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The type and frequency of disturbances experienced by soil microbiomes is expected to increase given predicted global climate change scenarios and intensified anthropogenic pressures on ecosystems. While the direct effect of multiple disturbances to soil microbes has been explored in terms of function, their effect on the recovery of microbial community composition remains unclear. Here, we used soil microcosm experiments and multiple model disturbances to explore their short-term effect on the recovery of soil microbiota after identical or novel stresses. Soil microcosms were exposed to a heat shock to create an initial effect. Upon initial community recovery (25 days after stress), they were subjected to a second stress, either a heat or a cold shock, and they were monitored for additional 25 days. To carefully verify the bacterial response to the disturbances, we monitored changes in community composition throughout the experiment using 16S rRNA gene transcript amplicon sequencing. The application of a heat shock to soils with or without the initial heat shock resulted in similar successional dynamics, but these dynamics were faster in soils with a prior heat shock. The application of a cold shock had negligible effects on previously undisturbed soils but, in combination with an initial heat shock, caused the largest shift in the community composition. Our findings show that compounded perturbation affects bacterial community recovery by altering community structure and thus, the community's response during succession. By altering dominance patterns, disturbance legacy affects the microbiome's ability to recover from further perturbation within the 25 days studied. Our results highlight the need to consider the soil's disturbance history in the development of soil management practices in order to maintain the system's resilience.

Purpose: To evaluate whether patients with prostate cancer have worse functional urinary recovery with focal brachytherapy (FBT) at the base versus the apex of the prostate., Methods and Materials: The functional outcomes of patients treated with FBT at the base of the prostate were compared with those of patients treated with FBT at the apex. Urinary symptoms, continence, and erectile dysfunction were measured using the International Prostate Symptom Score (IPSS), International Continence Score (ICS), and International Index of Erectile Function (IIEF-5) questionnaires, respectively, at baseline and at 6, 12, and 24 months after treatment., Results: Twenty-eight and 13 patients were treated with FBT at the apex and the base, respectively, of the prostate. A significant difference between groups was found in the IPSS score at 6 months (mean IPSS: apex 6.4 ± 4.7, base 10.6 ± 5.7; p = 0.02), but not at baseline or at 12 and 24 months after treatment. On multivariate analysis, only FBT at the base of the prostate remained an independent predictor of worsening urinary symptoms (odds ratio, 5.8; p = 0.04)., Conclusions: At 6 months after FBT, significantly less urinary toxicity was found in patients who underwent FBT at the apex versus the base of the prostate. Continence and sexual side effects were minimal in all patients., (Copyright © 2017 American Brachytherapy Society. Published by Elsevier Inc. All rights reserved.)

Introduction and Objective: Focal cryotherapy emerged as an efficient option to treat favorable and localized prostate cancer (PCa). The purpose of this video is to describe the procedure step by step., Materials and Methods: We present the case of a 68 year-old man with localized PCa in the anterior aspect of the prostate., Results: The procedure is performed under general anesthesia, with the patient in lithotomy position. Briefly, the equipament utilized includes the cryotherapy console coupled with an ultrasound system, argon and helium gas bottles, cryoprobes, temperature probes and an urethral warming catheter. The procedure starts with a real-time trans-rectal prostate ultrasound, which is used to outline the prostate, the urethra and the rectal wall. The cryoprobes are pretested and placed in to the prostate through the perineum, following a grid template, along with the temperature sensors under ultrasound guidance. A cystoscopy confirms the right positioning of the needles and the urethral warming catheter is installed. Thereafter, the freeze sequence with argon gas is started, achieving extremely low temperatures (-40ºC) to induce tumor cell lysis. Sequentially, the thawing cycle is performed using helium gas. This process is repeated one time. Results among several series showed a biochemical disease-free survival between 71-93% at 9-70 month- follow-up, incontinence rates between 0-3.6% and erectile dysfunction between 0-42% (1-5)., Conclusions: Focal cryotherapy is a feasible procedure to treat anterior PCa that may offer minimal morbidity, allowing good cancer control and better functional outcomes when compared to whole-gland treatment., Competing Interests: Conflict of interest: None declared., (Copyright® by the International Brazilian Journal of Urology.)

Background: Laparoscopic adrenalectomy is the gold standard surgical approach for small benign adrenal tumors [1]. Several surgical approaches were developed in order to overcome the difficulty to access the adrenal glands, located in the upper retroperitoneum space [2-4]. Laparoendoscopic single-site posterior retroperitoneoscopic adrenalectomy (LESS-PRA) is an emerging technique that reduced the multiple trocar-related trauma and improved cosmetic outcomes while minimizing postoperative morbidity [5-8]. The aim of this study was to describe our step-by-step technique for LESS-PRA and to compare our perioperative outcomes with the conventional 3-port lateral retroperitoneoscopic adrenalectomy (LRA)., Methods: A retrospective review was carried out from February 2008 to January 2016 that included 100 patients with adrenal tumors smaller than 4 cm. Study exclusion criteria were defined as tumor size greater than 4 cm, patients older than 80 years, and body mass index (BMI) greater than 40. A total of 20 patients underwent LESS-PRA and 80 patients underwent 3-port lateral retroperitoneoscopic laparoscopic adrenalectomy. Patient's demographic data and perioperative outcomes were compared and statistically analyzed. The cosmetic satisfaction was evaluated with a visual analog scale., Results: Estimated blood loss was higher in LRA (100 vs. 50 ml; p = 0.35). Operative time was longer in LESS-PRA than LRA (100.0 vs. 60 min; p < 0.001). Analgesic time necessary for LRA was longer than LESS-PRA (40 vs. 24 h; p < 0.001). Cosmetic satisfaction score was higher in LESS-PRA (9.5 vs. 8.6; p = 0.03). There were no significant differences in perioperative complications and length of hospital stay. No conversion to conventional laparoscopic or open surgery was necessary., Conclusion: LESS-PRA presented comparable functional and perioperative outcomes to LRA for small adrenal tumors. Although LESS-PRA was associated with longer operative time, it provided inferior estimated blood loss, analgesic time, and improved cosmetic satisfaction.

Advances in sequencing technologies allow deeper studies of the soil protist diversity and function. However, little attention has been given to the impact of the chosen soil DNA extraction procedure to the overall results. We examined the effect of three acknowledged DNA recovery methods, two manual methods (ISOm-11063, GnS-GII) and one commercial kit (MoBio), on soil protist community structures obtained from different sites with different land uses. Results from 18S rRNA gene amplicon sequencing suggest that DNA extraction method significantly affect the replicate homogeneity, the total number of operational taxonomic units (OTUs) recovered and the overall taxonomic structure and diversity of soil protist communities. However, DNA extraction effects did not overwhelm the natural variation among samples, as the community data still strongly grouped by geographical location. The commercial DNA extraction kit was associated with the highest diversity estimates and with a corresponding higher retrieval of Excavata, Cercozoa and Amoebozoa-related taxa. Overall, our findings indicate that this extraction offers a compromise between rare and dominant taxa representation, while providing high replication reproducibility. A comprehensive understanding of the DNA extraction techniques impact on soil protist diversity can enable more accurate diversity assays., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

The response of bacterial communities to environmental change may affect local to global nutrient cycles. However the dynamics of these communities following disturbance are poorly understood, given that they are often evaluated over macro-ecological time scales and end-point measurements. In order to understand the successional trajectory of soil bacterial communities following disturbances and the mechanisms controlling these dynamics at a scale relevant for these organisms, we subjected soil microcosms to a heat disturbance and followed the community composition of active bacteria over 50 days. The disturbance imposed a strong selective pressure that persisted for up to 10 days, after which the importance of stochastic processes increased. Three successional stages were detected: a primary response in which surviving taxa increased in abundance; a secondary response phase during which community dynamics slowed down, and a stability phase (after 29 days), during which the community tended towards its original composition. Phylogenetic turnover patterns indicated that the community experienced stronger deterministic selection during recovery. Thus, soil bacterial communities, despite their extreme diversity and functional redundancy, respond to disturbances like many macro-ecological systems and exhibit path-dependent, autogenic dynamics during secondary succession. These results highlight the role of autogenic factors and successional dynamics in microbial recovery.

