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We describe a strategy for the development of a rational approach of neoplastic disease therapy based on the demonstration that scale-free networks are susceptible to specific attacks directed against its connective hubs. This strategy involves the (i) selection of up-regulated hubs of connectivity in the tumors interactome, (ii) drug repurposing of these hubs, (iii) RNA silencing of non-druggable hubs, (iv) in vitro hub validation, (v) tumor-on-a-chip, (vi) in vivo validation, and (vii) clinical trial. Hubs are protein targets that are assessed as targets for rational therapy of cancer in the context of personalized oncology. We confirmed the existence of a negative correlation between malignant cell aggressivity and the target number needed for specific drugs or RNA interference (RNAi) to maximize the benefit to the patient's overall survival. Interestingly, we found that some additional proteins not generally targeted by drug treatments might justify the addition of inhibitors designed against them in order to improve therapeutic outcomes. However, many proteins are not druggable, or the available pharmacopeia for these targets is limited, which justifies a therapy based on encapsulated RNAi.

Early stages of cervical cancer in young women need conservative treatments. Electrosurgical therapies (LLETZ, LEEP, SWETZ, NETZ) have been recommended for these women. However, there are recommendations to perform a second excision when the specimen margins are not free of disease. This can lead to some important complications. This article aims to verify the frequency of residual invasive or microinvasive disease after the excisional procedure in women with IA1CC. Data on women with IA1CC diagnosed between 1990 and 2022, were retrieved from medical records. Post-treatment disease was detected during a second surgical procedure or postoperative follow-up. Among the 69 included women, three (4.3 percent; CI95 percent 0-9.2) had residual microinvasive lesions, while none showed invasive disease during a second procedure or follow-up. Only the age of 37 years or more was significantly related to the presence of preinvasive or microinvasive residual lesions. Nearly 80 percent of the women who underwent a second procedure showed no residual lesions. The absence of invasive disease in a second procedure or during the follow-up of these women and the large proportion of women with no residual lesion questions the need for a new surgical procedure even when the surgical margins of the initial specimen are involved.

Background: Managing cervical intraepithelial neoplasia grade 2 (CIN2) is challenging, considering the CIN2 regression rate, perinatal risks associated with excisional procedures, and insufficient well-established risk factors to predict progression., Objectives: To determine the ability of p16INK4a and Ki-67 staining in biopsies diagnosed with CIN2 to identify patients with higher-grade lesions (CIN3 or carcinoma)., Design and Setting: Cross-sectional study conducted at a referral center for treating uterine cervical lesions., Methods: In 79 women, we analyzed the correlation of p16INK4a and Ki-67 expression in CIN2 biopsies with the presence of a higher-grade lesions, as determined via histopathology in surgical specimens from treated women or via two colposcopies and two cytological tests during follow-up for untreated women with at least a 6-month interval. The expression of these two biomarkers was verified by at least two independent pathologists and quantified using digital algorithms., Results: Thirteen (16.8%) women with CIN2 biopsy exhibited higher-grade lesions on the surgical excision specimen or during follow-up. p16INK4a expression positively and negatively predicted the presence of higher-grade lesions in 17.19% and 86.67% patients, respectively. Ki-67 expression positively and negatively predicted the presence of higher-grade lesions in 40% and 88.24% patients, respectively., Conclusions: Negative p16INK4a and Ki67 immunohistochemical staining can assure absence of a higher-grade lesion in more than 85% of patients with CIN2 biopsies and can be used to prevent overtreatment of these patients. Positive IHC staining for p16INK4a and Ki-67 did not predict CIN3 in patients with CIN2 biopsies.

Long-term memory (LTM) formation is dependent on neurochemical changes that guarantee that a recently formed memory (short-term memory [STM]) remains in the specific neural circuitry via the consolidation process. The persistence of recognition memory has been evidenced by using behavioral tagging in young adult rats, but it has not been effective on aging. Here, we investigated the effects of treatment with a standardized extract of Ginkgo biloba (EGb) associated with novelty on the consolidation of object location memory (OLM) and its persistence after weak training of spatial object preference in young adult and aged rats. The object location task used in this study included two habituation sessions, training sessions associated or not associated with EGb treatment and contextual novelty, and short-term or long-term retention testing sessions. Altogether, our data showed that treatment with EGb associated with novelty close to the time of encoding resulted in STM that lasted for 1 h and persisted for 24 h for both young adult and aged rats. In aged rats, the cooperative mechanisms induced robust long-term OLM. Our findings support and extend our knowledge about recognition memory in aged rats and the modulating effects of EGb treatment and contextual novelty on the persistence of memory., (© 2023 de Andrade et al.; Published by Cold Spring Harbor Laboratory Press.)

