The role of the p33:p33/p92 interaction domain in RNA replication and intracellular localization of p33 and p92 proteins of Cucumber necrosis tombusvirus

Replication of plus-stranded RNA viruses is performed by the viral replicase complex, which, together with the viral RNA, must be targeted to intracellular membranes, where replication takes place in membraneous vesicles/spherules. Tombusviruses code for two overlapping replication proteins, the p33 auxiliary protein and the p92 polymerase. Using replication-competent fluorescent protein-tagged p33 of Cucumber necrosis virus (CNV), we determined that two domains affected p33 targeting to peroxisomal membranes in yeast: an N-proximal hydrophobic trans-membrane sequence and the C-proximal p33:p33/p92 interaction domain. On the contrary, only the deletion of the p33:p33/p92 interaction domain, but not the trans-membrane sequence, altered the intracellular targeting of p92 protein in the presence of wt p33 and DI-72(+) RNA. Moreover, unlike p33, p92 lacking the trans-membrane sequence was still functional in supporting the replication of a replicon RNA in yeast, whereas the p33:p33/p92 interaction domain in both p33 and p92 was essential for replication. In addition, p33 was also shown to facilitate the recruitment of the viral RNA to peroxisomal membranes and that p33 is colocalized with (+) and (-)-stranded viral RNAs. Also, FRET and pull-down analyses confirmed that p33 interacts with other p33 molecules in yeast cells. Based on these data, we propose that p33 facilitates the formation of multimolecular complexes, including p33, p92, viral RNA, and unidentified host factors, which are then targeted to the peroxisomal membranes, the sites of CNV replication.     

Eculizumab treatment for rescue of renal function in IgA nephropathy

Background

Immunoglobulin A (IgA) nephropathy is a chronic glomerulonephritis with excessive glomerular deposition of IgA1, C3 and C5b-9, which may lead to renal failure.

Case Diagnosis/Treatment

We describe the clinical course of an adolescent with rapidly progressive disease leading to renal failure in spite of immunosuppressive treatment. Due to refractory disease the patient was treated with eculizumab (anti-C5) for 3 months in an attempt to rescue renal function. Treatment led to clinical improvement with stabilization of the glomerular filtration rate and reduced proteinuria. Discontinuation of treatment led to a rapid deterioration of renal function. This was followed by a single dose of eculizumab, which again reduced creatinine levels temporarily.

Conclusions

Early initiation of eculizumab therapy in patients with progressive IgA nephropathy may have a beneficial effect by blocking complement-mediated renal inflammation.     

Survival of patients with head and neck squamous cell carcinoma in association with human papillomavirus and p53 polymorphism

Abstract

Survival of patients with head and neck squamous cell carcinoma (HNSCC) is dependent on many factors — stage of the disease, treatment regimen, operation technique etc. Many authors discuss on association of survival with various biomarkers as HPV infection, p53 mutation and polymorphism or p16 expression. The objective of our study was to analyze the survival of HNSCC patients in association with HPV infection and p53 polymorphism.

Methods

39 patients with primary diagnosed HNSCC were investigated. HPV DNA was detected using PCR with general primers MY09/11; p53 polymorphism was analyzed using single nucleotide polymorphism assay by PCR.

Results

Of the 39 patients, 12 (30.8%) had detectable HPV. After p53 polymorphism analysis heterozygous Prol/Arg type was found in 34 cases (87.2%). Survival was higher in laryngeal cancer patients and in patients when tumour was classified as T_1–2. Somewhat higher survival was in the HPV positive patients, however difference was not statistically significant (P = 0.7). Only significant factor influencing survival in our study group was site of primary tumour (P < 0.05).

Conclusion

HNSCC patients’ survival in our study depend on primary tumour site; HPV infection and p53 SNP was not associated with better survival.

     

Spectrophotometric analysis of tooth discolouration induced by mineral trioxide aggregate after final irrigation with sodium hypochlorite: An in vitro study

The aim of this study was to evaluate specific chromatic alterations induced by white mineral trioxide aggregate after final irrigation with sodium hypochlorite. Sixty specimens were prepared mechanically and filled with mineral trioxide aggregate after different final irrigation protocols: Group 1 – distilled water, Group 2 – sodium hypochlorite followed by distilled water, Group 3 – sodium hypochlorite, only. Colour changes were recorded with a spectrophotometer at baseline, and then after 1, 2 and 4 months. The Commision Internationale de l’éclairage colour system was used and the total colour changes ΔE were calculated. In groups where sodium hypochlorite was used, parameter L* decreased significantly after the first month (Group 2 (P < 0.006), Group 3 (P < 0.009)). Group 3 demonstrated greater colour change ΔE than Group 1, after the first month (P < 0.02). In conclusion, white mineral trioxide aggregate can lead to tooth discolouration by contact with sodium hypochlorite residues in dentinal tubules.     