Introduction: The local inflammatory process after prostate biopsies can have a negative impact on functional outcomes of radical prostatectomy. There is no evidence in literature demonstrating its impact on radical prostatectomy., Objectives: To evaluate the impact of the number of TRUS core biopsies in the surgical morbidity and rate of positive margin on robot assisted radical prostatectomy (RARP)., Material and Methods: A prospectively maintained database of 2,054 RARPs in a single institution. Patients were further grouped into 2 groups based on the number of TRUS biopsy cores (G1≤12 cores; G2>12 cores). Multivariable logistic regression model was applied to analyze the impact of number of cores on complications., Results: A total number of 1,042 patients in the group 1 (≤12 cores) and 1,012 patients in the group 2 (>12 cores) were included. The rate of perioperative complications increased with higher number of biopsies (G1 6.4 vs. G2 8.5%; P=.03), but high grade complication (Clavien 3-4) were similar (G1 1.4 vs. G2 2.2%; P=.16). Positive surgical margin rates were similar in both groups (G1 11.8 vs. 9.98%; P=.2). At the multivariable logistic regression analysis shown that G2 had a 39% (OR 0.645) higher rate to experience perioperative complications during RARP., Conclusion: Higher number of TRUS biopsy cores (>12) is associated to higher blood loss and perioperative complications during RARP. Careful preoperative evaluation for those patients underwent multiple biopsies or saturation protocols is mandatory. Application of longer intervals (>6 weeks) between biopsy and surgery may be advisable to minimize potential risks of surgical complications in patients may benefit from RARP. Further studies are still necessary to confirm these results., (Copyright © 2016 AEU. Publicado por Elsevier España, S.L.U. All rights reserved.)

Prostate cancer (PCa) is stratified into different risk categories based on the patient's prognosis. High-risk disease was formerly characterized by an increased risk of metastasis and lethality, requiring complex treatments. Surgery was recently highlighted to have a pivotal role for the treatment of such cases, even as monotherapy. In the past, open radical prostatectomy was performed for most patients with high-risk PCa; however, robot-assisted radical prostatectomy (RARP) emerged as a reasonable option because it provided optimal outcomes for low- and intermediate-risk PCa. Robust studies are lacking to properly assess the role of RARP for high-risk PCa. We summarize this knowledge and present a literature review on the perioperative recovery and functional and oncologic outcomes of RARP for the treatment of patients with high-risk PCa.

SisPorto 4.0 is the most recent version of a program for the computer analysis of cardiotocographic (CTG) signals and ST events, which has been adapted to the 2015 International Federation of Gynaecology and Obstetrics (FIGO) guidelines for intrapartum foetal monitoring. This paper provides a detailed description of the analysis performed by the system, including the signal-processing algorithms involved in identification of basic CTG features and the resulting real-time alerts.

Objective: To evaluate whether intrapartum fetal monitoring with computer analysis and real-time alerts decreases the rate of newborn metabolic acidosis or obstetric intervention when compared with visual analysis., Methods: A randomized clinical trial carried out in five hospitals in the United Kingdom evaluated women with singleton, vertex fetuses of 36 weeks of gestation or greater during labor. Continuous central fetal monitoring by computer analysis and online alerts (experimental arm) was compared with visual analysis (control arm). Fetal blood sampling and electrocardiographic ST waveform analysis were available in both arms. The primary outcome was incidence of newborn metabolic acidosis (pH less than 7.05 and base deficit greater than 12 mmol/L). Prespecified secondary outcomes included operative delivery, use of fetal blood sampling, low 5-minute Apgar score, neonatal intensive care unit admission, hypoxic-ischemic encephalopathy, and perinatal death. A sample size of 3,660 per group (N=7,320) was planned to be able to detect a reduction in the rate of metabolic acidosis from 2.8% to 1.8% (two-tailed α of 0.05 with 80% power)., Results: From August 2011 through July 2014, 32,306 women were assessed for eligibility and 7,730 were randomized: 3,961 to computer analysis and online alerts, and 3,769 to visual analysis. Baseline characteristics were similar in both groups. Metabolic acidosis occurred in 16 participants (0.40%) in the experimental arm and 22 participants (0.58%) in the control arm (relative risk 0.69 [0.36-1.31]). No statistically significant differences were found in the incidence of secondary outcomes., Conclusion: Compared with visual analysis, computer analysis of fetal monitoring signals with real-time alerts did not significantly reduce the rate of metabolic acidosis or obstetric intervention. A lower-than-expected rate of newborn metabolic acidosis was observed in both arms of the trial., Clinical Trial Registration: ISRCTN Registry, http://www.isrctn.com, ISRCTN42314164.

Twenty-seven animals (½ Angus - ½ Nellore) were fed for four months with one of the following diets: without addition of essential oils (E0.0), with 3.5 (E3.5) or 7 (E7.0) g/animal/day of an essential oil blend (oregano, garlic, lemon, rosemary, thyme, eucalyptus and sweet orange). Chemical composition, fatty acid profile and meat color were evaluated in Longissimus muscle. In addition, the effects of aging (one, seven and 14days) on the meat water holding capacity, texture and lipid oxidation were evaluated. Essential oils had no effect on chemical and fatty acid composition, meat color, water holding capacity or texture, but an inclusion of 3.5g/day decreased lipid oxidation. The addition of 7.0g/animal/day had a pro-oxidant effect on meat during aging and resulted in higher values for lipid oxidation at 14days of aging. Aging significantly affected thawing losses and texture. A dose of 3.5g/animal/day could be recommended in feedlot animals, but greater doses could have a pro-oxidant effect., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Copper has been intensively used in industry and agriculture since mid-18(th) century and is currently accumulating in soils. We investigated the diversity of potential active bacteria by 16S rRNA gene transcript amplicon sequencing in a temperate grassland soil subjected to century-long exposure to normal (∼15 mg kg(-1)), high (∼450 mg kg(-1)) or extremely high (∼4500 mg kg(-1)) copper levels. Results showed that bioavailable copper had pronounced impacts on the structure of the transcriptionally active bacterial community, overruling other environmental factors (e.g. season and pH). As copper concentration increased, bacterial richness and evenness were negatively impacted, while distinct communities with an enhanced relative abundance of Nitrospira and Acidobacteria members and a lower representation of Verrucomicrobia, Proteobacteria and Actinobacteria were selected. Our analysis showed the presence of six functional response groups (FRGs), each consisting of bacterial taxa with similar tolerance response to copper. Furthermore, the use of FRGs revealed that specific taxa like the genus Nitrospira and several Acidobacteria groups could accurately predict the copper legacy burden in our system, suggesting a potential promising role as bioindicators of copper contamination in soils., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Creatinine is eliminated by the kidneys through a combination of glomerular filtration and active transport. Drug-induced increases in serum creatinine (SCr) and/or reduced creatinine renal clearance are used as a marker for acute kidney injury. However, inhibition of active transport of creatinine can result in reversible and, therefore, benign increases in SCr levels. Herein, the transporters involved in creatinine clearance are discussed, in addition to limitations of using creatinine as a biomarker for kidney damage., (© 2016 American Society for Clinical Pharmacology and Therapeutics.)

