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Background: The natural history of magnetic resonance imaging (MRI) in pre-ataxic stages of spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is not well known. We report cross-sectional and longitudinal data obtained at this stage., Methods: Baseline (follow-up) observations included 32 (17) pre-ataxic carriers (SARA < 3) and 20 (12) related controls. The mutation length was used to estimate the time to onset (TimeTo) of gait ataxia. Clinical scales and MRIs were performed at baseline and after a median (IQR) of 30 (7) months. Cerebellar volumetries (ACAPULCO), deep gray-matter (T1-Multiatlas), cortical thickness (FreeSurfer), cervical spinal cord area (SCT) and white matter (DTI-Multiatlas) were assessed. Baseline differences between groups were described; variables that presented a p < 0.1 after Bonferroni correction were assessed longitudinally, using TimeTo and study time. For TimeTo strategy, corrections for age, sex and intracranial volume were done with Z-score progression. A significance level of 5% was adopted., Results: SCT at C1 level distinguished pre-ataxic carriers from controls. DTI measures of the right inferior cerebellar peduncle (ICP), bilateral middle cerebellar peduncles (MCP) and bilateral medial lemniscus (ML), also distinguished pre-ataxic carriers from controls, and progressed over TimeTo, with effect sizes varying from 0.11 to 0.20, larger than those of the clinical scales. No MRI variable showed progression over study time., Discussion: DTI parameters of the right ICP, left MCP and right ML were the best biomarkers for the pre-ataxic stage of SCA3/MJD. TimeTo is an interesting timescale, since it captured the longitudinal worsening of these structures., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

Biodistribution assays are integral to gene therapy commercialization and have traditionally used real-time qPCR. Droplet digital PCR (ddPCR), however, has distinct advantages including higher sensitivity and absolute quantification but is underused because of lacking regulatory guidance and meaningful examples in the literature. We report a fit-for-purpose model process to validate a good laboratory practice (GLP)-compliant ddPCR assay for AVGN7, a Smad7 gene therapeutic for muscle wasting. Duplexed primer/probe sets for Smad7 and mouse TATA-box binding protein were optimized using gBlock DNA over a dynamic range of 10-80,000 copies/reaction in 250 ng mouse gDNA. Linearized plasmid and mouse gDNA were used for validation, which determined precision, accuracy, ruggedness/robustness, selectivity, recovery, specificity, dilution linearity, and stability. Inter-run precision and accuracy met previously established criteria with bias between -5% and 15%, coefficient of variation (CV) less than 19%, and total error within 8%-35%. The limit of detection was 2.5 copies/reaction, linearity was confirmed at 40-80,000 copies/reaction, specificity was demonstrated by single droplet populations and assay stability was demonstrated for benchtop, refrigerated storage, and repeated freeze-thaw cycles. The procedural road map provided exceeds recently established standards. It is also relevant to many IND-enabling processes, as validated ddPCR assays can be used in biodistribution studies and with vector titering and manufacturing quality control., Competing Interests: B.D.R. is the founder, CEO, and majority stock owner of AAVogen, Inc., which develops the proprietary gene therapeutic for muscle wasting (AVGN7) that is discussed herein. S.K.H. directs AAVogen’s preclinical development and regulatory affairs, while the other authors are staff scientists of Smithers. Neither S.K.H. nor the other authors have any financial interests to disclose., (© 2023 The Author(s).)

Background: Little is known about preclinical stages of Machado-Joseph disease, a polyglutamine disorder characterized by progressive adult-onset ataxia., Objective: We aimed to describe the longitudinal progression of clinical and oculomotor variables in the preataxic phase of disease., Methods: Carriers and noncarriers were assessed at three visits. Preataxic carriers (Scale for Assessment and Rating of Ataxia score < 3) expected to start ataxia in ≤4 years were considered near onset (PAN). Progressions of ataxic and preataxic carriers, considering status at the end of the study, were described according to the start (or its prediction) of gait ataxia (TimeToAfterOnset) and according to the study time., Results: A total of 35 ataxics, 38 preataxics, and 22 noncarriers were included. The "TimeToAfterOnset" timeline showed that Neurological Examination Scale for Spinocerebellar Ataxias (NESSCA; effect size, 0.09), Inventory of Non-Ataxia Symptoms (INAS0.07), and the vestibulo-ocular reflex gain (0.12) progressed in preataxic carriers, and that most slopes accelerate in PAN, turning similar to those of ataxics. In the study time, NESSCA (1.36) and vertical pursuit gain (1.17) significantly worsened in PAN, and 6 of 11 PANs converted to ataxia. For a clinical trial with 80% power and 2-year duration, 57 PANs are needed in each study arm to detect a 50% reduction in the conversion rate., Conclusions: NESSCA, INAS, vestibulo-ocular reflex, and vertical pursuit gains significantly worsened in the preataxic phase. The "TimeToAfterOnset" timeline unveiled that slopes of most variables are small in preataxics but increase and reach the ataxic slopes from 4 years before the onset of ataxia. For future trials in preataxic carriers, we recommend recruiting PANs and using the conversion rate as the primary outcome. © 2022 International Parkinson and Movement Disorder Society., (© 2022 International Parkinson and Movement Disorder Society.)

In feed manufacturing, the need for an effective combination of ingredients to produce a complete feed for animals is well known. However, limited quantitative data illustrate the effect of low homogeneity on the performance of animals. This study was conducted to determine the coefficient of variation (CV) of feed mixtures produced with different mixing times and the effects of these feeds with varying homogeneity consumed by 1-40-day-old broilers on their feed intake, body weight gain, feed conversion, mortality, and body weight uniformity. The mixing times used were 30, 60, 90, and 120 s, and the CV obtained from the feeds ranged from 49.5% to 5.4%. A linear decrease was observed in the feed CV with increasing mixing time, but sometimes, a longer mixing time did not present the lowest CV. The influence of the lack of feed homogeneity on the performance of broilers was mainly observed in body weight gain and, in some cases, in feed conversion. The effects on feed intake were more consistent for broilers older than 22 days, increasing body weight gain without affecting feed conversion. Our results suggest that feeds with CVs up to 22.6% had no adverse effects on the performance of broilers older than 12 days, and the lack of uniformity in broiler body weight was not influenced by feed homogeneity., Competing Interests: None, (© 2022 The Authors. Published by Elsevier Ltd.)

