for determining the HER-2/Neu amplification for both methods. The concordance rate and agreement were determined between the two methods. Ninety specimens of women with a mean age of 50.93 +/- 10.64 (29-84) years were assessed. There were 84 cases with invasive ductal carcinoma and 6 cases with invasive lobular carcinoma. Lymph nodes were involved in 50 cases and there were vascular and neural involvement in 40 and 35 cases, respectively. Her-2/Neu was not detected in 27 cases (30%) with weak and strong amplifications in 47 (52.2%) and 16 (17.8%) cases of FNA specimens, respectively. Her-2/Neu was not detected in 29 cases (32.2%) with weak and strong amplifications in 42 (46.7%) and 19 (21.1%) cases of histologic specimens, respectively. The concordance rate was 70% between the two methods. The agree ment was statistically significant between the two methods, as well (kappa = 0.51, p < 0.001). HER-2/neu gene amplification can be reliably estimated by immunohistochemistry on breast cancer FNAs and a good correlation has been found between this and results on histological sections.

Flora, life forms and chorotypes of plants of Salok protected area (North Khorassan Province Iran).

Salok protected Area is located in the 37 degrees 15' to 37 degrees 08' of the North latitude and 57 degrees 16' to 57 degrees 06' of East longitude, in West North Esfarayen in North khorassan province. In this research 52 families, 174 genera and 213 species were identified. The largest plant family is Asteraceae (34 species), Poaceae (18 species), Brassicaceae (17 species) and Fabaceae (17 species), respectively. Chief life forms are Hemicryptopytes (49.29%), Therophytes 23.47% and cryptophytes (12.67%). The most of plants chorotype with 62.91% is influenced by Irano-Touranina elements. Among 213 identified species of this region and 15 species endemic of Iran contain 0.87% of total endemic species of Iran's flora.

Human papillomavirus infection in lung vs. oral squamous cell carcinomas: a polymerase chain reaction study.

The role of Human Papillomavirus (HPV) has been suspected in pathogenesis of various malignancies; however, the available data are not conclusive. This study aimed to determine and compare the frequency of HPV infection in oral and lung Squamous Cell Carcinoma (SCC) by a sensitive method. Sixty specimens of oral and lung SCC (30 cases each one) were reevaluated in Tabriz Imam Reza Centre in a 24 month period. Following genomic DNA extract, the Polymerase Chain Reaction (PCR) amplification was performed in presence of specific MY11 and MY09 primers for HPV infection. Three cervical specimens and a combination of PCR solution lacking DNA plus healthy persons' DNA samples were employed as positive and negative controls, respectively. The oral group was significantly older than the lung group (68.90 vs. 56.67 y, p < 0.001) with more males in the latter (83.3 vs. 60%; p = 0.04). Percentages of HPV infection in the oral and lung groups were comparable (20 vs. 10%, respectively; p = 0.47). Majority of patients with HPV infection were older than 60 years (88.9%) or male (88.9%). In the oral group, all these cases were well differentiated and the majority was of lower lip origin (83.3%). In the lung group, 66.7% of these specimens were moderately differentiated and the origin was bronchus in all cases. In conclusion, the rate of HPV infection in lung and oral SCC samples is rather lower than the previous reports in the literature. This rate is apparently higher in the oral than the lung SCC specimens.

Expression of P27, Ki67 and P53 in squamous cell carcinoma, actinic keratosis and Bowen disease.