Introduction: Retzus-sparing robotic-assisted radical prostatectomy(RARP) is a newly approach that preserve the Retzus structures and provide better recovery of continence and erectile function. In Brazil, this approach has not yet been pre¬viously reported., Objective: Our goal is to describe Step-by-Step the Retzus-sparing RARP surgical technique and report our first Brazilian experience., Methods: We present a case of a 60-year-old white man with low risk prostate cancer. Surgical materials were four arms Da Vinci robotic platform system, six transperitoneal portals, two prolene wires and Polymer Clips. This surgical tech¬nique was step-by-step described according to Galfano et al. One additional step was added as a modification of Galfano et al. Primary technique description: The closure of the Denovellier fascia., Results: We have operated one patient with this technique. The operative time was 180minutes, console time was135 min, the blood loss was 150ml, none perioperative or postoperative complications was found, hospital stay of 01 day. The anatomopathological classification revealed a pT2aN0M0 specimen with free surgical margins. The patient achieved continence immediately after bladder stent retrieval. Full erection reported after 30 days of surgery., Conclusion: Retzus-sparing RARP approach is feasible and reproducible. However, further comparative studies are neces¬sary to demonstrate potential benefits in continence and sexual outcomes over the standard approaches., Competing Interests: Conflicts of Interest: None declared., (Copyright® by the International Brazilian Journal of Urology.)

In humans, creatinine is formed by a multistep process in liver and muscle and eliminated via the kidney by a combination of glomerular filtration and active transport. Based on current evidence, creatinine can be taken up into renal proximal tubule cells by the basolaterally localized organic cation transporter 2 (OCT2) and the organic anion transporter 2, and effluxed into the urine by the apically localized multidrug and toxin extrusion protein 1 (MATE1) and MATE2K. Drug-induced elevation of serum creatinine (SCr) and/or reduced creatinine renal clearance is routinely used as a marker for acute kidney injury. Interpretation of elevated SCr can be complex, because such increases can be reversible and explained by inhibition of renal transporters involved in active secretion of creatinine or other secondary factors, such as diet and disease state. Distinction between these possibilities is important from a drug development perspective, as increases in SCr can result in the termination of otherwise efficacious drug candidates. In this review, we discuss the challenges associated with using creatinine as a marker for kidney damage. Furthermore, to evaluate whether reversible changes in SCr can be predicted prospectively based on in vitro transporter inhibition data, an in-depth in vitro-in vivo correlation (IVIVC) analysis was conducted for 16 drugs with in-house and literature in vitro transporter inhibition data for OCT2, MATE1, and MATE2K, as well as total and unbound maximum plasma concentration (Cmax and Cmax,u) data measured in the clinic., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Introduction: In special situations such as malrotated or ectopic kidneys and UPJ stenosis treatment of renal lithiasis can be challenging. In these rare cases laparoscopy can be indicated., Objective: Describe the Laparoscopic-assisted rigid nephroscopy performed via transpyelic approach and report the feasibility., Patients and Methods: We present two cases of caliceal lithiasis. The first is a patient that ESWL and previous percutaneous lithotripsy have failed, with pelvic kidney where laparoscopic dissection of renal pelvis was carried out followed by nephroscopy utilizing the 30 Fr rigid nephroscope to remove the calculus. Ideal angle between the major axis of renal pelvis and the rigid nephroscope to allow success with this technique was 60-90 grades. In the second case, the kidney had a dilated infundibulum., Results: The operative time was 180 minutes for both procedures. No significant blood loss or perioperative complications occurred. The bladder catheter was removed in the postoperative day 1 and Penrose drain on day 2 when patients were discharged. The convalescence was completed after 3 weeks. Patients were stone free without symptons in one year of follow-up., Conclusions: Laparoscopic-assisted rigid nephroscopy performed via tranpyelic approach can be done safely with proper patient selection and adherence to standard laparoscopic surgical principles. This approach is an alternative in cases where flexible endoscope is not available and when standard procedure is unlikely to produce a stone-free status., Competing Interests: Conflicts of Interest: None declared., (Copyright© by the International Brazilian Journal of Urology.)

Objective. To compare United States and European Higher Education Area (EHEA) undergraduate pharmacy curricula in terms of patient-centered care courses. Methods. Websites from all pharmacy colleges or schools in the United States and the 41 countries in the EHEA were retrieved from the FIP Official World List of Pharmacy Schools and investigated. A random sample of schools was selected and, based on analyses of course descriptions from syllabi, each course was classified into the following categories: social/behavioral/administrative pharmacy sciences, clinical sciences, experiential, or other/basic sciences. Results. Of 147 schools of pharmacy, 59 were included (23 in US and 36 in the EHEA). Differences existed in the percentages of credits/hours in all of the four subject area categories. Conclusion. Institutions in EHEA countries maintain a greater focus on basic sciences and a lower load of clinical sciences in pharmacy curricula compared to the United States. These differences may not be in accordance with international recommendations to educate future pharmacists focused on patient care.

Introduction: The management of esophageal strictures has evolved from surgical treatment to the endoscopic dilation and, more recently, esophageal stenting., Clinical Case: We describe a case of a two-year-old boy with a double stenosis of the esophagus resulting from accidental ingestion of strong alkaline liquid. After several unsuccessful endoscopic dilations for three years and even topical mitomicin, it was decided to place a dynamic stent developed by the Digestive Surgery and Endoscopic Unit of the Bambino Gesù Hospital, Rome. The stent is a custom silicon device built coaxially on a nasogastric tube that is inserted after stricture dilations, by endoscopic guidance, and then fixed outside the nose. The device was removed after seven weeks with good clinical outcome (no dysphagia more than a year of follow-up)., Conclusion: This case confirms that the dynamic stent is a simple device that may avoid aggressive surgical substitution in cases of refractory strictures.