Although health-related quality of life (HRQoL) has been increasingly valued in healthcare and in clinical trials, there is scarce information about it in spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD). This study describes the HRQoL results obtained from ataxic SCA3/MJD subjects, and their non-ataxic offspring included in the BIGPRO (Biomarkers and genetic modifiers in a study of presymptomatic and symptomatic SCA3/MJD carriers) study. Demographic data, clinical scales, and HRQoL instruments EQ-5D-3L and SF-36 were collected. Subjects at 50% risk were genotyped in a double-blind manner. The time left until the onset of the disease was estimated for mutation carriers with a SARA < 3 and combined with disease duration of ataxic subjects (TimeToAfterOnset). Analyses were performed using PASW Statistics version 18.0, R version 4.0.0, and G*Power 3.1, and p < 0.05 was considered statistically significant. Twenty-three ataxic carriers, 33 pre-ataxic carriers, and 21 controls were enrolled. Significant differences between ataxic carriers and controls were seen in EQ-VAS, EQ-5D Index, and in some domains of EQ-5D-3L and SF-36. EQ-5D Index showed the best effect size between ataxic and controls (Cohen's d = 2.423). Stepwise changes were seen in pre-ataxic subjects, although not statistically significant. TimeToAfterOnset correlated with EQ-5D Index, EQ-VAS, and SF-36 Physical functioning, Role Physical, Pain, and General Health. EQ-5D Index and EQ-VAS correlated with clinical scales in the ataxic group. These results suggest that HRQoL worsens among carriers since pre-ataxic stages and that they might encompass the underlying disease process. In this cohort, SF-36 Physical Functioning, SF-36 General health, and especially EQ-5D Index and EQ-VAS were the best HRQoL instruments to be used as ancillary evidence to support biological and social meanings for future interventions., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: The pathological burden of spinocerebellar ataxia type 3, also known as Machado-Joseph disease (SCA3/MJD), accumulates before the beginning of symptoms. Our study aims at validating biomarkers for disease progression since pre-ataxic periods. We report on baseline findings of clinical scales and oculomotor neurophysiology., Methods: Ataxic (Scale for the Assessment and Rating of Ataxia > 2.5) and at 50% risk subjects were included. The latter were subdivided into noncarriers, pre-ataxic carriers near (PAN), or pre-ataxic carriers far from (PAFF) ataxia onset (AO), with 4 years from the predicted age at onset being the cutoff. The subjects were assessed by Neurological Examination Score for Spinocerebellar Ataxia (NESSCA), International Cooperative Ataxia Rating Scale (ICARS), Inventory of Non-Ataxic Signs (INAScount), Composite Cerebellar Functions Score and SCA Functional Index, and video-oculography, including the regression slope of vestibulo-ocular reflex gain (VORr), main sequence of volitional and reflexive vertical saccades, slow-phase velocity of central and gaze-evoked (SPV-GE) nystagmus, and vertical pursuit gain. Correction for multiple comparisons was performed; the threshold for statistical significance was P < 0.05., Results: A total of 35 ataxic, 14 PAN, 24 PAFF, and 22 noncarriers were included. All variables showed significant differences between groups and correlated to time to onset or time after onset, among all 73 SCA3/MJD carriers; none significantly changed with age in controls. NESSCA, ICARS, INAScount, VORr, main sequence of volitional saccades, and SPV-GE not only distinguished PAN from controls but also correlated with time left to AO., Conclusions: Clinical scales and video-oculography variables were already altered in pre-ataxic SCA3/MJD carriers and worsened with time. NESSCA, ICARS, INAScount, VORr, main sequence of vertical volitional saccades, and SPV-GE are good candidates to measure preclinical changes in SCA3/MJD. © 2021 International Parkinson and Movement Disorder Society., (© 2021 International Parkinson and Movement Disorder Society.)

Purpose: CTLA4, PTPN22, and CD40 are immune-regulatory genes strongly associated with GD, as well as PPARG, but their clinical significance in different populations is still uncertain., Methods: We genotyped 282 Brazilian GD patients (234 women and 48 men, 39.80 ± 11.69 years old), including 144 patients with GO, and 308 healthy control individuals (246 women and 62 men, 36.86 ± 12.95 years old)., Results: A multivariate analysis demonstrated that the inheritance of the GG genotype rs3087243 of CTLA4 (OR = 2.593; 95% CI = 1.630-4.123; p < 0.0001) and the CC genotype of rs3789607 of PTPN22 (OR = 2.668; 95% CI = 1.399-5.086; p = 0.0029) consisted in factors independent of the susceptibility to GD. The inheritance of polymorphic genotypes of rs5742909 of CTLA4 was associated with older age at the time of diagnosis (42.90 ± 10.83 versus 38.84 ± 11.81 years old; p = 0.0105), with higher TRAb levels (148.17 ± 188.90 U/L versus 112.14 ± 208.54 U/L; p = 0.0229) and the need for higher therapeutic doses of radioiodine (64.23 ± 17.16 versus 50.22 ± 16.86; p = 0.0237). The inheritance of the CC genotype of rs1883832 CD40 gene was more frequent among women (69.65%) than men (52.00%; p = 0.0186). The polymorphic genotype of PPARG gene (rs1801282) was associated with TPOAb positivity (p = 0.0391), and the GG genotype of rs2476601 of PTPN22 gene was associated with positivity for both TgAb (p = 0.0360) and TPOAb (p < 0.0001). Both polymorphic genotypes rs2476601 and rs3789607 of the PTPN22 gene were more frequent among nonsmoking patients (p = 0.0102 and p = 0.0124, respectively)., Conclusions: Our data confirm the important role of CTLA4 polymorphisms in GD susceptibility; demonstrate the role of PTPN22 polymorphisms in patients' clinical features; and suggest these genes may influence the severity of the disease.