This study aims at evaluating the expression of P27, Ki67 and P53 in Squamous Cell Carcinoma (SCC), Actinic Keratosis (AK) and Bowen Disease (BD) specimens. In an analytic-descriptive setting, skin biopsy specimens of 45 patients were evaluated in three 15-case groups including BD, AK and SCC specimens. Fifteen normal skin biopsy specimens were obtained and used as the control group. Immunohistochemical staining was performed in all the specimens and the expression rates and patterns of Ki67, P27 and P53 were determined. The results were compared between the four groups. Ki67 was expressed in 0.8, 23.7, 12.3 and 19.3% of the cells in the normal skin, AK, BD and SCC groups, respectively. No significant difference was seen between the three pathological conditions regarding the expression rate of Ki67. P27 was positive in 23.4, 26.2, 25.9 and 4.5% of specimens in the normal skin, AK, BD and SCC groups, respectively. This rate was significantly the lowest in the SCC group. P53 expression was detected in 26.6, 41.8 and 54.6% of the assessed cells in the AK, BD and SCC groups, respectively. There was no expression of P53 in the normal skin specimens. This rate was significantly the highest again in the SCC group. Based on these results, the quantitative and qualitative (pattern of distribution) evaluation of the expressions of Ki67, P27 and P53 may be helpful in differentiating malignant and premalignant epidermal lesions, particularly in unsatisfactory or fragmented specimens.

A rare case of intramedullary cervical spinal cord meningioma and review of the literature.

STUDY DESIGN: Case report. SETTINGS: Department of Neurosurgery, Imam Hospital, Faculty of Medicine, Tabriz University of Medical Sciences, Daneshgah Street, Tabriz, East Azerbayjan, Iran. REPORT: A 21-year-old man presented with progressive paraesthesia and weakness in the four limbs, with disturbances in the vibration and positional senses. Magnetic resonance imaging revealed a cervical intramedullary tumour from the cervicomedullary junction to C3. Radical resection of the tumour was performed and histology revealed a meningioma. His clinical outcome after a 3-year follow-up is relatively good. CONCLUSION: Intramedullary cervical meningiomas are very rare and have been reported only five times before. Because of its tendency to recur after surgery and because of a possible aggressive behaviour, meticulous histopathological examination is mandatory to predict the evolution and plan the follow-up. Outcome is mainly related to the type of tumour and the complete removal.

Copper binding to the PrP isoforms: a putative marker of their conformation and function.

We show here that PrP(C), the normal isoform of the prion protein (PrP(Sc)), could be retained by a Cu(2+)-loaded resin through two different binding sites. Contrarily, PrP(Sc) was not retained at all by such resin. This constitutes a new prion-specific property of PrP(Sc), which in addition to protease resistance and beta-sheet content, may result from its aberrant conformation.

A protease-resistant prion protein isoform is present in urine of animals and humans affected with prion diseases.

Prion protein (PrP)(Sc), the only known component of the prion, is present mostly in the brains of animals and humans affected with prion diseases. We now show that a protease-resistant PrP isoform can also be detected in the urine of hamsters, cattle, and humans suffering from transmissible spongiform encephalopathies. Most important, this PrP isoform (UPrP(Sc)) was also found in the urine of hamsters inoculated with prions long before the appearance of clinical signs. Interestingly, intracerebrally inoculation of hamsters with UPrP(Sc) did not cause clinical signs of prion disease even after 270 days, suggesting it differs in its pathogenic properties from brain PrP(Sc). We propose that the detection of UPrP(Sc) can be used to diagnose humans and animals incubating prion diseases, as well as to increase our understanding on the metabolism of PrP(Sc) in vivo.

Prion protein with an E200K mutation displays properties similar to those of the cellular isoform PrP(C).

Creutzfeldt-Jakob disease (CJD) in Libyan Jews, linked to the E200K mutation in PRNP (E200KCJD), is the most prevalent of the inherited prion diseases. As other prion diseases, E200KCJD is characterized by the brain accumulation of PrP(Sc), a pathologic conformational isoform of a normal glycoprotein denominated PrP(C). To investigate whether the E200K mutation is enough to de novo confer PrP(Sc) properties to mutant PrP, as suggested by experiments in Chinese hamster ovary cells, we examined the biochemical behavior of E200KPrP in brains and fibroblasts from sporadic as well as homozygous and heterozygous E200KCJD patients, asymptomatic transgenic mice carrying the E200K mutation, as well as in normal and scrapie-infected mouse neuroblastoma cells expressing E200KPrP. E200KPrP was examined for protease sensitivity, solubility in detergents, releasibility by phosphoinositol phospholypase-C and localization in cholesterol enriched membrane microdomains (rafts). In all tissues except in brains of CJD patients and ScN2a cells, E200KPrP displayed properties similar to those of PrP(C). Our results indicate that the E200K mutation does not automatically convey the properties of PrP(Sc) to new PrP molecules. A conversion process occurs mainly in the prion disease affected brain, suggesting the presence of a tissue-specific or age-dependent factor, in accord with the late onset nature of inherited CJD.