Five systems for computer analysis of foetal monitoring signals are currently available, incorporating the evaluation of cardiotocographic (CTG) or combined CTG with electrocardiographic ST data. All systems have been integrated with central monitoring stations, allowing the simultaneous monitoring of several tracings on the same computer screen in multiple hospital locations. Computer analysis elicits real-time visual and sound alerts for health care professionals when abnormal patterns are detected, with the aim of prompting a re-evaluation and subsequent clinical action, if considered necessary. Comparison between the CTG analyses provided by the computer and clinical experts has been carried out in all systems, and in three of them, the accuracy of computer alerts in predicting newborn outcomes was evaluated. Comparisons between these studies are hampered by the differences in selection criteria and outcomes. Two of these systems have just completed multicentre randomised clinical trials comparing them with conventional CTG monitoring, and their results are awaited shortly. For the time being, there is limited evidence regarding the impact of computer analysis of foetal monitoring signals on perinatal indicators and on health care professionals' behaviour., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objective: To compare fetal heart rate (FHR) signals acquired simultaneously by an external ultrasound probe and a scalp electrode during the second stage of labor., Methods: This was a prospective observational study in a labor ward of a tertiary care university hospital. The population was women in labor with uneventful singleton pregnancies at term. Simultaneous external and internal FHR monitoring was performed in 67 consecutively recruited women during the second stage of labor. Cases were subsequently excluded if the trace length was under 40 min, cesarean birth occurred, or the interval between trace-end and birth exceeded 5 min, leaving a total of 33 traces for analysis. The last 40-60 min of these traces were analyzed by a computer system (Omniview-SisPorto 3.5; Speculum, Lisbon, Portugal) to quantify cardiotocographic parameters. Paired sample t-test and Bland-Altman limits of agreement (LoA) were used for statistical analysis, setting significance at 0.05. The main outcome measures were signal loss, FHR baseline, periodic events, and percentage of periodic events coinciding with contractions., Results: A higher signal loss was observed with external monitoring [10% vs. 4%; P<0.001, LoA=(-6, 18)]. No differences were found in mean FHR baseline [129 bpm vs. 130 bpm, P=0.245, LoA=(-15, 12)], but more accelerations [12 vs. 8, P<0.001, LoA=(-5, 13)] and less decelerations [8 vs. 10, P<0.001, LoA=(-8, 4)] were detected with external monitoring. With this method there were also more accelerations (66% vs. 55%, P=0.036) and less decelerations (68% vs. 81%, P=0.017) coinciding with contractions., Conclusions: External FHR monitoring during the second stage of labor results in higher signal loss, increased number of accelerations, and decreased number of decelerations when compared with internal monitoring.

Recent reports suggest that N-methyl-d-aspartate receptor (NMDAR) blockade by MK-801 decreases tumor growth. Thus, we investigated whether other ionotropic glutamate receptor (iGluR) antagonists were also able to modulate the proliferation of melanoma cells. On the other hand, the antiestrogen tamoxifen (TAM) decreases the proliferation of melanoma cells, and is included in combined therapies for melanoma. As the efficacy of TAM is limited by its metabolism, we investigated the effects of the NMDAR antagonist MK-801 in combination with TAM and its active metabolites, 4-hydroxytamoxifen (OHTAM) and endoxifen (EDX). The NMDAR blockers MK-801 and memantine decreased mouse melanoma K1735-M2 cell proliferation. In contrast, the NMDAR competitive antagonist APV and the AMPA and kainate receptor antagonist NBQX did not affect cell proliferation, suggesting that among the iGluR antagonists only the NMDAR channel blockers inhibit melanoma cell proliferation. The combination of antiestrogens with MK-801 potentiated their individual effects on cell biomass due to diminished cell proliferation, since it decreased the cell number and DNA synthesis without increasing cell death. Importantly, TAM metabolites combined with MK-801 promoted cell cycle arrest in G1. Therefore, the data obtained suggest that the activity of MK-801 and antiestrogens in K1735-M2 cells is greatly enhanced when used in combination., (© 2013 Published by Elsevier Inc.)

Purpose: The saltatory pattern, characterized by wide and rapid oscillations of the fetal heart rate (FHR), remains a controversial entity. The authors sought to evaluate whether it could be associated with an adverse fetal outcome., Material and Methods: The authors report a case series of four saltatory patterns occurring in the last 30 minutes before birth in association with cord artery metabolic acidosis, obtained from three large databases of internally acquired FHR tracings. The distinctive characteristics of this pattern were evaluated with the aid of a computer system., Results: All cases were recorded in uneventful pregnancies, with normal birthweight singletons, born vaginally at term. The saltatory pattern lasted between 23 and 44 minutes, exhibited a mean oscillatory amplitude of 45.9 to 80.0 beats per minute (bpm) and a frequency between four and eight cycles per minute., Conclusions: A saltatory pattern exceeding 20 minutes can be associated with the occurrence of fetal metabolic acidosis.

This paper reports, for the first time, the concentrations of selected phthalates in drinking water consumed in Portugal. The use of bottled water in Portugal has increased in recent years. The main material for bottles is polyethylene terephthalate (PET). Its plasticizer components can contaminate water by leaching, and several scientific studies have evidenced potential health risks of phthalates to humans of all ages. With water being one of the most essential elements to human health and because it is consumed by ingestion, the evaluation of drinking water quality, with respect to phthalate contents, is important. This study tested seven commercial brands of bottled water consumed in Portugal, six PET and one glass (the most consumed) bottled water. Furthermore, tap water from Lisbon and three small neighbor cities was analyzed. Phthalates (di-n-butyl phthalate ester (DnBP), bis(2-ethylhexyl) phthalate ester (DEHP), and di-i-butyl phthalate ester (DIBP)) in water samples were quantified (PET and glass) by means of direct immersion solid-phase microextraction and ionic liquid gas chromatography associated with flame ionization detection or mass spectrometry due to their high boiling points and water solubility. The method utilized in this study showed a linear range for target phthalates between 0.02 and 6.5 μg L(-1), good precision and low limits of detection that were between 0.01 and 0.06 μg L(-1), and quantitation between 0.04 and 0.19 μg L(-1). Only three phthalates were detected in Portuguese drinking waters: dibutyl (DnBP), diisobutyl (DIBP), and di(ethylhexyl) phthalate (DEHP). Concentrations ranged between 0.06 and 6.5 μg L(-1) for DnBP, between 0.02 and 0.16 μg L(-1) for DEHP, and between 0.1 and 1.89 μg L(-1) for DIBP. The concentration of DEHP was found to be up to five times higher in PET than in glass bottled water. Surprisingly, all the three phthalates were detected in glass bottled water with the amount of DnBP being higher (6.5 μg L(-1)) than in PET bottled water. These concentrations do not represent direct risk to human health. Regarding potable tap water, only DIBP and DEHP were detected. Two of the cities showed concentration of all three phthalates in their water below the limits of detection of the method. All the samples showed phthalate concentrations below 6 μg L(-1), the maximum admissible concentration in water established by the US Environmental Protection Agency. The concentrations measured in Portuguese bottled waters do not represent any risk for adult's health.