Mutations in the mitochondrial fusion protein mitofusin (MFN) 2 cause the chronic neurodegenerative condition Charcot-Marie-Tooth disease type 2A (CMT2A), for which there is currently no treatment. Small-molecule activators of MFN1 and MFN2 enhance mitochondrial fusion and offer promise as therapy for this condition, but prototype compounds have poor pharmacokinetic properties. Herein, we describe a rational design of a series of 6-phenylhexanamide derivatives whose pharmacokinetic optimization yielded a 4-hydroxycyclohexyl analogue, 13 , with the potency, selectivity, and oral bioavailability of a preclinical candidate. Studies of 13 cis - and trans -4-hydroxycyclohexyl isostereomers unexpectedly revealed functionality and protein engagement exclusively for the trans form, 13B . Preclinical absorption, distribution, metabolism, and excretion (ADME) and in vivo target engagement studies of 13B support further development of 6-phenylhexanamide derivatives as therapeutic agents for human CMT2A.

Background: Fetal alcohol syndrome (FAS) is a disorder caused by alterations in embryo-fetal development due to prenatal alcohol exposure. It is estimated that between 0.5 and 2 per 1,000 individuals are born with FAS every year. In Brazil, there are few studies addressing the extent of the problem of FAS/fetal alcohol spectrum disorders (FASD); these studies are confined to limited geographic areas. Therefore, we decided to perform a health needs assessment for FAS/FASD in Brazil., Methods: To estimate the prevalence of FAS and FASD in Brazil, we used information from the literature, which estimates between 0.5 and 2/1,000 births per year for FAS and 10 to 50/1,000 for FASD., Results: We estimated that approximately 1,500 to 6,000 children are born with FAS every year. Considering the whole population, the prevalence would be 95,377 to 380,000 affected people. However, when we consider FASD as a whole, we estimate that between 1,900,000 and 9,500,000 Brazilians might suffer the more severe consequences of alcohol exposure during pregnancy and be living with FASD., Conclusion: The results of the current study indicate that FAS and FASD are prevalent disorders in Brazil, and more policies targeting alcohol intake during pregnancy must be developed., (© 2020 by the Research Society on Alcoholism.)

The objective of this study was to evaluate the use of a lyophilized and gamma-sterilized allogeneic freeze-dried bone wedge as a spacer for advancement of a modified tibial tuberosity (mTTA) in 16 knees that were clinically diagnosed with cranial cruciate ligament disease. Patients underwent radiography before the surgical procedure as well as immediately after surgery and at 30, 60, 90 and 120 days post-surgery, and their locomotion was evaluated at the same time points except for the immediate postoperative period. The surgical wounds were evaluated for signs of infection and rejection of the bone implant. Locomotion was graded on a scale of 0-5, with 0 indicating no limping and 5 indicating limb functional impotence. The "tibial-tibial bone-tibial implant" interfaces were evaluated radiographically, and each interface was assigned scores of 0-3, with 0 indicating no contact between the implant and adjacent bone and 3 indicating a bone bridge throughout the interface. The patients showed good clinical and radiographic recovery. The lyophilized bone spacer allowed for easy storage and transport and rapid and satisfactory execution of mTTA while showing resistance to drilling and fixation with screws in 87.5% of cases and a mean surgical time of 45.9 minutes. No immunogenic reactions were observed in 93.7% of the cases. One patient presented infection of the surgical focus, which showed remission after antimicrobial therapy. All patients showed functional recovery of the operated limb, with the number of clinically healthy patients being higher than those with claudication at 120 days (p ≤ 0.05). In all patients, it was possible to verify the incorporation of the bone implant into the tibia. Bone union occurred progressively, and the degrees of bone union observed on radiographs at postoperative days 60, 90, and 120 were significantly greater (p < 0.05) than those observed in the immediate postoperative period and at 30 days., Competing Interests: The authors have declared that no competing interests exist.

Spinocerebellar ataxia type 7 (SCA7) is a polyglutamine disease that progressively affects the cerebellum, brainstem, and retina. SCA7 is quite rare, and insights into biomarkers and pre-clinical phases are still missing. We aimed to describe neurologic and ophthalmological findings observed in symptomatic and pre-symptomatic SCA7 subjects. Several neurologic scales, visual acuity, visual fields obtained by computer perimetry, and macular thickness in optical coherence tomography (mOCT) were measured in symptomatic carriers and at risk relatives. Molecular analysis of the ATXN7 was done blindly in individuals at risk. Thirteen symptomatic carriers, 3 pre-symptomatic subjects, and 5 related controls were enrolled. Symptomatic carriers presented scores significantly different from those of controls in most neurologic and ophthalmological scores. Gradual changes from controls to pre-symptomatic and then to symptomatic carriers were seen in mean (SD) of visual fields - 1.34 (1.15), - 2.81 (1.66). and - 9.56 (7.26); mOCT - 1.11 (2.6), - 3.48 (3.54), and - 7.73 (2.56) Z scores; and "Spinocerebellar Ataxia Functional Index (SCAFI)" - 1.16 (0.28), 0.65 (0.56), and - 0.61 (0.44), respectively. Visual fields and SCAFI were significantly correlated with time to disease onset (pre-symptomatic)/disease duration (symptomatic carriers). Visual fields, mOCT, and SCAFI stood out as candidates for state biomarkers for SCA7 since pre-symptomatic stages of disease.

Aim: Conjugated critical reagents are a pillar of ligand binding analysis. Although good practices for characterization have already been discussed, little is known about how assays are affected by specific conjugation parameters. Results: Here we developed, characterized and screened a toolset of bioconjugates that provided new insights about the optimization of conjugated critical reagent attributes. Biotinylated and sulfo-tagged trastuzumab were utilized as capture and detection antibodies, respectively, in an antidrug antibody (ADA) assay. The optimal conjugation window was identified by functional assessment. Excess of unlabeled biotin, but not sulfo-tag, affected the assay performance. An increase in the assay baseline was observed when sulfo-tagged trastuzumab underwent increasing freeze-thaw cycles. Conclusion: Upfront systematic screening and characterization of conjugated critical reagent attributes benefit assay robustness.