Further observations on the impact of a veterinary programme in Afghanistan on seasonal livestock mortality.

This paper reports further results of a two year livestock mortality survey in Afghanistan, where a war of more than a decade had completely disrupted the veterinary field services. A questionnaire-based survey to measure the impact of a veterinary field programme indicated that average annual mortality in cattle, sheep, and goats was substantially lower in districts that received veterinary services (covered districts) than in districts without any veterinary services (control districts). The impact of the programme varies according to the season and the age group of the animals involved. The programme lacked impact in winter, in particular in adult small ruminants. The highest impact was noted in the spring and autumn for adult goats, and summer and autumn for adult sheep. The impact of the programme was also limited (in small ruminants even completely absent) during the suckling period in young animals. The highest impact was noted in the post-weaning period in small ruminants, when approximately four times more lambs and kids died in the control districts than in the covered districts. It was concluded that the major impact of the veterinary programme was achieved when animals are in contact with other flocks during grazing seasons, when parasitic and infectious diseases are present and against which the veterinary programme is directed. Additional inputs-including extension activities-are therefore required to improve the effect of the programme in the winter and in the neonatal period.

The anti-prion activity of Congo red. Putative mechanism.

PrPSc, an abnormal conformational isoform of the normal prion protein, PrPC, is the only known component of the prion, a proteinacious agent that causes fatal neurodegenerative disorders in humans and other animals. The hallmark properties of PrPSc are its insolubility in nondenaturing detergents and its resistance to digestion by proteases. Anions such as Congo red (CR) have been shown to reduce the accumulation of PrPSc in a neuroblastoma cell line permanently infected with prions as well as to delay disease onset in rodents when administrated prophylactically. The mechanism by which such anti-prion agents operate is unknown. We show here that in vitro incubation with CR renders native PrPSc resistant to denaturation by boiling SDS. This resulted from PrPSc conformation, since neither the properties of PrPC nor those of predenatured PrPSc were changed by the addition of CR. CR-PrPSc could only be denatured by the addition of acidic 3 M guanidine thiocyanate. Since in vitro conversion experiments have suggested that partial denaturation may be required for PrPSc to serve as template in the PrPC --> PrPSc conversion, we propose that CR inhibits prion propagation by overstabilizing the conformation of PrPSc molecules.

Differential allelic expression of PrP mRNA in carriers of the E200K mutation.

Creutzfeldt-Jakob disease (CJD) linked to the E200K mutation of the prion protein (PrP) gene presents with a wide range of age at disease onset. Since most patients are heterozygous for the mutation, we tested whether differential expression of mutant versus wild-type (wt) PrP may affect the age at disease onset in carriers of the mutation. We measured wt and mutant PrP protein and mRNA in Epstein-Barr virus (EBV)-transformed B cells of either E200K CJD patients or healthy E200K carriers. Our results suggests that while in most healthy carriers the expression of wt PrP was higher than that of E200K PrP, most of the E200K CJD patients express equal levels of both PrP proteins. Similar results were obtained for either PrP protein or PrP mRNA. These results suggest that preferential expression of PrP from the wt allele may modulate the outbreak of the disease in carriers of prion mutations. This notion is consistent with the results obtained in transgenic mice carrying a human PrP gene, which suggest that endogenous PrP protects mice from contracting scrapie after inoculation with human CJD brain. Similar mechanisms may prevail in other inherited diseases with variable phenotypes.