Background: According to the Brazilian Guidelines on Cervical Cancer Screening, women with cytopathologic diagnosis of high-grade intraepithelial lesion, abnormal colposcopic findings, fully visible squamocolumnar junction and age 25 years or older should be treated at the first visit ("see and treat-S&T"). The main limitation to this approach is the risk of overtreatment, identified by histology without preinvasive lesion. The objectives of this study were to identify the overtreatment rate in women undergoing S&T in cervical cancer prevention at a referral center with extensive experience with the method and to detect possible factors associated with this rate., Methods: This was a cross-sectional study that analyzed records from a database with 616 women submitted to S&T from 1996 to 2017. Negative histology was defined as the following histopathologic results: human papillomavirus without cervical intraepithelial neoplasia (CIN), inflammatory, low-grade squamous intraepithelial lesion, and CIN 1., Results: Of the 616 women, there were 52 (8.44%, 95%CI 6.25-10.64%) with a histopathologic report without preinvasive cervical lesion. No statistical association was found between this outcome and age or a significant downward trend over time., Conclusion: The overtreatment rate in this study can be considered low and consistent with the acceptable rates reported in the literature, reinforcing the prevailing Brazilian guideline, in which the benefits of immediate treatment outweigh the risk of losses following biopsy., (© 2021. The Author(s).)

Background: Cervical cancer screening in Brazil is done using Pap smears. Women who are most likely to have a preinvasive lesion or cervical cancer are immediately referred for colposcopy., Objective: The aim of this study was to evaluate the diagnostic performance of endocervical cytological tests in diagnosing preinvasive cervical lesions in women with initial high-grade squamous intraepithelial lesions (HSIL), or atypical squamous cells in which high-grade lesions could not be ruled out (ASC-H), or atypical glandular cells (AGC), and whose colposcopy did not show any abnormalities, with no fully visible transformation zone (types 2 and 3)., Design and Setting: Retrospective observational study conducted in Rio de Janeiro, Brazil., Methods: Data from women who came to the cervical pathology outpatient clinic between January 2012 and April 2017 were analyzed. The results from endocervical cytological tests were compared with the final diagnosis, which was obtained through examination of a surgical specimen or, among women who did not undergo an excisional procedure, after cytological and colposcopic follow-up for two years., Results: We included 78 women. The sensitivity of endocervical cytological tests was 72.7%; specificity 98.5%; positive and negative predictive values 88.9% and 95.6%, respectively; and positive and negative likelihood ratios 48.7 and 0.28., Conclusion: Endocervical cytological tests are simple, inexpensive and noninvasive, and form a reliable method for determining management among patients with HSIL, ASC-H and AGC cytological findings and negative colposcopic findings without visualization of the squamocolumnar junction.