The prevalence of hearing problems in the Western world has, due to aging of the population, doubled over the past 30 years. Thereby, noise-induced hearing loss is an important factor that worsens over time in addition to age-related hearing loss. Hearing loss is usually measured as an elevation of a person's hearing thresholds, expressed in decibel (dB). However, recent animal studies have unraveled a type of permanent cochlear damage, without an elevation of hearing thresholds. This subtle damage is linked to a permanent and progressive degeneration of auditory fibers that occurs in association with damage of the inner hair cell synapse. Afferent neuronal degeneration has been suggested to be involved in hyperacusis (over sensitivity to sound) and tinnitus (a phantom sound percept). Hyperacusis and tinnitus are potentially devastating conditions that are still incurable. The main risk factors to develop tinnitus or hyperacusis are hearing loss, social stress and age. Both tinnitus and hyperacusis have been discussed in the context of a pathological increased response gain in subcortical brain regions as a reaction to deprivation of sensory input. Novel studies confirm the involvement of peripheral deafferentation for tinnitus and hyperacusis, but suggest that the disorder results from different brain responses to different degrees of deafferentation: while tinnitus may arise as a failure of the brain to adapt to deprived peripheral input, hyperacusis may result from an 'over-adaptive' increase in response gain. Moreover, moderate and high stress levels at the time of acoustic trauma have been suggested to play a pivotal role in the vulnerability of the cochlea to acoustic damage and therefore for the development of tinnitus and hyperacusis., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The taxonomic and biogeographic affinities of Strabomantis aramunha from the Campos Rupestres of Brazil are intriguing. A unique skull morphology of females suggest affinities with the broad-headed eleutherodactylines of Northwestern South America in the genus Strabomantis. Male and juvenile morphology nonetheless suggest S. aramunha could be related to members of the recently described genus Haddadus from eastern Brazil. We assess the affinities of S. aramunha using molecular phylogenetic analyses of mitochondrial (12S, tRNAval, 16S, cyt b) and nuclear sequences (RAG-1and rhodopsin). Bayesian inference, likelihood, and parsimony analysis recover a highly supported clade with S. aramunha and H. binotatus as sister taxa. Accordingly, we transfer S. aramunha to Haddadus, and provide a new generic definition of the later. The distribution of species in Haddadus (highlands of the Espinhaço mountain Range and coastal eastern Brazil) is now concordant with the general pattern observed for other species in the area.

There is an ever-increasing global demand from consumers for high-quality foods with major emphasis placed on quality and safety attributes. One of the main demands that consumers display is for minimally processed, high-nutrition/low-energy natural foods with no or minimal chemical preservatives. The nutritional value of raspberry fruit is widely recognized. In particular, red raspberries are known to demonstrate a strong antioxidant capacity that might prove beneficial to human health by preventing free radical-induced oxidative stress. However, food products that are consumed raw, are increasingly being recognized as important vehicles for transmission of human pathogens. Food irradiation is one of the few technologies that address both food quality and safety by virtue of its ability to control spoilage and foodborne pathogenic microorganisms without significantly affecting sensory or other organoleptic attributes of the food. Food irradiation is well established as a physical, nonthermal treatment (cold pasteurization) that processes foods at or nearly at ambient temperature in the final packaging, reducing the possibility of cross contamination until the food is actually used by the consumer. The aim of this study was to evaluate effects of gamma radiation on raspberries in order to assess consequences of irradiation. Freshly packed raspberries (Rubus idaeus L.) were irradiated in a (60)Co source at several doses (0.5, 1, or 1.5 kGy). Bioburden, total phenolic content, antioxidant activity, physicochemical properties such as texture, color, pH, soluble solids content, and acidity, and sensorial parameters were assessed before and after irradiation and during storage time up to 14 d at 4°C. Characterization of raspberries microbiota showed an average bioburden value of 10(4) colony-forming units (CFU)/g and a diverse microbial population predominantly composed of two morphological types (gram-negative, oxidase-negative rods, 35%, and filamentous fungi, 41%). The inactivation studies on the raspberries mesophilic population indicated a one log reduction of microbial load (95% inactivation efficiency for 1.5 kGy), in the surviving population mainly constituted by filamentous fungi (79-98%). The total phenolic content of raspberries indicated an increase with radiation doses and a decrease with storage time. The same trend was found for raspberries' antioxidant capacity with storage time. Regarding raspberries physicochemical properties, irradiation induced a significant decrease in firmness compared with nonirradiated fruit. However, nonirradiated and irradiated fruit presented similar physicochemical and sensory properties during storage time. Further studies are needed to elucidate the benefits of irradiation as a raspberries treatment process.

This unit describes two basic protocols for the detection of the proliferation of neural stem cells (NSC). The first one addresses cell proliferation in cultures, starting with primary cell cultures isolated from the mouse subventricular zone (SVZ), in which SVZ-derived NSC are kept in culture as neurospheres. By using this culture system, we are able to study different stages of adult neurogenesis, such as proliferation, differentiation, migration, and survival. Thus, in the first basic protocol, we describe two different techniques to evaluate cell proliferation based on EdU incorporation: (a) immunocytochemistry and (b) flow cytometry. EdU, a new thymidine analog, which is detected by a reproducible and sensitive method based on click chemistry, does not require DNA denaturation, as is the case with BrdU. Thus, co-labeling of EdU with other specific antibodies of extracellular or intracellular targets, as well as other DNA dyes, is possible. In the second basic protocol, we describe an in vivo assay to evaluate proliferation of NSC in the dentate gyrus of hippocampus of adult mice, by both BrdU and EdU detection. With this approach, it is also possible to study different stages of adult neurogenesis, by co-labeling thymidine analogs with other specific markers, such as doublecortin (DCX) or neuronal nuclei protein (NeuN).

Objectives: The aim of this study was to compare the single-dose effects of thiazide-type diuretics cicletanine and hydrochlorothiazide (HCTZ), on natriuresis and kaliuresis in prehypertensive and treatment-naïve, stage 1 hypertensive patients and to explore the impact of GRK4 gene polymorphisms on thiazide-induced urinary electrolyte excretion., Methods: The study was a randomized, double-blind, placebo-controlled, three-period, four-treatment, balanced incomplete block, cross-over study in male patients assigned to treatment sequences consisting of placebo, cicletanine 50 mg, cicletanine 150 mg, and HCTZ 25 mg, doses used to treat hypertension. Cumulative urine samples were collected predosing and over 24 h after dosing in each period to compare urine electrolyte excretion profiles of potassium (UKV), sodium (UNaV), magnesium, calcium, phosphate, chloride, and pH among groups. Each treatment was administered to 18 different patients in each period, and an equal number of patients had less than and at least three GRK4 allele variants., Results: Compared with placebo, mean UKV was significantly increased with HCTZ 25 mg (12.7 mmol/day; P ≤ 0.001), cicletanine 50 mg (4.6 mmol/day; P = 0.026), and cicletanine 150 mg (5.5 mmol/day; P = 0.011), and mean UNaV was significantly increased with HCTZ 25 mg (102.2 mmol/day; P ≤ 0.001), cicletanine 50 mg (21.7 mmol/day; P = 0.005), and cicletanine 150 mg (57.9 mmol/day; P ≤ 0.001)., Conclusion: All treatments had more natriuresis, diuresis, and kaliuresis than placebo, and both doses of cicletanine had less kaliuresis than HCTZ. These findings suggest that cicletanine is a favorable and well tolerated option for the treatment of hypertension with an improved safety profile compared with HCTZ.