Detection of 14-3-3 protein in the CSF of genetic Creutzfeldt-Jakob disease.

The 14-3-3 protein, a protein involved in signal transduction, is present in the CSF of patients with Creutzfeldt-Jakob disease (CJD) and not in patients with other dementing diseases. We show here that this is also true for patients with E200K CJD, but not for healthy carriers of the mutation.

Effect of scrapie infection on the activity of neuronal nitric-oxide synthase in brain and neuroblastoma cells.

Nitric-oxide synthase (NOS) is responsible for the synthesis of nitric oxide which serves as a neural messenger in the central nervous system. NOS activity was markedly inhibited in brains of mice and hamsters and neuroblastoma cells infected with scrapie (ScN2a). The decrease in activity was in accordance with decreased NADPH-diaphorase-positive cells and decreased staining of NOS-positive cells demonstrated by specific anti-NOS antibodies. However, the specific nNOS mRNA in ScN2a was elevated when compared with normal neuroblastoma cells (N2a). Immunoblotting of fractions from these cell lines with an anti-nNOS monoclonal antibody revealed a band of nNOS from N2a and two bands with a lower molecular weight in ScN2a cells. Furthermore, NOS in ScN2a cells was insoluble in nondenaturing detergents. This insolubility is one of the landmark properties of PrPSc. It is, therefore, possible that nNOS in scrapie-infected cells and brains is aberrantly folded, resulting in an insoluble and inactive enzyme.

Livestock mortality in Afghanistan in districts with and without a veterinary programme.

This paper reports on livestock mortality in Afghanistan where a war has been going on for more than a decade, with complete disruption of the veterinary field services. The study attempted to measure the impact of a veterinary field programme carried out mainly by paravets. The study also provides valuable data on the impact of diseases in the absence of any veterinary intervention. The veterinary programme, implemented by a Dutch non-governmental organisation (NGO) for approximately 3 years, consisted essentially of vaccination against major infectious diseases and administration of anthelmintic drugs for nematodes and liver flukes. Veterinary personnel were also involved in curative treatments. Livestock mortality figures were collected by questionnaire over a period of 2 years from more than 700 farmers in randomly selected villages in 4 covered districts with a veterinary programme and 4 control districts without a veterinary programme. The average annual mortality rates for calves, lambs and kids respectively were 16.2%, 17.3% and 19.1% in the covered areas, against 21.5%, 25.2% and 24.6% in the control areas. Adult mortality figures were 3.8%, 7.4% and 5.4% in the covered areas, against 5.3%, 13.6% and 15.6% in the control areas for cattle, sheep and goats respectively. The survey indicated significant differences (P < 0.01 in cattle, P < 0.001 in small ruminants) in livestock mortality between the control and covered areas and it is concluded that these differences were attributable to the presence or absence of the animal health programme.

Insoluble wild-type and protease-resistant mutant prion protein in brains of patients with inherited prion disease.

We studied prion proteins (PrP) in skin and brains of Libyan Jews carrying the E200K mutation who died of familial Creutzfeldt-Jakob disease (CJD). Unexpectedly, studies with brain showed that PrP molecules encoded both by the wild-type (wt) and mutant alleles exhibit altered properties characteristic of the prion protein associated with prion diseases (PrPSc). Using monospecific antisera, we found that wtPrP was insoluble in the brains of three patients who were heterozygous for the E200K mutation, whereas mutant PrP was both insoluble and protease-resistant. Our results argue that both wild-type and mutant PrP undergo conformational changes and are particularly intriguing, because the normal isoform PrPc is soluble in nondenaturing detergents and is readily digested by proteases, whereas PrPSc is insoluble and resistant to proteolytic digestion. Our findings indicate that insoluble wtPrP represents a conformational intermediate, the first to be identified, within a pathway in which PrPc is converted to PrPSc.