The cause of epidermodysplasia verruciformis is infection by human papillomavirus, usually types 5 or 8, and it exhibits a high potential for malignant transformation. The diagnostic histologic features of epidermodysplasia verruciformis are not always present and can be mimicked by non-viral diseases. The purpose of this study was to interrogate such lesions for new potential biomarkers to aid in the diagnostic accuracy. HPV DNA was high copy and localized to the upper half of the lesion in cells with cytologic features that included perinuclear halos, blue-grey cytoplasm, and hyper/parakeratosis. Serial section analyses demonstrated that there was increased expression of importin-β, exportin-5, Mcl1, p16, Ki67 and PDL1 in 13/13 epidermodysplasia verruciformis lesions. Each of these proteins localized primarily to the less differentiated cells in the parabasal aspect of the lesion. Only Ki67 and exportin-5 were expressed in the normal epithelia, though much less so, in 13/13 aged matched controls. It is concluded that the host response to HPV 5/8 infection in epidermodysplasia verruciformis includes the up regulation of several proteins including p16, Ki67, importin-β, exportin-5, Mcl1, and PDL1. Thus, these proteins may serve as new biomarkers of this disease that can aid in cases that are equivocal for epidermodysplasia verruciformis on histologic examination., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Importin-β, exportin-5, p16, Ki-67, Mcl1, PDL1, and cFLIP are each over-expressed in the majority of CIN 1 lesions. These biomarkers, plus HPV E6/E7 RNA, were analyzed in carcinoma-in-situ (CIS), microinvasive, and squamous cell carcinoma (SCC) of the uterine cervix and cervical carcinoma cell lines. Only p16 and Ki-67 continued to be over-expressed in CIS, with a concomitant marked increase in E6/E7 RNA. There was a highly significant increase in PDL1 expression and decrease in Ki-67 (each p < 0.001) in microinvasive cancer compared to CIS whereas p16 and E6/E7 remained stable. As the lesion progressed to SCC, p16 and E6/E7 RNA remained strongly overexpressed with a concomitant over expression of importin-β and Ki67. HPV positive Caski cells showed significant elevations of p16, importin-β, exportin-5 and PDL1 compared to the HPV negative cervical cancer cell line C33A, consistent with viral induction of these biomarkers. The data suggest that PDL1 may be a useful biomarker to differentiate CIS from microinvasive cancer and, thus, anti-PDL1 therapy may inhibit the progression of CIS to the invasive stage., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Acute human papillomavirus (HPV) infection of the cervix (cervical intraepithelial neoplasia, CIN) is marked by high copy episomal viral DNA and L1/L2 capsid protein expression (productive infection) in the cells towards the surface that facilitate sexual viral transmission. Viral DNA is low copy and not associated with viral capsid protein expression in the less differentiated lower part of the CIN (nonproductive infection). The purpose of this study was to examine the host response in these two areas. Serial section and co-localization analyses demonstrated that in 29/33 (88%) of cases the NF-κB pathway was activated and localized to the suprabasal nonproductively infected cells in the CIN lesions. There was a concomitant increased expression of importin-β, exportin-5, Mcl1, p16, Ki67 and cFLIP in 32/33 (96%) of CIN lesions that likewise localized primarily to the nonproductively infected cells. Only Ki67 and exportin-5 were expressed, though much less so, in the adjacent, normal squamous epithelia. The viral proteins E1^E4 and L1 were localized to productively infected cells whereas E6/E7 protein/RNA was rarely present in early CIN. It is concluded that the host viral response to acute cervical HPV infection includes strong increased expression of proteins besides p16 and Ki67. These include importin-β, exportin-5, Mcl1, and cFLIP in cells with low copy and relatively quiescent viral DNA that, in turn, may serve as new biomarkers of this disease., (Copyright © 2018 Elsevier Inc. All rights reserved.)

This work focused on immunohistochemistry markers of acute viral infections. Viral infected cells were detected by in situ based methods (reovirus, rabies virus) or cytologic changes (human papillomavirus, molloscum contagiosum virus, herpes simplex virus). Two proteins involved in nuclear trafficking, importin-β and exportin-5, were detected in the infected cells for each virus and not in the control tissues. A wide variety of other proteins, including caspase-3, and bcl-2 family members (bcl2, bclX, MCL1, BAK, BAX, BIM, BAD) showed wide variations in expression among the different viral infections. Specificity of the importin-β and exportin-5 signals varied greatly with different commercially available peroxidase conjugates. It is concluded that immunohistochemistry detection of importin-β and exportin-5 may be useful markers of acute viral infection, which suggests that increased nuclear trafficking may be an important concomitant of viral proliferation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In this work, a natural zeolite, modified and loaded with iron (NZ-A-Fe) as a heterogeneous catalyst, was characterized for its suitability as a permeable reactive barrier (PRB) material for treatment of aromatic hydrocarbons in groundwater. Adsorption and oxidation processes were analyzed. Batch adsorption tests for benzene, toluene and xylene (BTX) aqueous concentrated solutions were performed at neutral pH. Kinetic adsorption was described with the pseudo-second-order model. Experiments were performed using a stirred batch reactor with near 11 mM initial BTX concentration applying NZ-A-Fe as solid catalyst and H 2 O 2 as an oxidant. BTX removal reached 80% in 600 min in these experimental conditions. Catalytic oxidation was described with a pseudo-first-order kinetic model. No significant iron leaching was detected during all the experiences. These investigations show that coupling adsorption with catalytic oxidation with this novel system is a promising procedure to simultaneously remove BTX from moderately concentrated aqueous solution at neutral pH in groundwater.