In 1990, the first Teratogen Information Service in Brazil (SIAT) was implemented in the Medical Genetics Service at Hospital de Clinicas de Porto Alegre. SIAT is a free-to-use information service both to health professionals and the general population, especially to women who are pregnant or planning pregnancy. The main objective of this paper is to present the activities of SIAT in its initial years (1990-2006), compared to those in the last decade (2007-2017). In addition we review the scientific contribution of SIAT in the field of human teratogenesis. Since 1990, SIAT received 10,533 calls. Use of medications were the main reason for concern, accounting for 74% of all questions, followed by other chemical exposures (occupational, cosmetics, environmental), and maternal infectious diseases. Among its main contributions to scientific knowledge was the collaboration for the identification of two new human teratogens: misoprostol in the 1990s and Zika virus in 2015/16. In conclusion, SIAT is still evolving, as is the Medical Genetics Service that hosts it. Through its 27 years of existence more than 300 undergraduate and graduate students have rotated at SIAT. Presently, SIAT is expanding the research to experimental teratogenesis and to investigation of molecular mechanisms of teratogens.

Leaf-cutting ants belonging to the genus Atta (Formicidae: Myrmicinae) are important pests in agricultural and forest environments. In the present study, we evaluated the formicidal activity of the essential oil of Pogostemon cablin and its nanoformulation on the leaf-cutting ants: Atta opaciceps (Borgmeier, 1939), Atta sexdens (Linnaeus, 1758), and Atta sexdens rubropilosa Forel, 1908. The nanoformulation was developed by magnetic stirring using polyoxyethylene (36%), pure ethanol (36%), essential oil of P. cablin (18%), and water (10%). Bioassays of acute toxicity by fumigation and behavioral bioassays in treated arenas, with and without choice, were performed. The essential oil of P. cablin and its nanoformulation demonstrated efficient insecticidal activity and irritability to ant species. The concentration required to kill 50% of workers varied from 1.06 to 2.10 μL L -1 , with a mean time to death of less than or equal to 42 h. The essential oil of P. cablin and its nanoformulation reduced the displacement and velocity speed of the workers of A. opaciceps and A. sexdens rubropilosa in totally treated arenas. In the bioassays with choices, the three species of ants walked less and at a greater speed on the treated side of arena. This work demonstrates the potential of the essential oil of P. cablin and its nanoformulation to the generation of new formicidal products.

The cysteine desulfurase Nfs1/Isd11 uses the amino acid cysteine as the substrate and its activity is absolutely required for contributing persulfide sulfur to the essential process of iron-sulfur (Fe-S) cluster assembly in mitochondria. Here we describe a novel regulatory process involving phosphorylation of Nfs1 in mitochondria. Phosphorylation enhanced cysteine desulfurase activity, while dephosphorylation decreased its activity. Nfs1 phosphopeptides were identified, and the corresponding phosphosite mutants showed impaired persulfide formation. Nfs1 pull down from mitochondria recovered an associated kinase activity, and Yck2, a kinase present in the pull down, was able to phosphorylate Nfs1 in vitro and stimulate cysteine desulfurase activity. Yck2 exhibited an eclipsed distribution in the mitochondrial matrix, although other cellular localizations have been previously described. Mitochondria lacking the Yck2 protein kinase (∆yck2) showed less phosphorylating activity for Nfs1. Compared with wild-type mitochondria, ∆yck2 mitochondria revealed slower persulfide formation on Nfs1 consistent with a role of Yck2 in regulating mitochondrial cysteine desulfurase activity. We propose that Nfs1 phosphorylation may provide a means of rapid adaptation to increased metabolic demand for sulfur and Fe-S clusters within mitochondria., (Copyright © 2017 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Mitofusins (MFNs) promote fusion-mediated mitochondrial content exchange and subcellular trafficking. Mutations in Mfn2 cause neurodegenerative Charcot-Marie-Tooth disease type 2A (CMT2A). We showed that MFN2 activity can be determined by Met 376 and His 380 interactions with Asp 725 and Leu 727 and controlled by PINK1 kinase-mediated phosphorylation of adjacent MFN2 Ser 378 Small-molecule mimics of the peptide-peptide interface of MFN2 disrupted this interaction, allosterically activating MFN2 and promoting mitochondrial fusion. These first-in-class mitofusin agonists overcame dominant mitochondrial defects provoked in cultured neurons by CMT2A mutants MFN2 Arg 94 →Gln 94 and MFN2 Thr 105 →Met 105 , as demonstrated by amelioration of mitochondrial dysmotility, fragmentation, depolarization, and clumping. A mitofusin agonist normalized axonal mitochondrial trafficking within sciatic nerves of MFN2 Thr 105 →Met 105 mice, promising a therapeutic approach for CMT2A and other untreatable diseases of impaired neuronal mitochondrial dynamism and/or trafficking., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Friedreich's ataxia (FA) is an autosomal recessive disease caused by an intronic GAA triplet expansion in the FXN gene, leading to reduced expression of the mitochondrial protein frataxin. FA is estimated to affect 1 in 50 000 with a mean age of death in the fourth decade of life. There are no approved treatments for FA, although experimental approaches, which involve up-regulation or replacement of frataxin protein, are being tested. Frataxin is undetectable in serum or plasma, and whole blood cannot be used because it is present in long-lived erythrocytes. Therefore, an assay was developed for analyzing frataxin in platelets, which have a half-life of 10 days. The assay is based on stable isotope dilution immunopurification two-dimensional nano-ultra high performance liquid chromatography/parallel reaction monitoring/mass spectrometry. The lower limit of quantification was 0.078 pg frataxin/μg protein, and the assay had 100% sensitivity and specificity for discriminating between controls and FA cases. The mean levels of control and FA platelet frataxin were 9.4 ± 2.6 and 2.4 ± 0.6 pg/μg protein, respectively. The assay should make it possible to rigorously monitor the effects of therapeutic interventions on frataxin expression in this devastating disease.

