Circulating microRNAs associated with liver fibrosis in chronic hepatitis C patients.

A major challenge in hepatitis C research is the detection of early potential for progressive liver disease. MicroRNAs (miRNAs) are small RNAs that regulate gene expression and can be biomarkers of pathological processes. In this study, we compared circulating miRNAs identified in hepatitis C virus (HCV)-infected patients presenting two extremes of liver disease: mild/moderate fibrosis and cirrhosis. The patients in the cirrhosis group subsequently developed hepatocellular carcinoma (HCC). We identified 163 mature miRNAs in the mild/moderate fibrosis group and 171 in the cirrhosis group, with 144 in common to both groups. Differential expression analysis revealed 5 upregulated miRNAs and 2 downregulated miRNAs in the cirrhosis group relative to the mild/moderate fibrosis group. Functional analyses of regulatory networks (target gene and miRNA) identified gene categories involved in cell cycle biological processes and metabolic pathways related to cell cycle, cancer, and apoptosis. These results suggest that the differentially expressed circulating miRNAs observed in this work (miR-215-5p, miR-483-5p, miR-193b-3p, miR-34a-5p, miR-885-5p, miR-26b-5p and miR -197-3p) may be candidates for biomarkers in the prognosis of liver disease.

Minimally Invasive Adhesive Rehabilitation for a Patient With Tooth Erosion: Seven-year Follow-up.

Tooth wear is a multifactorial condition of growing concern. In clinical practice, it is often a challenge for prevention and treatment since many etiological factors may be involved. This case report describes an esthetic rehabilitation of a young patient presenting tooth wear due to erosion. The etiological factor of this case was the patient sucking on lemons, an acidic fruit. The main complaint of the patient was the appearance of short maxillary incisors. The treatment involved orthodontic and restorative dentistry techniques. First of all, an orthodontic device was used to increase the vertical dimension of occlusion and create an adequate space for the direct restorations. The posterior teeth were restored with two direct composite resin techniques; the anterior teeth were then restored using a balanced occlusion. The seven-year follow-up of the case is presented. Replicas of the restorations were made and visualized under scanning electron microscopy up to the 12-month evaluation. In the clinical follow-up at seven years, maintenance of the results and restorations can be clearly seen.

Proximal giant neurofilamentous axonopathy in mice genetically engineered to resist calpain and caspase cleavage of α-II spectrin.

We use 1,2-diacetylbenzene (1,2-DAB) to probe molecular mechanisms of proximal giant neurofilamentous axonopathy (PGNA), a pathological hallmark of amyotrophic lateral sclerosis. The spinal cord proteome of rodents displaying 1,2-DAB PGNA suggests a reduction in the abundance of α-II spectrin (Spna2), a key protein in the maintenance of axonal integrity. Protein immunoblotting indicates that this reduction is due to Spna2 degradation. We investigated the importance of such degradation in 1,2-DAB PGNA. Spna2 mutant mice lacking a calpain- and/or caspase-sensitive domain (CSD), thus hypothetically resistant to 1,2-DAB, and wild-type littermates, were treated with 1,2-DAB, 35 mg/kg/day, or saline control, for 3 weeks. 1,2-DAB induced motor weakness and PGNA, irrespective of the genotype. Spna2-calpain breakdown products were not detected in mutant mice, which displayed a normal structure of the nervous system under saline treatment. Intriguingly, treatment with 1,2-DAB reduced the abundance of the caspase-specific 120-kDa Spna2 breakdown products. Our findings indicate that degradation of Spna2 by calpain- and/or caspase is not central to the pathogenesis of 1,2-DAB axonopathy. In addition, the Spna2-CSD seems to be not required for the maintenance of the cytoskeleton integrity. Our conceptual framework offers opportunities to study the role of calpain-caspase cross talk, including that of the protease degradomics, in models of axonal degeneration.

Densitometry, radiography, and histological assessment of collagen as methods to evaluate femoral bones in an experimental model of osteoporosis.