A major concern associated with ZIKV infection is the increased incidence of microcephaly with frequent calcifications in infants born from infected mothers. To date, postmortem analysis of the central nervous system (CNS) in congenital infection is limited to individual reports or small series. We report a comprehensive neuropathological study in ten newborn babies infected with ZIKV during pregnancy, including the spinal cords and dorsal root ganglia (DRG), and also muscle, pituitaries, eye, systemic organs, and placentas. Using in situ hybridization (ISH) and electron microscopy, we investigated the role of direct viral infection in the pathogenesis of the lesions. Nine women had Zika symptoms between the 4th and 18th and one in the 28th gestational week. Two babies were born at 32, one at 34 and 36 weeks each and six at term. The cephalic perimeter was reduced in four, and normal or enlarged in six patients, although the brain weights were lower than expected. All had arthrogryposis, except the patient infected at 28 weeks gestation. We defined three patterns of CNS lesions, with different patterns of destructive, calcification, hypoplasia, and migration disturbances. Ventriculomegaly was severe in the first pattern due to midbrain damage with aqueduct stenosis/distortion. The second pattern had small brains and mild/moderate (ex-vacuo) ventriculomegaly. The third pattern, a well-formed brain with mild calcification, coincided with late infection. The absence of descending fibres resulted in hypoplastic basis pontis, pyramids, and cortico-spinal tracts. Spinal motor cell loss explained the intrauterine akinesia, arthrogryposis, and neurogenic muscle atrophy. DRG, dorsal nerve roots, and columns were normal. Lympho-histiocytic inflammation was mild. ISH showed meningeal, germinal matrix, and neocortical infection, consistent with neural progenitors death leading to proliferation and migration disorders. A secondary ischemic process may explain the destructive lesions. In conclusion, we characterized the destructive and malformative consequences of ZIKV in the nervous system, as reflected in the topography and severity of lesions, anatomic localization of the virus, and timing of infection during gestation. Our findings indicate a developmental vulnerability of the immature CNS, and shed light on possible mechanisms of brain injury of this newly recognized public health threat.

Controversy still exists over whether the benefits of the available HPV vaccines outweigh the risks and this has suppressed uptake of the HPV vaccines in comparison to other vaccines. Concerns about HPV vaccine safety have led some physicians, healthcare officials and parents to withhold the recommended vaccination from the target population. The most common reason for not administering the prophylactic HPV vaccines are concerns over adverse effects. The aim of this review is the assessment of peer-reviewed scientific data related to measurable outcomes from the use of HPV vaccines throughout the world with focused attention on the potential adverse effects. We found that the majority of studies continue to suggest a positive risk-benefit from vaccination against HPV, with minimal documented adverse effects, which is consistent with other vaccines. However, much of the published scientific data regarding the safety of HPV vaccines appears to originate from within the financially competitive HPV vaccine market. We advocate a more independent monitoring system for vaccine immunogenicity and adverse effects to address potential conflicts of interest with regular systematic literature reviews by qualified individuals to vigilantly assess and communicate adverse effects associated with HPV vaccination. Finally, our evaluation suggests that an expanded use of HPV vaccine into more diverse populations, particularly those living in low-resource settings, would provide numerous health and social benefits.