Background: The development of antihypertensives requires efficient and accurate tools for identifying pedal edema. Methodologies used to gauge the potential of an agent to induce pedal edema in short-term (<4-week) clinical trials have not been reported in the literature., Objective: The purpose of this study was to identify a robust and practical method for measuring drug-induced pedal edema for use in the clinical development of antihypertensives. The efficacy of segmental bioimpedance in the detection of increased pedal edema was compared with that of clinical pitting assessment, ankle circumference, and water displacement volumetry., Methods: The study population consisted of male and female healthy subjects and patients with stage 1 or 2 hypertension who were otherwise healthy. Participants were randomly assigned to receive amlodipine 10 mg or placebo once daily in this 6-week, double-blind, parallel-group study. Amlodipine was used as a means of inducing ankle edema, and not for the treatment of hypertension. Patients with hypertension were required to undergo a washout of antihypertensive therapies. Edema was evaluated using segmental bioimpedance at 10 kHz, clinical pitting assessment, ankle circumference, and water displacement at weeks 2, 4, and 6. The ANOVA model used included treatment and baseline values as covariates, with treatment pairs compared via t tests derived from the model., Results: A total of 47 individuals were randomized (49% male; 29 [62%] with hypertension; mean [SD] age, 59 [5.9] years; baseline body mass index, 28.6 kg/m(2) [2.8]; blood pressure 146.6 [10.7]/93.5 [6.5] and 139.3 [8.3]/89.5 [4.5] in individuals with and without hypertension, respectively; amlodipine 10 mg, n = 24; placebo, n = 23). At weeks 2, 4, and 6, statistically significant treatment differences in changes from baseline were detected using water displacement (mean [90% CI] treatment differences, +39.0 g [+17.9 to +60.1], +61.9 g [+36.1 to +87.6], and +72.2 g [+42.3 to +102.1], respectively; all, P ≤ 0.001), ankle circumference (+4.74 mm [+2.38 to +7.11; P < 0.001], +2.92 mm [+0.33 to +5.49; P = 0.032], and +5.16 mm [+2.21 to +8.11; P = 0.002]), and bioimpedance (-11.7 Ω [-18.1 to -5.4], -18.3 Ω [-26.2 to -10.4], and -20.9 Ω [-29.7 to -12.0]; all, P≤0.001), but no significant differences were detected using clinical assessment of pitting., Conclusion: In this population of healthy subjects and patients with hypertension, segmental bioimpedance was comparable to water displacement and ankle circumference and outperformed clinical assessment of pitting for the detection of ankle edema, supporting the use of segmental bioimpedance as a drug-development tool to objectively quantify amlodipine-induced pedal edema., (Copyright © 2012 Elsevier HS Journals, Inc. All rights reserved.)

Glioblastomas (GBM) are the most lethal subtype of astrocytomas, with a mean patient survival rate of 12 months after diagnosis. The gold standard treatment of GBM, which includes surgery followed by the combination of radiotherapy and chemotherapy with temozolomide (TMZ), increases the survival rate to 14.6 months. The success of TMZ appears to be limited by the occurrence of chemoresistance that allows glioma cells to escape from death signaling pathways. However, the mechanism of TMZ action is yet to be clarified although some controversial results have been reported. Therefore, our aim was to evaluate the occurrence of apoptosis and autophagy in glioma cells treated with TMZ and to correlate TMZ action with the survival pathways Pi3K/Akt and ERK1/2 MAP kinase. Cell proliferation was evaluated by incorporation of bromodeoxyuridine. Apoptosis was studied by flow cytometry as well as by fluorescence confocal microscopy in order to evaluate the sub G0/G1 percentage of cells and chromatin condensation. The expression of the autophagy-associated protein, LC3, as well as Akt and ERK1/2 was performed by Western blotting. In TMZ-treated GBM cells the expression of LC3, the autophagy-associated protein was increased and only a reduced percentage of cells underwent apoptosis. In addition, we showed that the phosphorylation status of Pi3K/Akt and ERK1/2 MAP kinase was maintained during the treatment with TMZ, suggesting that glioma cells escape from TMZ-induced cell death due to these signaling pathways. The chemoresistance of U-118 cells to TMZ was partially eradicated when cells were simultaneously treated with specific inhibitors of Pi3K/Akt and ERK1/2 MAP kinase signaling pathways and TMZ. Therefore, we hypothesized that in order to induce glioma cell death it is essential to evaluate the activation of the survival pathways and establish a combined therapy using TMZ and inhibitors of those signaling pathways.

Background: The aim of this study was to assess the usefulness of a new miniaturized echocardiographic system (MS) to perform bedside echocardiography in initial outpatient cardiology consultations, in addition to physical examination., Methods: One hundred eighty-nine patients referred for initial cardiology outpatient consultations at two tertiary hospitals in two countries were studied. Each patient was submitted to physical examination followed by MS assessment. Scanning time, the number of examinations with abnormal results after physical examination and the MS, and the information obtained by physical examination alone and followed by the MS (in terms of its importance in reaching a diagnosis, in the necessity of performing routine echocardiography, and in the decision to release the patient from the outpatient clinic) were assessed., Results: The scanning time with the MS was 180 ± 86 seconds. Its use after physical examination led to diagnoses in 141 patients (74.6%) and to an additional 37 patients (19.6%) being released from the outpatient clinic. After physical examination followed by MS assessment, only 64 patients (33.9%) were sent to the echocardiography lab. The MS modified the decision of whether to send a patient to the echocardiography lab, with referral determined by the MS in 27 patients (14.3%) and no referral determined by the MS in 58 patients (30.7%)., Conclusions: The new MS caused a negligible increase in the duration of consultations. It showed additive clinical value over physical examination, increasing the number of diagnoses, reducing the use of unnecessary routine echocardiography, increasing the number of adequate echocardiographic studies, and determining a large number of releases from the outpatient clinic., (Copyright © 2010 American Society of Echocardiography. Published by Mosby, Inc. All rights reserved.)

Antihypertensive drugs can have different effects on central and brachial blood pressures, which may affect outcomes. Nitric oxide donors have acute effects on central blood pressure but have not been assessed with renin-angiotensin system blockade. Thirteen patients with prehypertensive/Stage 1 hypertension were randomized to five single-dose treatments separated by ≤4 days using a double-blind, crossover study design: angiotensin receptor blocker (ARB) losartan 100 mg, isosorbide mononitrate (ISMN) 60 mg, losartan 100 mg + ISMN 15 mg, losartan 100 mg + ISMN 60 mg, and placebo. Central and brachial blood pressures were measured throughout 10 hours. Mean placebo-subtracted decrease from baseline in augmentation index (AIx) approximately 1% for losartan 100 mg, 26% for ISMN 60 mg, 19% for losartan 100 mg + ISMN 15 mg, and 24% for losartan 100 mg + ISMN 60 mg. Administered with losartan 100 mg or alone, ISMN lowered AIx, demonstrating that acute effects of a nitrate donor are much larger than those of an ARB even when administered with an ARB. Differences from placebo were statistically significant except for losartan 100 mg. AIx is a good biomarker of acute hemodynamic effects of nitric oxide in prehypertensive/Stage 1 hypertension., (2010 American Society of Hypertension. Published by Elsevier Inc. All rights reserved.)

This article reports the use of the GsuI restriction enzyme to differentiate genotypes of Bovine Coronavirus (BCoV), based on an 18-nucleotide deletion of S1-coding region found in one of the two genotypes. It was concluded that this assay can be used as a rapid tool for BCoV genotypes differentiation.

Thromboembolism is a major cause of death in cancer patients. The association between paraneoplastic hypercoagulability of oncological patients and long-term central venous catheters (CVC) may result in CVC associated thrombosis. Patent Foramen Ovale (PFO), especially when associated with atrial septal aneurysm (ASA) is a risk factor for paradoxical embolism. We report a case of paradoxical embolism with stroke in an oncological patient with a huge CVC thrombus playing "ping-pong" with an hypermobile ASA with a PFO. We review the management of hypercoagulability in oncologic patients and discuss the potential role of routine transthoracic echocardiography before the implantation of long term central venous catheters to identify predisposing conditions to paradoxical embolism and select patients for anticoagulant therapy.