SUMMARY: All methods to detect experimental loss of bone present technique limitations. The sensitivities of image and histological analyses to detect the effects of teriparatide in rats with bone loss after ovariectomy were evaluated. All methods were qualitatively valid. INTRODUCTION: The standardization of methods to assess bone loss after ovariectomy is crucial to establish the degree of experimental osteoporosis. In general, methods per image or histological techniques are used. To validate these two ways to determine the degree of bone loss in ovariectomized rats, we evaluated the sensitivities of bone densitometry, conventional radiography, and histological analysis of the area occupied by collagen, detecting the effects of teriparatide treatment in the femur of ovariectomized rats with bone loss. METHODS: Wistar rats were divided into three groups: a control group, in which the animals were only subjected to laparotomy; an ovariectomized group, in which bilateral removal of the ovaries was performed; and an ovariectomized + teriparatide group, in which bilateral removal of the ovaries was performed, and the animals were treated with 3 µg/100 g/day of teriparatide. Three months following the ovariectomy, bone densitometry, radiographic densitometry, and histological analysis of the area occupied by collagen fibers were carried out in the femur diaphysis. RESULTS: The bone densitometry revealed 11.2% reduction in femur density; in the conventional radiography, the loss of bone mass was 14.5%, and with the histological analysis, a 40.9% reduction in the area occupied by collagen was detected in the femur diaphysis. CONCLUSIONS: In conclusion, histological analysis could not be quantitatively compared with the methods of bone densitometry and conventional radiography; however, all of these methods were qualitatively valid for assessing the loss of bone stemming from ovariectomy and the therapeutic effect of teriparatide in the prevention of osteoporosis.

Composition and color stability of carbon monoxide treated dried porcine blood.

Color stability of swine blood was studied over 12 weeks of storage in plastic bags, after pH (7.40, 6.70, or 6.00) adjustment, saturation with carbon monoxide (CO) and spray-drying. CO-treated dried blood presented a redder color and higher reflectance between 610 and 700 nm, compared to a brownish-red color and lower reflectance of untreated samples. As indicated by reflectance spectra, blood pH adjustment did not influence (P>0.05) the initial color of dried blood but influenced (P<0.05) its color stability (browning index). During storage, CO-treated blood showed a reduction in reflectance percentages as well as in CIE L(*) and a(*) values, which was more pronounced in polyethylene (OTR=4130 cm(3)/m(2)/day/atm) packaged samples. After 12 weeks of storage, CO-treated samples packaged in high OTR bags presented color indexes similar to those of the untreated dried samples. CO-treated samples packaged in nylon-polyethylene (OTR=30-60 cm(3)/m(2)/day/atm) bags showed a smaller rate of discoloration and color difference (DeltaE(*)) between the CO-treated and untreated samples. Even with some darkening, packaging CO-treated dry blood in low OTR bags still gives an acceptable reddish color after 12 weeks of storage while untreated dry blood has a brownish color just after drying.

DNA brain damage after stress in rats.

OBJECTIVE: The purpose of this study was to verify the presence of DNA brain lesion after acute stress in rats. METHOD: Adult male Wistar rats were divided into 3 groups according to the stressor (control, forced swimming or restraint), and sampled at 2 time points: immediately or 1week after stress. Trunk blood and the brain areas (prefrontal cortex, amygdala and hippocampus) were extracted for DNA analysis by the comet assay. The cells were classified according to the damage index and damage frequency based on the comet tail size. RESULTS: Immediately after the stress, DNA damage was detected in the amygdala area and in the hippocampus after restraint and forced swimming. In the prefrontal cortex, DNA was damaged after forced swimming. However, no alteration was seen in blood. Seven days after the stress, DNA damage was still identified in the hippocampus after forced swimming and restraint, whereas no alteration was detected in the other brain areas or in blood. CONCLUSION: One week after a single stressful event, a reversible DNA damage was identified in the prefrontal cortex and in the amygdala, whereas DNA damage in the hippocampus still remained.

Evaluation of memory immune response to mycobacterium extract among household contact of tuberculosis cases.

The human immune response to tuberculosis (TB) is especially mediated by T CD4(+)lymphocytes. However, more studies are needed in order to understand the exact role of each cytokine in the mechanisms for cures. In this article, our aim was to analyze the production of TNF-alpha, IL-10, and IFN-gamma in peripheral blood mononuclear cells (PBMCs) among the household contacts of common primary TB cases, with or without histories of active TB infection, who were negative to parasitological and HIV tests. In order to characterize the cytokine production, PBMCs from these groups were stimulated with whole-protein extract of M. tuberculosis (WPE) antigen (rAgTb) for 24 and 48 hr. The culture supernatants were collected and IFN-gamma, TNF-alpha, and IL-10 were assayed using capture ELISA. There were no statistical differences between primary TB cases and their household contacts with or without previous histories of lung TB. Our results suggest that T memory cells, T regulatory cells, and the Th1/Th2 dichotomy may be responsible for the results described in this article. Further studies are currently underway.

Eosinophil cationic protein damages protoscoleces in vitro and is present in the hydatid cyst.