Occurrence of kidney diseases and patterns of glomerular disease based on a 10-year kidney biopsy material: A retrospective single-centre analysis in Estonia

Abstract Objective. Kidney biopsy is an important diagnostic tool in assessing glomerular damage. This study aimed to determine the occurrence of glomerular disease during the past decade at a single centre, to assess potential changes in the structure of primary glomerulopathies over time, and to define gender- and age-related differences. Material and methods. A total of 578 consecutive native kidney biopsies during the period 2000-2010 was retrospectively reviewed at Tartu University Hospital, Estonia. Biopsies were evaluated according to clinical data with standard histological methods. Results. The patients' mean age was 39.9 ± 17.9 (range 4-87) years. Less than half of informative kidney biopsies (n = 547) comprised primary glomerulopathies (45.4%), the patients' mean age was 38.7 ± 17.7 (4-79) years and the predominant group comprised male patients. Secondary glomerulopathies made up 22.3%, tubulointerstitial diseases 8.2% and other conditions 24.1%. Among primary glomerulopathies, inflammatory damage to glomeruli dominated (63.4%), whereas immunoglobulin A (IgA) nephropathy was the most common disease (35.5%). Non-inflammatory diseases of glomeruli made up 34.6%, among which the most common was focal and segmental glomerulosclerosis (16.1%), followed by minimal change disease (14.1%). Membranoproliferative glomerulonephritis was a rare form of glomerular damage among primary glomerulopathies (7.7%). Comparison between male and female cases in the primary glomerulopathies group revealed a statistically significant difference in their frequency (p = 0.01). Conclusions. Inflammatory glomerulopathies mostly prevailed in the spectrum of primary glomerulopathies. IgA nephropathy was the most common glomerulopathy. Comparing the data with those from a 15-year earlier period at the same centre, a change towards non-inflammatory glomerulopathies was noticed.     

State of HPV16 integration in Lithuanian women with cervical neoplasia

Cervical cancer morbidity and mortality in Lithuania is one of the biggest in the European Union. The main risk factor of cervical cancer is human papillomavirus (HPV). The deletion of the HPV E2 gene influences HPV DNA integration into the cell genome, as well as a rapid progression of cervical lesions. The purpose of this study is to determine HPV, its types, and HPV 16 integration in different grades of cervical intraepithelial neoplasias (CIN). 253 women with cytological lesions were involved in the study. After a histology, 31 women were diagnosed with CIN I, 35 with CIN II, and 51 with CIN III. The biggest prevalence of HPV infection was detected in women younger than 25 years old (69.7%) and in women with CIN II (90.9%). HPV 16 was detected in 67.8% of all cases, with the highest prevalence in CIN III (84.4%). A partial integration form was detected in 65.0% of HPV 16 infected women, a complete virus integration in 26.5%, and an episomal form in 8.4% of cases. Our study concludes that in all the cases confirmed using a histology, the partial virus integration form of CIN was identified the most. It was less frequently detected in CIN I cases (60.0%), but more frequently in CIN II and CIN III cases (72.8 and 69.3%, respectively).     

Evaluation of proteasomal gene polymorphisms in Lithuanian patients with asthma

Objective: To investigate polymorphisms of proteasomal genes PSMA6 (rs1048990 and rs2277460), PSMC6 (rs2295826 and rs2295827) and PSMA3 (rs2348071) in Lithuanian patients with asthma. Methods: One-hundred forty-six asthma patients and 150 control subjects were studied. DNA was extracted from peripheral blood samples. Five single nucleotide polymorphisms (SNP’s) of the three proteasomal genes were analyzed using allele-specific amplification or the cleaved amplified polymorphic sequence method. Results: While certain alleles and genotypes of PSMA6 rs2277460 and rs1048990 and PSMA3 rs2348071 SNP’s occurred more frequently in asthma patients than in controls, no statistically significant differences in alleles or genotypes of PSMA6, PSMC6 or PSMA3 were observed between asthma patients and control subjects. However, when male and female study subjects were considered separately, we found that the CG genotype of PSMA6 rs1048990 is significantly more frequent in male asthma patients compared to male control subjects. Conclusions: We found no significant differences in frequencies of selected five proteasomal gene PSMA6, PSMC6 and PSMA3 SNP’s between asthma patients and control subjects overall. Among male subjects, however, the CG PSMA6 rs1048990 genotype was significantly more frequent in asthma patients compared with control subjects.