The discovery of the human papillomavirus (HPV) vaccine illustrates the power of in situ-based pathologic analysis in better understanding and curing diseases. The 2 available HPV vaccines have markedly reduced the incidence of cervical intraepithelial neoplasias, genital warts, and cervical cancer throughout the world. Concerns about HPV vaccine safety have led some physicians, health care officials, and parents to refuse providing the recommended vaccination to the target population. The aims of the study were to discuss the discovery of HPV vaccine and review scientific data related to measurable outcomes from the use of HPV vaccines. The strong type-specific immunity against HPV in humans has been known for more than 25 years. Multiple studies confirm the positive risk benefit of HPV vaccination with minimal documented adverse effects. The most common adverse effect, injection site pain, occurred in about 10% of girls and was less than the rate reported for other vaccines. Use of HPV vaccine should be expanded into more diverse populations, mainly in low-resource settings., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Niemann-Pick type C disease (NPC) is a neurodegenerative genetic disorder caused by accumulation of lipids, especially cholesterol, in the perinuclear space. U18666A is a cholesterol transport-inhibiting agent, being used to mimic NPC, mainly in fibroblasts. The objective of this study was to observe the effect of the drug U18666A, which causes the accumulation of cholesterol in the cytoplasm of astrocytes from newborn rats, on some lysosomal hydrolase activities. Filipin staining and fluorescence microscopy, through CellM software, were used for visualization and quantification of cholesterol. The dose of U18666A that provided the greatest accumulation of cholesterol was that of 0.25 µg/mL in incubation for 48 h. Primary rat astrocytes incubated with the drug (NPC) showed a significantly higher amount of cholesterol than those without U18666A (controls). The measurement of activity of enzymes sphingomyelinase and beta-glucosidase in astrocytes of rats with NPC was significantly lower than that of control astrocytes, which is consistent with the disease in humans. The activity of the enzyme beta-galactosidase showed no significant difference between both groups. We concluded that U18666A appears to be an excellent intracellular cholesterol transport-inhibiting agent affecting some metabolic pathways in astrocytes of young rats, which mimics NPC in these animals. Just like the change in the activity of lysosomal enzymes has been demonstrated, other biochemical parameters of the cell can be tested with this animal model, thus contributing to a better understanding of the disease.

Acroangiodermatitis is an angioproliferative disease usually related to chronic venous insufficiency, and it is considered a clinical and histological simulator of Kaposi's sarcoma (KS). Immunohistochemistry is the suitable method to differentiate between these two entities. It reveals the following immunostaining profile: immunopositivity with anti-CD34 antibody is restricted to the vascular endothelium in acroangiodermatitis, and diffuse in the KS (endothelial cells and perivascular spindle cells); immunopositivity with anti-HHV-8 only in KS cases. We report the case of an HIV seropositive patient without apparent vascular disease, who presented violaceous and brownish erythematous lesions on the feet, and whose histopathology and immunohistochemistry indicated the diagnosis of acroangiodermatitis.

Myxopapillary ependymomas are almost exclusively seen at the conus medullaris/filum terminale/cauda equina region, usually as solitary space-occupying lesions. The authors report the case of a 14-year-old boy with double concomitant myxopapillary ependymoma, proximal and caudal on the filum terminale in which a totally gross removal was achieved in two stages. This presentation is rare and, so far, we have known just three similar cases that were previously reported in children. The true nature of these lesions is controversial, and while some argue that they are related to metastatic seeding, others consider them independent lesions developing synchronously. A review on dissemination of spinal myxopapillary ependymomas was done.

