Detection and genotyping of oocysts of Cryptosporidium parvum by real-time PCR and melting curve analysis

Several real-time PCR procedures for the detection and genotyping of oocysts of Cryptosporidium parvum were evaluated. A 40-cycle amplification of a 157-bp fragment from the C. parvum beta-tubulin gene detected individual oocysts which were introduced into the reaction mixture by micromanipulation. SYBR Green I melting curve analysis was used to confirm the specificity of the method when DNA extracted from fecal samples spiked with oocysts was analyzed. Because C. parvum isolates infecting humans comprise two distinct genotypes, designated type 1 and type 2, real-time PCR methods for discriminating C. parvum genotypes were developed. The first method used the same beta-tubulin amplification primers and two fluorescently labeled antisense oligonucleotide probes spanning a 49-bp polymorphic sequence diagnostic for C. parvum type 1 and type 2. The second genotyping method used SYBR Green I fluorescence and targeted a polymorphic coding region within the GP900/poly(T) gene. Both methods discriminated between type 1 and type 2 C. parvum on the basis of melting curve analysis. To our knowledge, this is the first report describing the application of melting curve analysis for genotyping of C. parvum oocysts.     

A new model for the induction of tumours in the forestomach of rats by N-methyl-N-nitrosourea

BACKGROUND AND AIM: Experimental carcinogenesis models provide a useful tool in the study of the aetiopathogenesis and treatment of gastric cancer. We developed a model based on the administration of N-methyl-N-nitrosourea (NMU) in Wistar rats for the induction of maximal yield of gastric carcinomas with a short latency period, and being exclusively localized at the gastric level. METHODS: A gastric antiperistaltic fistula was performed in 90 Wistar rats classified into eight different groups. Fifteen days after surgery 5, 10, 15 or 20 mg of NMU/100 g were administered through the fistula once a week for a 3- to 5-week period. Before the administration of NMU, a pyloric blockade was made in order to obtain a temporary isolation of the stomach. At 20 weeks, animals were sacrificed and organs were removed for histological study. RESULTS: All rats treated with 15 mg NMU/100 g once a week for 5 weeks, after pyloric blockade maintained for 1 h, developed well-differentiated carcinomas in the forestomach. Carcinomas were multiple in 11% of cases and appeared with papillomatous lesions in 33% of rats. No tumours were observed in any other organs. In the other groups, no gastric carcinomas were diagnosed. CONCLUSION: The high incidence of carcinomas in the forestomach, the absence of tumours in other organs and the short latency period represent valuable criteria for the use of our model in chemotherapeutic investigations, as well as in the study of cancer evolution without interferences caused by tumour development in other organs.     

Monoamine oxidase inhibitory activities of the scorpion Mesobuthus gibbosus (Buthidae) venom peptides.

In the present study, crude venom of Mesobuthus gibbosus (Buthidae), a scorpion distributed all over Anatolia was isolated and purified by the Sephadex G-50 gel filtration and high pressure liquid chromatographic (HPLC) separation. Two of the five fractions (fractions 4 and 5) obtained from the Sephadex G-50 filtration and detected as lethal on mice and Musca domestica larvae in in vivo toxicity tests, were independently subjected to the HPLC separation. Only one of seven fractions (fraction 5.5*) obtained from the HPLC separation of the fraction 5 was found to be extremely lethal. Sodium dodecylsulfate polyacrylamide gel electrophoretic (SDS-PAGE) analysis of the crude venom and its chromatographic fractions demonstrated that crude venom consisted of peptides with molecular weights of 6500-210,000 Da. The neurotoxic fraction 5.5* appeared as a single band of 28,000 Da and two bands of 6200 and 22,000 Da in SDS-PAGE under non-reducing and reducing conditions, respectively, suggesting that it might consist of two chains attached by a disulfide bridge. Fractions 5 and 5.5* inhibited monoamine oxidase A (MAO-A) of rat liver reversibly and non-competitively, in a concentration-dependent manner. Fraction 5.5* appeared as a potent and specific MAO-A inhibitor with a Ki value of 0.12 mg venom proteinml(-1). The inhibitory effect of venom peptide 5.5* on MAO-A was found to be dependent on the preincubation time suggesting that the peptide binds to some site other than the substrate-binding site. Results of the present study demonstrated that M. gibbosus venom contains a peptide with specific MAO-A inhibitory activity which may be responsible for the anxiogenic effects of the scorpion venoms on animals and humans.     

Vascular Endothelial Growth Factor Levels in Childhood Acute Lymphoblastic and Myeloblastic Leukemia

Angiogenesis has been associated with the growth, dissemination and metastasis and has been shown to be a prognostic. Although there are some data suggesting that angiogenesis may have a role in the pathophysiology of leukemia, its role in patient prognosis is yet to be defined. We analyzed the expression level of vascular endothelial growth factor (VEGF), an angiogenesis promoter and its possible- prognostic value in bone marrow samples at the time of diagnosis and remission of acute childhood leukemia patients. Besides 46 patients diagnosed as ALL or AML, 16 children were also included as a control group in the study. Our data have demonstrated that VEGF levels of AML patients were found higher than the control group statistically (P = 0.022). However we could not find any significant difference between VEGF levels of diagnosis and remission in both AML and ALL groups by blastic VEGF expression (P > 0.05). In this study the higher levels of VEGF in AML patients is one of the main findings although we were not able to assess any role of VEGF in predicting prognosis in pediatric leukemia patients by evaluating blastic cell VEGF expression. These results have demonstrated that the relationship between angiogenesis or angiogenesis promoters and hematological malignancies is not clear and simple as different methods or different cells beside different angiogenesis promotors are involved to these studies. So that not only tumor cells and their cytokines but also surrounding cells and their cytokines must be taken into consideration with the standardized study methods in the further studies to obtain a promising treatment approach.     