Eosinophils are locally recruited during the establishment and chronic phases of cystic hydatidosis. This study provides evidence that eosinophil cationic protein (ECP), one of the major components of eosinophil granules, can damage Echinococcus granulosus protoscoleces (PSC). The toxicity of ECP was investigated in vitro by following parasite viability in the presence of this protein. ECP was found to damage PSC at micromolar concentrations; the effect was blocked by specific antibodies and heparin, and was more severe than the one caused by similar concentrations of RNase A, suggesting that the cationic nature of ECP, and not its ribonuclease activity, is involved in toxicity. This observation may highlight the capacity of eosinophils to control secondary hydatidosis, derived from PSC leakage from a primary cyst. To further assess the relevance of the previous result during infection, the presence of eosinophil proteins was investigated in human hydatid cysts. ECP was found to be strongly associated with the laminated layer of the cyst wall, and present at micromolar concentrations in the hydatid fluid. Overall, these results demonstrate that eosinophils degranulate in vivo at the host-parasite interface, and that the released ECP reaches concentrations that could be harmful for the parasite.

[Management of lymphoid hyperplasia of the ocular adnexa]. / Manejo de las hiperplasias linfoides orbitarias.

OBJECTIVE: Lymphoid Hyperplasia (LH) is a benign proliferative lymphocyte-rich process. Histologically, LH may be similar to a lymphoma. Its incidence has decreased because of new sophisticated diagnostic procedures and a better knowledge of lymphomas. Most of the cases are seen in patients between forty and sixty years of age. As there was no consensus about the best treatment, determining this was the objective of our study. METHOD: We describe three patients with a lymphoproliferative process affecting the ocular anexae, in whom histologic, immuno-histochemical and molecular studies were performed. PCR analysis allowed a definitive diagnosis to be made. RESULTS: Histologically, all three cases were diagnosed as LH; immuno-histochemically, one was diagnosed as an idiopathic orbital inflammation, and the other two, as LH. After PCR analysis, one of these latter two cases was definitively diagnosed as B-cell Non-Hodgkin's lymphoma. CONCLUSIONS: Today's molecular techniques allow us to make a definite diagnosis of lymphoma, because sometimes histology and immuno-histochemistry alone can result in a wrong diagnosis being made. LH of the ocular adnexae may be the first stage of a much more serious disease, and a benign hyperplasia at this site must lead to suspicion of lymphoma, which can then be diagnosed or excluded by the improved diagnostic procedures. Therefore, when we make a diagnosis of LH it is necessary to complete a full evaluation and institute a defined follow-up of the patient's clinical condition.

The efficacy of a fluoride varnish in reducing enamel demineralization adjacent to orthodontic brackets: an in vitro study.

OBJECTIVES: To test the hypothesis that fluoride varnish is effective in reducing demineralization (white spot) lesions adjacent to bonded orthodontic brackets. DESIGN: Two similar samples of extracted bovine incisors, with bonded orthodontic brackets, were separated into an experimental group (fluoride varnish was applied) and control group (no fluoride varnish) to examine the preventive effects of fluoride varnish. SETTING AND SAMPLE POPULATION: The dental clinic of the State University of Maringá--UEM (Maringá, Paraná, Brazil). Thirty-eight extracted bovine incisors with bonded orthodontic brackets. EXPERIMENTAL VARIABLE: Fluoride varnish was applied topically to half of the sample of extracted bovine teeth. No varnish was applied to the other half. OUTCOME MEASURE: The depths of enamel demineralization (white spot) lesions were measured from polarized light microscopy images using image analysis software. RESULTS: The teeth in both the experimental and control groups had been exposed to a cariogenic environment twice a day for 35 days. Those teeth that had been treated with two applications of fluoride varnish (one at the outset and another 15 days later) demonstrated about 38% less mean lesion depth than teeth where no varnish had been applied. CONCLUSION: Orthodontists may wish to consider the application of fluoride varnish during fixed orthodontic therapy to help reduce the development of enamel white spot lesions.

Glutathione peroxidase induction protects Saccharomyces cerevisiae sod1deltasod2delta double mutants against oxidative damage.