This study aimed to determine the enzymatic activity in dried blood samples collected on filter paper (DBS) for the diagnosis of the following diseases: Fabry, Pompe, Mucopolysaccharidosis type I (MPS I) and Mucopolysaccharosis type VI (MPS VI). DBS was used for high risk patientscreening, according to clinical suspicion. Plasma, leukocytes and cultured fibroblasts were used to confirm the diagnosis when necessary. Among the 529 DBS samples sent to the laboratory, 164 had abnormal results. Confirmatory materials of 73 individuals were rerouted. The frequency of diagnosis for lysosomal storage disorders was 5.9%. DBS is an alternative screening technique used in high risk populations, which should lead to earlier diagnosis for lysosomal storage disorders (LSDs), help patients get treatment sooner and improve the outcome of the disease., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The relevance of 5-HT1A and 5-HT2C receptors of the basolateral nucleus of the amygdala (BLA) in the mediation of anxiety-related defensive responses has long been acknowledged. Whereas strong evidence supports that activation of the latter receptors provokes anxiety, conflicting findings have been reported on the role played by the former binding site. In this study we further investigated the involvement of 5-HT1A receptors (5-HT1A-Rs) in the regulation of anxiety- and panic-related defensive behaviors. The results showed that intra-BLA injection of the 5-HT1A-R agonist 8-OH-DPAT (0.4-16nmol) in male Wistar rats impaired the acquisition of inhibitory avoidance in the elevated T-maze, increased the percentage of time spent in the lit compartment of the light-dark transition model and enhanced the number of punished drinking events in the Vogel conflict test, all changes compatible with an anxiolytic effect. This agonist also impaired escape expression in the elevated T-maze, suggestive of a panicolytic-like effect. 8-OH-DPAT-induced changes in the elevated T-maze and light-dark tests were blocked by previous local administration of the 5-HT1A-R antagonist WAY-100635 (0.37nmol) and were also observed after intra-BLA microinjection of the benzodiazepine receptor agonist midazolam (10-40nmol). Thus, stimulation of 5-HT1A-Rs in the BLA causes both anxiolytic- and panicolytic-like effects, what may have implications for the pathophysiology and treatment of generalized anxiety and panic disorders., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Unlabelled: Cell cycle protein expression plays an important role in the pathophysiology of cervical cancer. However, few studies have attempted to correlate the use of these biomarkers with the clinical progression of the tumor., Objectives: 1) To analyze the expression of Ki-67, p53 and p16(INK4a) in cervical cancer, 2) to correlate the relative expression of these proteins as well as clinical parameters with the stage of disease, and 3) to determine the HPV DNA prevalence and subtype distribution., Methods: Tissue Micro-Arrays (TMA) from patients with invasive cervical cancer (ICC) and controls were analyzed. HPV DNA detection was done by PCR and in situ hybridization. Ki-67, p53 and p16(INK4a) were analyzed by immunohistochemistry; clinical data was derived from the chart review., Results: Advanced tumor stage (III and IV) was strongly associated (p<0.005) with advanced age (>55 years old), with more than four pregnancies and with the lack of formal education. HPV DNA was found in 94.3% of cases with the most prevalent types being HPV16 (67.5%), followed by HPV33 (12.0%) and HPV35 (3.6%). High expression of Ki-67 and p16 was more common in the advanced FIGO stages (p = 0.023). Women with HPV16 tended to be younger (50.9 years; SE 1.9) compared to women with other types (59.9 years; SE 2.8)., Conclusion: We found that Ki-67 and p16 expression were independently associated with the tumor stage. We also noted that about 1/3 of the cervical cancers in this Brazilian cohort were not associated with HPV types directly targeted by the current HPV vaccines.

This study investigates the miniaturization of the screening technique using dried blood spots on filter paper (DBS) to measure GBA and CT activities, and GBA and β-galactosidase activities in leukocytes. 274 DBS from individuals with suspected GD were screened for 1.5 years. Of these, we confirmed the diagnosis in 13.5%. The miniaturization of the DBS and leukocyte techniques afforded to reduce costs and sample size appropriate for a reliable diagnosis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Although several studies have evaluated the role of p16(INK4a) as a diagnostic marker of cervical intraepithelial neoplasia (CIN) and its association with disease progression, studies regarding the role of p16(INK4a) in human immunodeficiency virus (HIV)-infected patients remain scarce. The present study was designed to determine the potential utility of p16(INK4a) as a diagnostic marker for CIN and invasive cervical cancer in HIV-positive and negative cervical specimens. An immunohistochemical analysis of p16(INK4a) was performed in 326 cervical tissue microarray specimens. Performance indicators were calculated and compared using receiving operating characteristics curve (ROC)/area under the curve. In HIV-1-negative women, the percentage of cells that was positive for p16(INK4a) expression was significantly correlated with the severity of CIN (p < 0.0001). A ROC curve with a cut-off value of 55.28% resulted in a sensitivity of 89%, a specificity of 81%, a positive predictive value of 91% and a negative predictive value of 78%. HIV-seropositive women exhibited decreased expression of p16(INK4a) in CIN2-3 specimens compared with HIV-negative specimens (p = 0.031). The ROC data underscore the potential utility of p16(INK4a) under defined conditions as a diagnostic marker for CIN 2-3 staging and invasive cervical cancer. HIV-1 infection, however, is associated with relatively reduced p16(INK4a) expression in CIN 2-3.

Furcal perforations are significant iatrogenic complications of endodontic treatment and could lead to endodontic failure. Mineral trioxide aggregate (MTA) has been regarded as an ideal material for perforation repair, retrograde filling, pulp capping, and apexification. This case report describes a furcal perforation in a maxillary first molar, which was repaired using MTA. The tooth was endodontically treated and coronally restored with resin composite. After 2 years, the absence of periradicular radiolucent lesions, pain, and swelling along with functional tooth stability indicated a successful outcome of sealing the perforation using MTA.