The c-abl proto-oncogene encodes a nonreceptor tyrosine kinase involved in many cellular processes, including signaling from growth factor and antigen receptors, remodeling the cytoskeleton, and responding to DNA damage and oxidative stress. Many downstream pathways are affected by c-Abl. Elevated c-Abl kinase activity can inhibit NF-kappaB activity by stabilizing the inhibitory protein IkappaB alpha, raising the possibility that c-Abl-deficient cells might have increased NF-kappaB activity. We examined the levels of NF-kappaB activity in primary mouse embryonic fibroblasts (MEFs) derived from wild-type and c-Abl knockout mice and found that the knockout MEFs indeed exhibited elevated NF-kappaB activity in response to stimulation as well as constitutively elevated NF-kappaB activity. Thus, endogenous c-Abl is a negative regulator of basal and inducible NF-kappaB activity. Examination of various points of NF-kappaB regulation revealed that unstimulated c-Abl knockout MEFs do not exhibit an increase in IkappaB alpha degradation, p65/RelA nuclear translocation, or DNA binding of NF-kappaB subunits. They do, however, show reduced levels of the histone deacetylase HDAC1, a negative regulator of basal NF-kappaB activity. Unstimulated c-Abl knockout MEFs are less responsive to induction of NF-kappaB activity by trichostatin A, an HDAC inhibitor, suggesting that c-Abl might play a role in the HDAC-mediated repression of basal NF-kappaB activity.

African swine fever virus (ASFV), a large enveloped DNA-containing virus, infects domestic and wild pigs, and multiplies in soft ticks, causing an economically relevant hemorrhagic disease. Evaluation of the nuclear import ability of ASFV p10 protein was the major purpose of the present work. Two approaches were used to determine if p10 protein is imported into the nucleus by an active process: a yeast-based nuclear import assay and the determination of the subcellular localization of p10 protein in mammalian cells by fluorescence microscopy. The results obtained clearly demonstrate that p10 protein is actively imported into the nucleus, both in yeast and mammalian cells. Experiments aiming at identifying the critical residues responsible for the nuclear import of ASFV p10 protein indicate that the amino acids comprised between the positions 71 and 77 are important, although not sufficient, for the protein active nuclear import. In ASFV-infected cells, the p10 protein strongly accumulates in the nucleus at late times post-infection, indicating that p10 protein may accomplish an important function inside the nucleus during the late phase of the viral replication cycle.

The replication of African swine fever virus DNA is initiated inside the nucleus of host cells, being followed by a longer cytoplasmic replication stage. In face of previous results demonstrating the nucleo-cytoplasmic shuttling activity of ASFV p37 structural protein when considered isolated from the virus infection, we performed a systematic analysis of the subcellular localization of p37 protein in ASFV-infected cells, aiming at identifying the role of the nuclear transport mediated by this protein in the viral replication cycle. We report that the p37 protein of the incoming virions is localized throughout the cell at early times post-infection, concentrated in distinct nuclear regions, while at later times the newly synthesized protein is detected exclusively in the cytoplasm of infected cells. Experiments using leptomycin B and siRNAs targeting the CRM1 receptor demonstrate that the subcellular localization of p37 protein is not affected by inhibition of the CRM1-mediated nuclear export pathway. Finally, results from in situ hybridization experiments show a co-localization of the ASFV DNA and p37 protein in specific nuclear regions at early times post-infection, and in viral factories at later times. Overall, these results support the involvement of p37 protein in the nuclear transport of the viral DNA during ASFV replication cycle.

A Gram-positive bacterium, designated B22(T), was isolated from potting soil produced in Portugal. This organism is a catalase-positive, oxidase-negative, motile, spore-forming, aerobic rod that grows optimally at 37 degrees C and pH 8.0-8.5. Optimal growth occurs in media containing 1 % (w/v) NaCl, although the organism can grow in 0-8 % NaCl. The cell wall peptidoglycan is of the A4alpha type with a cross-linkage containing d-Asp. The major respiratory quinone is menaquinone 7 and the major fatty acids are anteiso-15 : 0, anteiso-17 : 0 and iso-15 : 0. The DNA G+C content is 37.9 mol%. Phylogenetic analysis of 16S rRNA gene sequences revealed that strain B22(T) formed a new branch within the family Bacillaceae. The novel isolate is phylogenetically closely related to members of genera of moderately halophilic bacilli and formed a coherent cluster with species of the genera Salinibacillus, Virgibacillus, Oceanobacillus and Lentibacillus, supported by bootstrap analysis at a confidence level of 71 %. Strain B22(T) exhibited 16S rRNA gene pairwise sequence similarity values of 94.7-94.3 % with members of the genus Salinibacillus, 95.1-92.8 % with members of the genus Virgibacillus, 94.7-93.2 % with members of the genus Oceanobacillus and 93.1-92.3 % with members of the genus Lentibacillus. On the basis of phylogenetic analysis and physiological and biochemical characteristics, it is proposed that strain B22(T) represents a novel species in a new genus, Paucisalibacillus globulus gen. nov., sp. nov. Strain B22(T) (=LMG 23148(T)=CIP 108857(T)) is the type strain of Paucisalibacillus globulus.

Nucleocytoplasmic shuttling activity of the African swine fever virus p37 protein, a major structural protein of this highly complex virus, has been recently reported. The systematic characterization of the nuclear export ability of this protein constituted the major purpose of the present study. We report that both the N- and C-terminal regions of p37 protein are actively exported from the nucleus to the cytoplasm of yeast and mammalian cells. Moreover, experiments using leptomycin B and small interfering RNAs targeting the CRM1 receptor have demonstrated that the export of p37 protein is mediated by both the CRM1-dependent and CRM1-independent nuclear export pathways. Two signals responsible for the CRM1-mediated nuclear export of p37 protein were identified at the N terminus of the protein, and an additional signal was identified at the C-terminal region, which mediates the CRM1-independent nuclear export. Interestingly, site-directed mutagenesis revealed that hydrophobic amino acids are critical to the function of these three nuclear export signals. Overall, our results demonstrate that two distinct pathways contribute to the strong nuclear export of full-length p37 protein, which is mediated by three independent nuclear export signals. The existence of overlapping nuclear export mechanisms, together with our observation that p37 protein is localized in the nucleus at early stages of infection and exclusively in the cytoplasm at later stages, suggests that the nuclear transport ability of this protein may be critical to the African swine fever virus replication cycle.

Since oxidative stress has been implicated in the development of numerous diseases including cataract, this laboratory has created and investigated the stress response of murine immortal lens epithelial cell lines (alphaTN4-1) conditioned to withstand lethal peroxide concentrations. Two of a group of antioxidative defense (AOD) enzymes found in such cells to have markedly enhanced activity are catalase (CAT) and GSH S-transferase alpha2 (GST). In order to determine if enrichment of one or both of these AODs is sufficient to protect alphaTN4-1 cells from lethal H(2)O(2) levels, these cells were infected with adenovirus vectors capable of expressing these AODs at a high level. With this system, gene enrichment and increased enzyme activity were observed with both CAT and GST vectors. The percentage of cells infected ranged from about 50 to 90% depending on the multiplicity of infection (MOI). CAT but not GST protected the cells from H(2)O(2) stress. The CAT activity was increased from 15- to 150-fold and even at the lower levels protected the cells from H(2)O(2) concentrations as high as 200 microM or more (H(2)O(2) levels which rapidly kill non-enriched cells). Even when only about 50% of the cell population is infected as judged by GFP infection, the entire population appeared to be protected based on cell viability. The CAT enrichment appears to protect other intracellular defense systems such as GSH from being depleted in contrast to non-enriched cell populations where GSH is rapidly exhausted. The overall results suggest that enriching the cellular CAT gene level with an appropriate recombinant viral vector may be sufficient to protect in vivo systems from peroxide stress.