Thyroid cancer therapy is based on surgery followed by radioiodine treatment. The incorporation of radioiodine by cancer cells is mediated by sodium iodide symporter (NIS) (codified by the SLC5A5 gene), that is functional only when targeted to the cell membrane. We aimed to evaluate if NIS expression in thyroid primary tumors would be helpful in predicting tumor behavior, response to therapy and prognosis. NIS expression was addressed by qPCR and immunohistochemistry. In order to validate our data, we also studied SLC5A5 expression on 378 primary papillary thyroid carcinomas from The Cancer Genome Atlas (TCGA) database. In our series, SLC5A5 expression was lower in carcinomas with vascular invasion and with extrathyroidal extension and in those harboring BRAF V600E mutation. Analysis of SLC5A5 expression from TCGA database confirmed our results. Furthermore, it showed that larger tumors, with locoregional recurrences and/or distant metastases or harboring RAS , BRAF and/or TERT promoter ( TERT p) mutations presented significantly less SLC5A5 expression. Regarding immunohistochemistry, 12/211 of the cases demonstrated NIS in the membrane of tumor cells, those cases showed variable outcomes concerning therapy success, prognosis and all but one were wild type for BRAF , NRAS and TERT p mutations. SLC5A5 mRNA lower expression is associated with features of aggressiveness and with key genetic alterations involving BRAF , RAS and TERT p. Mutations in these genes seem to decrease protein expression and its targeting to the cell membrane. SLC5A5 mRNA expression is more informative than NIS immunohistochemical expression regarding tumor aggressiveness and prognostic features., (© 2018 The authors.)

Fe-S clusters are cofactors that participate in diverse and essential biological processes. Mitochondria contain a complete machinery for Fe-S cluster assembly. Cysteine desulfurase (Nfs1) is required generation of a form of activated sulfur and is essential for the initial Fe-S cluster assembly step. Using mass-spectometry we identified proteins that were copurified with Nfs1 using a pull-down strategy, including a novel protein kinase. Furthermore, we were able to identify phosphorylation sites on the Nfs1 protein. These data and analyses support the research article "Cysteine desulfurase is regulated by phosphorylation of Nfs1 in yeast mitochondria" by Rocha et al. (in press) [1].

FeS-clusters are utilized by numerous proteins within several biological pathways that are essential for life. In eukaryotes, the primary FeS-cluster production pathway is the mitochondrial iron-sulfur cluster (ISC) pathway. In Saccharomyces cerevisiae, de novo FeS-cluster formation is accomplished through coordinated assembly with the substrates iron and sulfur by the scaffold assembly protein "Isu1". Sulfur for cluster assembly is provided by cysteine desulfurase "Nfs1", a protein that works in union with its accessory protein "Isd11". Frataxin "Yfh1" helps direct cluster assembly by serving as a modulator of Nfs1 activity, by assisting in the delivery of sulfur and Fe(ii) to Isu1, or more likely through a combination of these and other possible roles. In vitro studies on the yeast ISC machinery have been limited, however, due to the inherent instability of recombinant Isu1. Isu1 is a molecule prone to degradation and aggregation. To circumvent Isu1 instability, we have replaced yeast Isu1 with the fly ortholog to stabilize our in vitro ISC assembly system and assist us in elucidating molecular details of the yeast ISC pathway. Our laboratory previously observed that recombinant frataxin from Drosophila melanogaster has remarkable stability compared to the yeast ortholog. Here we provide the first characterization of D. melanogaster Isu1 (fIscU) and demonstrate its ability to function within the yeast ISC machinery both in vivo and in vitro. Recombinant fIscU has physical properties similar to that of yeast Isu1. It functions as a stable dimer with similar Fe(ii) affinity and ability to form two 2Fe-2S clusters as the yeast dimer. The fIscU and yeast ISC proteins are compatible in vitro; addition of Yfh1 to Nfs1-Isd11 increases the rate of FeS-cluster formation on fIscU to a similar extent observed with Isu1. Finally, fIscU expressed in mitochondria of a yeast strain lacking Isu1 (and its paralog Isu2) is able to completely reverse the deletion phenotypes. These results demonstrate fIscU can functionally replace yeast Isu1 and it can serve as a powerful tool for exploring molecular details within the yeast ISC pathway.

PolyGly is present in many proteins in various organisms. One example is found in a transmembrane β-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins, the mature portion of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (mSS) and enhanced green fluorescent protein (EGFP). Both t75-mSS and t75-EGFP were imported into isolated chloroplasts and their n75 removed. Resultant c75-mSS was associated with the envelope at the intermembrane space, whereas c75-EGFP was partially exposed outside the envelope. Deletion of polyGly or substitution of tri-Ala for the critical tri-Gly segment within polyGly caused each passenger to be targeted to the stroma. Transient expression of t75-EGFP in Nicotiana benthamiana resulted in accumulation of c75-EGFP exposed at the surface of the chloroplast, but the majority of the EGFP passenger was found free in the cytosol with most of its c75 attachment removed. Results of circular dichroism analyses suggest that polyGly within c75 may form an extended conformation, which is disrupted by tri-Ala substitution. These data suggest that polyGly is distinct from a canonical stop-transfer sequence and acts as a rejection signal at the chloroplast inner envelope., Competing Interests: The authors have declared that no competing interests exist.