Saccharomyces cerevisiae mutants deficient in superoxide dismutase genes (sod1delta, sod2delta and the double mutant) were subjected to H2O2 stress in the stationary phase. The highest sensitivity was observed in the sod2delta mutant, while the sod1deltasod2delta double mutant was not sensitive. Sod mutants had lower catalase activity (44%) than wild-type cells, independent of H2O2 stress. Untreated cells of sod1deltasod2delta double mutants showed increased glutathione peroxidase activity (126%), while sod1delta had lower activity (77%) than the wild type. Glutathione levels in sod1delta were increased (200-260%) after exposure to various H2O2 concentrations. In addition, the highest malondialdehyde levels could be observed without H2O2 treatment in sod1delta (167%) and sod2delta (225%) mutants. In contrast, the level of malondialdehyde in the sod1deltasod2delta double mutant was indistinguishable from that of the wild type. These results suggest that resistance to H2O2 by sod1deltasod2delta cells depends on the induction of glutathione peroxidase and is independent of catalase, and that glutathione is a primary antioxidant in the defense against H2O2 in stationary phase sod1delta mutants.

Glutathione peroxidase induction protects Saccharomyces cerevisiae sod1deltasod2delta double mutants against oxidative damage

Saccharomyces cerevisiae mutants deficient in superoxide dismutase genes (sod1delta, sod2delta and the double mutant) were subjected to H2O2 stress in the stationary phase. The highest sensitivity was observed in the sod2delta mutant, while the sod1deltasod2delta double mutant was not sensitive. Sod mutants had lower catalase activity (44 percent) than wild-type cells, independent of H2O2 stress. Untreated cells of sod1deltasod2delta double mutants showed increased glutathione peroxidase activity (126 percent), while sod1delta had lower activity (77 percent) than the wild type. Glutathione levels in sod1delta were increased (200-260 percent) after exposure to various H2O2 concentrations. In addition, the highest malondialdehyde levels could be observed without H2O2 treatment in sod1delta (167 percent) and sod2delta (225 percent) mutants. In contrast, the level of malondialdehyde in the sod1deltasod2delta double mutant was indistinguishable from that of the wild type. These results suggest that resistance to H2O2 by sod1deltasod2delta cells depends on the induction of glutathione peroxidase and is independent of catalase, and that glutathione is a primary antioxidant in the defense against H2O2 in stationary phase sod1delta mutants.

Isodicentric Y chromosomes and secondary microchromosomes.

We report here on two mosaic patients with both an idic(Yp) and a microchromosome. FISH with the DYZ3 alphoid repeat demonstrated that the isodicentrics effectively exhibited two alphoid clusters whereas the small markers had a Y-centromere. These data, along with 4 previous observations, indicate that such microchromosomes effectively result from functional dicentricity of isodicentric Y-chromosomes and represent the excision of one centromere plus various amounts of adjacent chromatin. Other than a real infrequency of such a concurrence or a very low proportion of the cell line(s) containing the microchromosome, the paucity of observations points to a high rate of underdiagnosis as revealed by two idic(Y) instances in which the microchromosome was detected only in samples assessed by FISH.

In search of a 9q13 latent centromere in 9qh polymorphic inversions.

In search of a 9q13 latent centromere in 9qh polymorphic inversions: The presence of alphoid sequences in 9q13 has prompted the suggestion that such a region could harbor a latent centromere which under certain circumstances may appear as a neocentromere. We tested this hypothesis by means of FISH with a centromere 9-specific alphoid probe in lymphocyte metaphases from 13 unrelated individuals with a 9qh polymorphic inversion. Since all inverted chromosomes had the alphoid signal onto the primary constriction, it was not possible to identify any neocentromere . We believe, however, that the number of cases was not enough to conclude that all the polymorphic inversions of chromosome 9 are genuine.

inv(9)(p24q13) in three sterile brothers.

Only nine non-polymorphic constitutional pericentric inversions of chromosome 9 have been described. We report on a familial inv(9)(p24q13) associated with sterility in three brothers. The mother's chromosomes were normal in blood lymphocytes (n=130); the father was already deceased and his karyotype unknown. However, the presence of any of the maternal chromosomes 9 (as assessed by C-banding) in her carrier children is inconsistent with the assumption of maternal mosaicism. Two single sisters were also carriers. The same rearranged chromosome 9 in the three sterile brothers can hardly be regarded as a fortuitous association, especially when the breakpoints are almost identical to those of the sole inversion previously found in an azoospermic male. If their father was a carrier, the observed sterility may be the result of 'chromosome anticipation', a phenomenon already invoked for certain familial chromosomal rearrangements.

An extra idic(21)(q22.1) in a child with some features of Down's syndrome.