Collagen for brain repair:therapeutic perspectives

Biomaterials have increasingly become a focus of research on neuroprotection and neuroregeneration.Collagen,in terms of brain repair,presents many advantages such as being remarkably biocompatible,biodegradable,versatile and non-toxic.Collagen can be used to form injectable scaffolds and micro/nano spheres in order to:(i) locally release therapeutic factors with the aim of protecting degenerating neurons in neurodegenerative conditions such as Alzheimer's or Parkinson's diseases,(ii) encapsulate stem cells for safe delivery,(iii) encapsulate genetically modified cells to provide a long term source of trophic factors,(iv) fill in the voids from injury to serve as a structural support and provide a permissive microenvironment to promote axonal growth.This mini-review summarizes different applications of collagen biomaterial for central nervous system protection and repair,as well as the future perspectives.Overall,collagen is a promising natural biomaterial with various applications which has the potential to progress the development of therapeutic strategies in central nervous system injuries and degeneration.     

Factors Predicting Iliac Metastasis and Overall Survival in Malignant Melanoma of the Lower Extremities

Background : To analyse the patient-related, disease-related and treatment-related factors in a group of melanoma patients to assess their impact on iliac metastasis and on overall survival.

Methods : Medical records of thirty nine patients with lower extremity malignant melanoma were retrospectively reviewed to confirm all of the clinical data. Age and gender were recorded as patient-related factors. Tumor location, size, histology, ulceration status, and TNM stage, Breslow thickness, Clark level, presence of inguinal LN metastases, and locoregional metastases (local recurrences, in transit metastases and regional LN metastases) were evaluated as disease-related factors. Type of surgery (en block excision of primary tumor, en block excision of primary tumor and inguinal lymph node dissection, en block excision of primary tumor and ilioinguinal lymph node dissection) and postoperative chemotherapy were taken into account as treatment-related factors.

Results : The presence of inguinal lymph node metastases was significantly associated with iliac metastasis (p = 0.015). Tumor size (p = 0.046), tumor TNM stage (p = 0.009), Breslow thickness (p = 0.033), Clark level (p = 0.029), presence of in transit metastases (p = 0.010) and postoperative chemotherapy (p = 0.002) has been related to impaired overall survival rate.

Conclusions : Therapeutic lymph node dissection appears to carry a small but definite therapeutic benefit. Selection of appropriate patients for the more extensive procedure would be ideal, but at present there are no well proven selection criteria. The authors advocate therapeutic dissection when the inguinal lymph nodes are involved.     

Effects of impacted maxillary canines on root resorption of lateral incisors

Objectives

The aim of this study was to analyze the amount of root resorption of maxillary lateral incisors by relating the position, location, and angulation of the impacted canine using cone-beam computed tomography (CBCT).

Materials and methods

The study sample consisted of panoramic and CBCT radiographs of 46 patients with a unilateral impacted canine (16 males and 30 females; mean age: 19.53 ± 6.66 and 19.44 ± 5.77 years, respectively). Sector location and canine angulation were measured in panoramics. All tomographs were obtained using CBCT (NewTom 5G, QR, Verona, Italy) and three-dimensional (3D) reconstructions of the maxillary laterals assessed by Mimics 14.01 image analysis software.

Results

Upper lateral incisor volume was smaller on the impacted side (401.95 ± 83.69 mm³) than on the nonimpacted side (433.54 ± 92.6 mm³, P < 0.05). There were no significant differences of lateral root resorption volume when comparing the impacted canines being on the labial or palatal sides (P > 0.05), but impacted canine angulation was significantly steeper on the labial side (70.85°) than on the palatal side (46.09°, P < 0.05). The volume of root resorption of laterals when comparing the various positions of the canine in different sectors or canine angulation in 30^o intervals was not statistically significantly different (P > 0.05).

Conclusions

The impacted canines caused root resorption of lateral incisors. The angulation of the canine was steeper on the labial side than on the palatal side but root resorption of adjacent laterals was not different. There were no statistically significant differences in the amount of root resorption of the laterals when the canine was evaluated according to localization and angulation.

     

What might cervical vestibular–evoked myogenic potential abnormalities mean in essential tremor?

Aim/background

Essential tremor (ET) is one of the most common movement disorders. However, its pathogenesis is unclear. Human vestibular reflexes are essential not only for gait and posture but also for goal-directed voluntary movements. In this study, cervical vestibular–evoked myogenic potentials (cVEMPs), the electrophysiological equivalent of the vestibulo-collic reflex was studied in ET patients to understand the interaction between the tremor network and the vestibular neural pathways.

Methods

cVEMPs were recorded in 40 ET patients and 40 age and sex-matched healthy controls (HCs). The latencies of peaks p13 and n23 and peak-to-peak amplitude of p13-n23 were measured.

Results

There was no statistically significant difference between the p13 latencies of the HC and ET groups (p 0.79 and p 0.23 for the right and left sides respectively). n23 latency was shortened bilaterally in the ET group (p 0.009 and p 0.02 for the right and left sides respectively). p13-n23 amplitudes of the ET patients were bilaterally reduced when compared with the HC (p <0.001 and p 0.001 for the right and left sides respectively).

Conclusion

Information provided by vestibular afferents is crucial in the control of voluntary movements in humans. Despite this silent but significant effect, the role of the vestibular system in movement disorders is often overlooked. In this study, it was found that cVEMP responses reflecting the activity of the vestibulo-collic pathway were affected in ET which can be either caused by dysfunctional structures or pathways responsible from ET or an additional disorder of vestibular information processing in these patients.