Background: Activation of the mTOR pathway has been observed in thyroid cancer, but the biologic consequences regarding tumor behavior and patient prognosis remain poorly explored., Methods: We aimed to evaluate the associations of the mTOR pathway with clinicopathologic and molecular features and prognosis through the immunocharacterization of pmTOR and pS6 expression (as readouts of the pathway) in a series of 191 papillary thyroid carcinomas., Results: pmTOR expression was associated with distant metastases (P = .05) and persistence of disease (P = .05). Cases with greater expression of pmTOR were submitted to more 131 I treatments (r[102] = 0.2; P = .02) and a greater cumulative dose of radioactive iodine (r[100] = 0.3; P = .01). Positive pmTOR expression showed to be an independent risk factor for distant metastases (odds ratio = 18.2; 95% confidence interval 2.1-157.9; P = .01). In contrast, pS6 expression was associated with absence of extrathyroid extension (P = .001), well-defined tumor margins (P = .05), and wild-type BRAF status (P = .01). There was no correlation between the expression of pmTOR and pS6 expression (r[140] = 0.1; P = .3)., Conclusion: pmTOR expression is an indicator of aggressive, metastatic papillary thyroid carcinoma, being possibly implicated in refractoriness to therapy, while pS6 expression is associated with less aggressive pathologic features. Further studies are needed to understand better the biologic consequences of activation of the mTOR pathway in the behavior of thyroid cancer, namely the contribution of other pmTOR downstream effectors., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Fe-S clusters are critically important cofactors implicated in numerous cellular processes, including respiration, amino acid biosynthesis, cofactor biosynthesis, tRNA modification, DNA repair and regulation of gene expression. In the accompanying manuscript, Tanaka et al. show that reengineering of the isoprenoid biosynthetic pathway in E. coli (to bypass the usage of essential Fe-S cluster proteins by inserting the mevalonate pathway) can offset the indispensability of the Fe-S cluster biosynthetic systems. They show that the resulting Δisc Δsuf double mutants supplemented with mevalonate can grow slowly without detectable Fe-S cluster proteins. This result is astounding and raises interesting questions about what is essential and what is dispensable in the compendium of Fe-S cluster protein functions in this cell., (© 2015 John Wiley & Sons Ltd.)

Context: Telomerase promoter mutations (TERT) were recently described in follicular cell-derived thyroid carcinomas (FCDTC) and seem to be more prevalent in aggressive cancers., Objectives: We aimed to evaluate the frequency of TERT promoter mutations in thyroid lesions and to investigate the prognostic significance of such mutations in a large cohort of patients with differentiated thyroid carcinomas (DTCs)., Design: This was a retrospective observational study., Setting and Patients: We studied 647 tumors and tumor-like lesions. A total of 469 patients with FCDTC treated and followed in five university hospitals were included. Mean follow-up (±SD) was 7.8 ± 5.8 years., Main Outcome Measures: Predictive value of TERT promoter mutations for distant metastasization, disease persistence at the end of follow-up, and disease-specific mortality., Results: TERT promoter mutations were found in 7.5% of papillary carcinomas (PTCs), 17.1% of follicular carcinomas, 29.0% of poorly differentiated carcinomas, and 33.3% of anaplastic thyroid carcinomas. Patients with TERT-mutated tumors were older (P < .001) and had larger tumors (P = .002). In DTCs, TERT promoter mutations were significantly associated with distant metastases (P < .001) and higher stage (P < .001). Patients with DTC harboring TERT promoter mutations were submitted to more radioiodine treatments (P = .009) with higher cumulative dose (P = .004) and to more treatment modalities (P = .001). At the end of follow-up, patients with TERT-mutated DTCs were more prone to have persistent disease (P = .001). TERT promoter mutations were significantly associated with disease-specific mortality [in the whole FCDTC (P < .001)] in DTCs (P < .001), PTCs (P = .001), and follicular carcinomas (P < .001). After adjusting for age at diagnosis and gender, the hazard ratio was 10.35 (95% confidence interval 2.01-53.24; P = .005) in DTC and 23.81 (95% confidence interval 1.36-415.76; P = .03) in PTCs., Conclusions: TERT promoter mutations are an indicator of clinically aggressive tumors, being correlated with worse outcome and disease-specific mortality in DTC. TERT promoter mutations have an independent prognostic value in DTC and, notably, in PTC.

Calcium is an important second messenger in eukaryotic cells that regulates many different cellular processes. To elucidate calcium regulation in chloroplasts, we identified the targets of calcium-dependent phosphorylation within the stromal proteome. A 73 kDa protein was identified as one of the most dominant proteins undergoing phosphorylation in a calcium-dependent manner in the stromal extracts of both Arabidopsis and Pisum. It was identified as TKL (transketolase), an essential enzyme of both the Calvin-Benson-Bassham cycle and the oxidative pentose phosphate pathway. Calcium-dependent phosphorylation of both Arabidopsis isoforms (AtTKL1 and AtTKL2) could be confirmed in vitro using recombinant proteins. The phosphorylation is catalysed by a stroma-localized protein kinase, which cannot utilize GTP. Phosphorylation of AtTKL1, the dominant isoform in most tissues, occurs at a serine residue that is conserved in TKLs of vascular plants. By contrast, an aspartate residue is present in this position in cyanobacteria, algae and mosses. Characterization of a phosphomimetic mutant (S428D) indicated that Ser428 phosphorylation exerts significant effects on the enzyme's substrate saturation kinetics at specific physiological pH values. The results of the present study point to a role for TKL phosphorylation in the regulation of carbon allocation.

Context: Thyroglobulin (Tg) levels measured at the time of remnant ablation after thyroid hormone withdrawal (THW) were shown to have prognostic value in predicting disease-free status., Objectives: Our objectives were to determine whether stimulated Tg levels, measured at the time of remnant ablation performed under recombinant human TSH (rhTSH) stimulation, has value in predicting absence of detectable disease 1 year after radioiodine therapy and to compare the results obtained with this approach with a cohort of patients submitted to ablation after THW., Design: This was a prospective observational study., Setting and Patients: The study included 293 consecutive patients treated for a differentiated thyroid carcinoma with no initial evidence of distant metastasis. All patients were submitted to a total or near-total thyroidectomy, followed by ablation either under rhTSH (n = 151) or endogenous TSH stimulation (n = 142). Patients with positive Tg antibodies were excluded., Main Outcome Measures: The predictive value of Tg at ablation was assessed by receiver operating characteristic curve analysis., Results: In the rhTSH group, 96 patients (73.3%) were considered disease-free at 1 year. Stimulated Tg at ablation after rhTSH was found to be an independent prognostic indicator of disease persistence 12 months later. The highest-accuracy cutoff value for absence of detectable disease was defined as 7.2 ng/mL, with a negative predictive value of 90%. In the THW group, Tg at ablation also proved to have independent predictive value. Using the same threshold (7.2 ng/mL), the negative predictive value of Tg was 95% in the THW group., Conclusions: When rhTSH was used, stimulated Tg at ablation had independent predictive value for disease-free status 1 year later. A low stimulated Tg at rhTSH-aided ablation may be considered a favorable prognosis factor.