A 30-month-old boy with mental retardation, hypotonia, joint hyperlaxity, Brushfield spots, open mouth, distal axial triradius t", and ulnar loops on both forefingers was found to have a 47,XY, + psu idic(21)(q22.1).ish psu idic(21)(q22.1)(D13Z1/D21Z1 + + ,ETS2-) karyotype. The patient's phenotype, with only some Down's syndrome (DS) features, is probably related to his disomy for most or all of the critical region 21q22.2 q22.3 and agrees with the current notion that certain DS features may also result from 21q proximal duplications. The phenotypical comparison with 2 other patients with a similar extra idic(21) reveals some discrepancies, which may be related to the inherent clinical variability of similar imbalances: yet, a real difference between the tetrasomic segments cannot be excluded. Noticeably, all 3 patients with 21q proximal tetrasomy did not have cardiac defect and exhibited none or just one out of the five other DS phenotypic features attributed to a single gene or cluster on distal 21q22.

Single cell chromosome rearrangements in indivuduals with reproductive failure

Single cell chromosome rearrangements (SCCR) are incidental findings in cell cultures. Nevertheless, some authors have implicated them in habitual abortion. Ninety individuals classified in four groups were prospectively studied: A) individuals with spontaneous abortions, malformed children or molar pregnancies (N = 36); B) azoospermic males (N = 14); C) individuals with one or more children with either de novo or inherited, constitutional, chromosome abnormalities (N = 26), and D) individuals with healthy children and no reproductive failure (control group, N = 14). Lymphocyte chromosome preparations were stained for GTG bands, and 50-100 metaphases were scored per individual. The rearrangements observed were translocations, deletions, duplications, isochromosomes, rings, fragments and markers. Thirty-four individuals (21 males, 13 females) had a range of 1-5 SCCR. Four had rearrangements only of chromosomes 7 and 14. SCCR frequency (with the exception of rearrangements involving chromosomes 7 and 14) was 0.0063, while that of rearrangements between chromosomes 7 and 14 was 0.0010. Statistical intergroup comparisons c2 with Yates correction) did not show significant differences. Hence, the occurrence of SCCR in our sample was found to be independent of ascertainment mode and sex.

Distribution of Defense-Related Enzymatic Activities in the Quiescent Organs of Phaseolus vulgaris L. Seeds.

Analysis of quiescent seed extracts of Phaseolus vulgaris cultivars found high activities of chitinase, N,N'-diacetylchitobiase and ß-N-acetylhexosaminidase in whole seeds and their dissected organs (cotyledons, axis, and seed coat). Activities of these enzymes were compared in seeds of two cultivars phenotypically distinguishable by soft white (cv. Surattowonder) and hard brown (cv. Maisugata) seed coats. In both cultivars, chitinase activity was found high in all organs, chitobiase in the seed coat, and ß-N-acetylhexosaminidase mainly in the axis. In terms of specific activity, all three enzymes were extraordinarily higher in extracts of seed coat than the others, specially referring to the cultivar Surattowonder. Although the cultivars showed in general similar distribution patterns of activities among their seed organs, the discrepancies found between them seem to be expressing intrinsic attributes of their seed coats. The relationship between the two cultivars, the enzyme activities measured and defense mechanisms are discussed.

Centromeric association of a microchromosome in a Turner syndrome patient with a pseudodicentric Y.

A 12-year-old patient with Turner syndrome was found to have a complex mosaicism for a microchromosome (MC) and a psu dic(Y)(q11). The MC was smaller than Yp, appeared pale in G, C and late replicating bands, had a pair of small centromeric dots, was associated with other chromosomes in most metaphases, and was rather stable both in size and during mitosis. The psu dic(Y) was Cd-positive only at the active centromere, had two pericentromeric heterochromatic regions, and lacked the Yq12 band. No cells with both abnormal chromosomes were found. To evaluate the association of the MC with all ordinary chromosomes, 857 G-banded cells with the marker were screened. The MC was considered as "associated" whenever the distance between it and other chromosome(s) was equal to, or smaller than, 18p. Out of 848 associations registered, 489 (57.7%) were centromeric, 202 (23.8%) telomeric, and 157 (18.5%) interstitial; i.e., centromeric associations were overrepresented (P < 0.001) and showed a random distribution, except for an excessive involvement of chromosome 8. This association pattern, also exhibited by two similar MCs in human beings, the minute Y of a marsupial and certain B chromosomes in plants, probably reflects the Rabl orientation of chromosomes in interphase.

Mental retardation, microbrachycephaly, hypotelorism, palpebral ptosis, thin/long face, cleft lip, and lumbosacral/pelvic anomalies.

We describe 2 Brazilian sisters with a combination of clinical signs strongly suggesting a new autosomal recessive MCA/MR syndrome.