This study was designed to evaluate the effect of mycoplasma contamination on acid hydrolase activity and the action of the mycoplasma removal agent (MRA), in cultures of human fibroblasts from individuals with lysosomal diseases. For this purpose, we measured the activity of the b-galactosidase, arylsulphatase B (ASB), hexosaminidase A and a-glucosidase enzymes. The activity of the above mentioned enzymes in fibroblasts contaminated by mycoplasma was measured before and after the addition of the MRA. The results were then compared to the enzymatic activity in contamination-free cultures. Only the ASB enzyme showed significant alteration in activity both in the presence of mycoplasma and MRA. The remaining enzymes did not suffer significant interference by the presence of the two agents. Of the four enzymes tested, three did not suffer significant alterations by the presence of the mycoplasma nor from the MRA. However, the activity measured in the ASB enzyme increased significantly in the presence of mycoplasma and MRA and could lead to a doubtful diagnosis. Therefore, we suggest that contamination should be prevented by using aseptic techniques as well as the MRA in those fibroblast cultures that cannot be discarded.

In the last years, the role played by the medial nucleus of the amygdala (MeA) in the modulation of fear- and anxiety-related behaviors has been increasingly investigated. This nucleus plays an important role in the processing of predator odor-induced defensive reactions, i.e. freezing and risk-assessment behaviors. Immunohistochemical evidence also indicates that the MeA may be involved in the regulation of escape, a defensive behavior related to panic attacks. In this study, we further addressed this question by investigating the effects of the reversible inactivation of the nucleus on escape behavior generated in male Wistar rats by two different aversive stimuli, electrical stimulation of the dorsal periaqueductal gray matter (dPAG) and exposure to one of the open arms of the elevated T-maze. Results showed that intra-MeA administration of either the reversible sodium channel blocker lidocaine (34 nmol/0.2 microl) or the GABA(A) receptor agonist muscimol (0.22 nmol/0.2 microl) raised the threshold of aversive electrical stimulation, increasing the amount of current that applied to the dPAG evokes escape, an antiaversive effect. Local microinjection of muscimol (0.22 nmol/0.2 microl) inhibited escape behavior in the elevated T-maze, also suggesting an antiaversive effect. In this latter test, muscimol did not affect inhibitory avoidance, a behavior associated with generalized anxiety disorder. Muscimol effect in the elevated T-maze was independent of changes in general exploratory activity as measured in an open-field. Taken together, our data corroborate previous evidences suggesting that the MeA is involved in the modulation of escape. Dysfunction of this regulatory mechanism may be of relevance in the genesis/maintenance of panic disorder.

The precise nature of hormones and growth factors directly responsible for cartilage maturation is still largely unclear. Since longitudinal bone growth occurs through endochondral bone formation, excess or deficiency of most hormones and growth factors strongly influences final adult height. The structure and composition of the cartilaginous extracellular matrix have a critical role in regulating the behavior of growth plate chondrocytes. Therefore, the maintenance of the three-dimensional cell-matrix interaction is necessary to study the influence of individual signaling molecules on chondrogenesis, cartilage maturation and calcification. To investigate the effects of insulin on both proliferation and induction of hypertrophy in chondrocytes in vitro we used high-density micromass cultures of chick embryonic limb mesenchymal cells. Culture medium was supplemented with 1% FCS + 60 ng/ml (0.01 microM) insulin and cultures were harvested at regular time points for later analysis. Proliferating cell nuclear antigen immunoreactivity was widely detected in insulin-treated cultures and persisted until day 21 and [ 3H]-thymidine uptake was highest on day 14. While apoptosis increased in control cultures as a function of culture time, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-labeled cells were markedly reduced in the presence of insulin. Type II collagen production, alkaline phosphatase activity and cell size were also lower in insulin-treated cultures. Our results indicate that under the influence of 60 ng/ml insulin, chick chondrocytes maintain their proliferative potential but do not become hypertrophic, suggesting that insulin can affect the regulation of chondrocyte maturation and hypertrophy, possibly through an antiapoptotic effect.