Calcium plays an important role in the regulation of several chloroplast processes. However, very little is still understood about the calcium fluxes or calcium-binding proteins present in plastids. Indeed, classical EF-hand containing calcium-binding proteins appears to be mostly absent from plastids. In the present study we analyzed the stroma fraction of Arabidopsis chloroplasts for the presence of novel calcium-binding proteins using 2D-PAGE separation followed by calcium overlay assay. A small acidic protein was identified by mass spectrometry analyses as the chloroplast protein CP12 and the ability of CP12 to bind calcium was confirmed with recombinant proteins. CP12 plays an important role in the regulation of the Calvin-Benson-Bassham Cycle participating in the assembly of a supramolecular complex between phosphoribulokinase and glyceraldehyde 3-phosphate dehydrogenase, indicating that calcium signaling could play a role in regulating carbon fixation.

Reactivation of telomerase has been implicated in human tumorigenesis, but the underlying mechanisms remain poorly understood. Here we report the presence of recurrent somatic mutations in the TERT promoter in cancers of the central nervous system (43%), bladder (59%), thyroid (follicular cell-derived, 10%) and skin (melanoma, 29%). In thyroid cancers, the presence of TERT promoter mutations (when occurring together with BRAF mutations) is significantly associated with higher TERT mRNA expression, and in glioblastoma we find a trend for increased telomerase expression in cases harbouring TERT promoter mutations. Both in thyroid cancers and glioblastoma, TERT promoter mutations are significantly associated with older age of the patients. Our results show that TERT promoter mutations are relatively frequent in specific types of human cancers, where they lead to enhanced expression of telomerase.

More than 70 years of studies have indicated that chloroplasts contain a significant amount of calcium, are a potential storage compartment for this ion, and might themselves be prone to calcium regulation. Many of these studies have been performed on the photosynthetic light reaction as well as CO(2) fixation via the Calvin-Benson-Bassham cycle, and they showed that calcium is required in several steps of these processes. Further studies have indicated that calcium is involved in other chloroplast functions that are not directly related to photosynthesis and that there is a calcium-dependent regulation similar to cytoplasmic calcium signal transduction. Nevertheless, the precise role that calcium has as a functional and regulatory component of chloroplast processes remains enigmatic. Calcium concentrations in different chloroplast subcompartments have been measured, but the extent and direction of intra-plastidal calcium fluxes or calcium transport into and from the cytosol are not yet very well understood. In this review we want to give an overview over the current knowledge on the relationship between chloroplasts and calcium and discuss questions that need to be addressed in future research.

In addition to redox regulation, protein phosphorylation has gained increasing importance as a regulatory principle in chloroplasts in recent years. However, only very few chloroplast-localized protein kinases have been identified to date. Protein phosphorylation regulates important chloroplast processes such as photosynthesis or transcription. In order to better understand chloroplast function, it is therefore crucial to obtain a complete picture of the chloroplast kinome, which is currently constrained by two effects: first, recent observations showed that the bioinformatics-based prediction of chloroplast-localized protein kinases from available sequence data is strongly biased; and, secondly, protein kinases are of very low abundance, which makes their identification by proteomics approaches extremely difficult. Therefore, the aim of this study was to obtain a complete list of chloroplast-localized protein kinases from different species. Evaluation of protein kinases which were either highly predicted to be chloroplast localized or have been identified in different chloroplast proteomic studies resulted in the confirmation of only three new kinases. Considering also all reports of experimentally verified chloroplast protein kinases to date, compelling evidence was found for a total set of 15 chloroplast-localized protein kinases in different species. This is in contrast to a much higher number that would be expected based on targeting prediction or on the general abundance of protein kinases in relation to the entire proteome. Moreover, it is shown that unusual protein kinases with differing ATP-binding sites or catalytic centres seem to occur frequently within the chloroplast kinome, thus making their identification by mass spectrometry-based approaches even more difficult due to a different annotation.

The role of protein phosphorylation for adjusting chloroplast functions to changing environmental needs is well established, whereas calcium signalling in the chloroplast is only recently becoming appreciated. The work presented here explores the potential cross-talk between calcium signalling and protein phosphorylation in chloroplasts and provides the first evidence for targets of calcium-dependent protein phosphorylation at the thylakoid membrane. Thylakoid proteins were screened for calcium-dependent phosphorylation by 2D gel electrophoresis combined with phospho-specific labelling and PsaN, CAS, and VAR1, among other proteins, were identified repeatedly by mass spectrometry. Subsequently their calcium-dependent phosphorylation was confirmed in kinase assays using the purified proteins and chloroplast extracts. This is the first report on the protein targets of calcium-dependent phosphorylation of thylakoid proteins and provides ground for further studies in this direction.

Chloroplasts and mitochondria are central to crucial cellular processes in plants and contribute to a whole range of metabolic pathways. The use of calcium ions as a secondary messenger in and around organelles is increasingly appreciated as an important mediator of plant cell signaling, enabling plants to develop or to acclimatize to changing environmental conditions. Here, we have studied the four calcium-dependent mitochondrial carriers that are encoded in the Arabidopsis genome. An unknown substrate carrier, which was previously found to localize to chloroplasts, is proposed to present a calcium-dependent S-adenosyl methionine carrier. For three predicted ATP/phosphate carriers, we present experimental evidence that they can function as mitochondrial ATP-importers., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

An available enzyme-linked immunosorbent assay (ELISA) was studied for the detection of anti-B. canis antibodies in the sera of dogs using, indirect fluorescent antibody test (IFAT) as a reference test. ELISA uses a soluble antigenic preparation of B. canis and the optimal dilutions of the antigen, serum and conjugate were determined by check board titration, using positive and negative reference serum. The soluble antigen preparation of B. canis merozoites was 10 microg x mL(-1), with reference sera from positive and negative in a single dilution of 1:100, and conjugated to 1:4.000. A total of 246 serum samples were collected from dogs during the rabies vaccination campaign in Jaboticabal, São Paulo, Brazil and examined for the presence of antibodies against B. canis by ELISA and IFAT. Under these conditions, the average absorbance of negative serum was 0.129 + or - 0.025, resulting in a cut-of value of 0.323 (ELISA level 3) and the average absorbance of positive reference serum was 2.156 + or - 1.187. The serological positive samples tested for B. canis by ELISA and IFAT were 67.89% (n = 167) and 59.35% (n = 146), respectively. These results suggest that ELISA described may prove to be an effective serological test to diagnose canine babesiosis.