African swine fever virus (ASFV), a large icosahedral deoxyvirus, is the causative agent of an economically relevant hemorrhagic disease that affects domestic pigs. The major purpose of the present study was to investigate the nuclear transport activities of the ASFV p37 and p14 proteins, which result from the proteolytic processing of a common precursor. Experiments were performed by using yeast-based nucleocytoplasmic transport assays and by analysis of the subcellular localization of different green fluorescent and Myc fusion proteins in mammalian cells. The results obtained both in yeast and mammalian cells clearly demonstrated that ASFV p14 protein is imported into the nucleus but not exported to the cytoplasm. The ability of p37 protein to be exported from the nucleus to the cytoplasm of both yeast and mammalian cells was also demonstrated, and the results clearly indicate that p37 nuclear export is dependent on the interaction of the protein with the CRM-1 receptor. In addition, p37 was shown to exhibit nuclear import activity in mammalian cells. The p37 protein nuclear import and export abilities described here constitute the first report of a nucleocytoplasmic shuttling protein encoded by the ASFV genome. Overall, the overlapping results obtained for green fluorescent protein fusions and Myc-tagged proteins undoubtedly demonstrate that ASFV p37 and p14 proteins exhibit nucleocytoplasmic transport activities. These findings are significant for understanding the role these proteins play in the replication cycle of ASFV., (Copyright 2004 American Society for Microbiology)

Clathrin-mediated endocytosis has been described as the primary internalization pathway for many viruses, including the influenza virus. However, caveolae, an alternative clathrin-independent endocytotic pathway, has also been described as mediating the entry of some molecules, including viruses. To address the question of pathway selection by the influenza virus, we have investigated whether the virus is internalized via clathrin-coated pits and/or caveolae in Madin Darby canine kidney (MDCK) cells. By applying pharmacological manipulations to selectively disrupt the cell internalization pathways, we found that, in MDCK cells, the influenza virus may be internalized via caveolae in addition to entry by clathrin-mediated endocytosis. However, a small contribution by another mode of entry, as recently proposed, cannot be excluded.

We performed a detailed kinetic analysis of influenza virus fusion with the endosomal and plasma membranes of Madin Darby canine kidney (MDCK) cells and provided a comparison of the kinetic parameters obtained for both cases at 20 degrees C and 37 degrees C. Using our mass action kinetic model, we determined that the fusion rate constant, f, for influenza virus with the endosomal membrane was 0.02 s(-1) at 37 degrees C and 0.0035 s(-1) at 20 degrees C. The analysis of the fusion kinetics of influenza virus with the plasma membrane yielded that the fusion rate constants were close to those deduced with the endosomal membrane. The systematic kinetic analysis performed in this study provides for the first time a biophysical support for studies on influenza virus-cell fusion where the acidic endosomal internal environment is simulated artificially by lowering the pH of the medium.

Dopaminergic (DA) neurons of substantia nigra in the midbrain control voluntary movement, and their degeneration is the cause of Parkinson's disease. The complete set of genes required to specifically determine the development of midbrain DA subgroups is not known yet. We report here that mice lacking the bicoid-related homeoprotein Pitx3 fail to develop DA neurons of the substantia nigra. Other mesencephalic DA neurons of the ventral tegmental area and retrorubral field are unaltered in their dopamine expression and histological organization. These data suggest that Pitx3-dependent gene expression is specifically required for the differentiation of DA progenitors within the mesencephalic DA system.

Fluorescence assays for viral membrane fusion employ lipidic probes whose kinetics of fluorescence dequenching should mimic the actual kinetics of membrane merging. We examined the fusion of influenza virus with CEM cells, erythrocyte ghosts or liposomes by monitoring the fluorescence dequenching of each one of the three probes, octadecylrhodamine B chloride (R18), N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (Rh-PE), or rac-2,3-dioleoylglycerol ester of rhodamine B (DORh-B), inserted into the virus membrane. Experimental conditions were designed to allow a clear distinction between membrane mixing and non-specific probe transfer. Fluorescence dequenching observed with Rh-PE was much slower than with R18, unless a particular experimental procedure was used. Using liposomes as a target membrane, the kinetics and extent of the decrease in resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) and Rh-PE, initially embedded in the liposome membrane, were matched by that of the dequenching of viral R18, but not of viral Rh-PE. DORh-B was found not to be appropriate to follow membrane merging. Our results indicate that on a time scale of several minutes R18 more accurately reflects the kinetics of membrane fusion. Nevertheless, control experiments should be performed to evaluate non-specific probe transfer of R18 molecules, whose contribution to fluorescence dequenching can become significant after long incubation times.

The defense of the ocular surfaces presents an unique challenge in that not only must integrity be maintained against microbial, inflammatory and physical assault, but it must be done while minimizing the risk of loss of corneal transparency. This puts severe limitations on the degree to which scarring or neovascularization can occur in the cornea secondary to any infectious, inflammatory, immunological or wound healing process. Moreover, this defense system must be equally effective under two extremes of conditions: those found in the open eye and the closed eye environments. It is our contention that these constraints have resulted in the evolution of a highly complex fail-safe defense system that utilizes distinctly different strategies in open and closed eye conditions. The extraordinary effectiveness of this system is evidenced by the fact that despite continued exposure to a microbe rich environment, the external ocular surfaces maintain a very low microbial titer and are highly resistant to breaching by all but a few pathogens. It is the intent of this review to provide a working model of this defense system as it operates under both open and closed eye conditions, to provide evidence in support of this model as well as highlight some of the many areas of uncertainty.

The requirement for the nonreceptor tyrosine kinase c-abl in the pathogenesis of retinopathy of prematurity (ROP) was examined using the mouse model for ROP and c-abl-deficient mice. Hyperoxia-induced retinal neovascularization was observed in wild-type and heterozygous mice but animals that were homozygous null for c-abl did not develop a vasoproliferative retinopathy in response to hyperoxia. Two gene products, endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF), have been implicated in the pathogenesis of ROP. The mRNA expression of ET-1 and VEGF was assessed in mice maintained in normoxia and in hyperoxia-exposed mice. ET-1 mRNA levels were unchanged in wild-type mice throughout the hyperoxia treatment, suggesting that ET-1 mRNA expression is not regulated by the increase in inspired oxygen. In wild-type mice maintained in room air, VEGF mRNA levels rose threefold from postnatal day 6 (P6) to P17. When wild-type mice were treated with the hyperoxia regimen, a fivefold decrease in VEGF mRNA expression was observed from P7 to P16. However, retinal VEGF expression in hyperoxia-treated homozygous null mice did not decrease and remained at control levels. These data suggest that c-abl is required for the hyperoxia-induced retinal neovascularization and hyperoxia-induced decrease in VEGF mRNA levels.