Purpose: In Canada, the incidence of childhood obesity has tripled within the past 20 years. The prevalence of obesity in the Timmins, Ontario, student population was studied to gain knowledge for program planning and resource allocation, and to compare Centers for Disease Control and Prevention (CDC) criteria with Cole's international criteria for childhood obesity., Methods: Anthropometric measurements of 801 students were taken. Students were chosen from randomly selected schools for each grade. Data were analyzed according to age, gender, and ethnicity. Data were also compared with other studies. Intragroup comparisons were performed using hypothesis testing for significance with the z table and chi-square test., Results: Overweight and obesity prevalence was 28% according to CDC criteria. No statistical difference was found between genders or among ethnic groups, or between this study and other Canadian studies. In comparison with the CDC criteria, Cole's international criteria indicated less obesity and increased overweight prevalence. These differences were not statistically significant., Conclusions: The findings suggest that in the northern Ontario community of Timmins, the prevalence of childhood obesity is of epidemic proportions. When the findings are shared with different agencies, this study will help the health unit to take necessary public health measures to curb the epidemic.

Chromophobe renal cell carcinoma (CRCC) may be grossly and microscopically confused with oncocytoma. It is now believed that many, if not all, of the so-called malignant oncocytomas or oncocytomas with metastases reported in the literature were indeed chromophobe renal cell carcinomas. CRCC is characteristically positive for colloidal iron and shows cytoplasmic microvesicles in electron microscopy. This study of CRCC is thought to be the first one done in Latin America. Of a total of 106 renal epithelial neoplasms, 7 (6.6%) fulfilled the criteria for chromophobe renal cell carcinoma. This frequency in Brazil is similar to that in other parts of the world. There was no difference in age, sex, and race distribution of CRCC compared to usual renal epithelial tumors. Grossly, the CRCC ranged in size from 3.5 to 20 cm (average: 10.2 cm) in greatest dimension. Most frequently, the tumor was brown on the cut surface. The growth pattern showed compact areas in all tumors and, in most of the cases, both clear and eosinophilic cellular subtypes were seen. The electron microscopic findings favor an origin of the microvesicles from outpouchings of the outer membrane of mitochondria. The strong positivity for colloidal iron in spite of the destruction of the cytoplasmic vesicles in paraffin-embedded specimens seems to indicate that the acid mucopolysaccharides are not located inside the microvesicles. By the time of diagnosis, only one case had regional lymph node metastases and this particular case was the only one mixed (associated with the usual renal cell carcinoma). The follow-up examination after nephrectomy showed that prognosis seems to be favorable in CRCC, except when the tumor coexists with the usual renal cell carcinoma.

Thirty bypass grafts to coronary arteries were made possible by manual core endarterectomy. The overall patency rate of the grafts was 57%, which is less than the patency rate of bypass grafts to nonendarterectomized vessels (75 to 80%). In the perioperative period and during follow-up to 29 months there was no increase in mortality or morbidity even when the endarterectomized vessel subsequently became occluded. These results represent early technical experience. Routine anticoagulant therapy in the postoperative period, to prevent early occlusion, was not used. The results of this and other studies suggest that coronary endarterectomy with bypass grafting is a useful procedure in situations where the coronary artery is so severely obstructed that standard saphenous vein bypass grafting cannot be performed; the procedure is superior to coronary endarterectomy alone.

Reconstruction of chest wall defects is performed by a variety of surgical procedures. Use of the pectoralis major myodermal flap following a Clagett procedure appears to be a reliable technique providing satisfactory results. The application of this technique demands a close interaction between the plastic surgeon and the thoracic surgeon.

Twenty-two instances of diaphragmatic rupture secondary to blunt thoracoabdominal trauma were seen at the Trauma Unit of the Health Sciences Centre, Winnipeg, Manitoba, Canada during a 30 year period. Diaphragmatic laceration occurred in the right leaf in 11, in the left in ten, and in both sides in one instance. In 14, the diagnosis was made and repair effected within 24 hours of presentation. Seven were diagnosed and treated from three days to several years after the injury. Two patients died soon after admission. They were victims of multiple intra-abdominal as well as intrathoracic injuries. Repair was generally effected through a laparotomy during the immediate post-traumatic period. Thoracotomy was used in those diagnosed after the latent interval. The diagnosis of diaphragmatic disruption should be considered in any patient suffering from blunt thoracoabdominal trauma. Usually a roentgenogram of the chest will confirm the suspected injury. In contradistinction to most of the reported series, our experience indicates that right diaphragmatic injuries are more common than what is usually thought.

Twenty patients undergoing a posterolateral thoracotomy for lung resection or a nonpulmonary procedure were divided into four groups. Group 1 was the control group. Patients in Group 2 had an intercostal nerve block at the time of closure. Those in Group 3 underwent a continuous intercostal nerve block for five days. Electronic pain control was used in Group 4. An additional group of patients underwent operation through an anterolateral thoracotomy (Group 5) and was compared with the control group. Breathing performance was evaluated daily for five days with bedside spirometry, and intergroup comparison was done utilizing the unpaired t test and analysis of variance. Forced expiratory volume in one second, expressed as percent of preoperative values, was significantly better in Group 3 (continuous intercostal nerve block) at 52.4 +/- 9.2% (standard deviation; p less than 0.05) and in Group 5 (anterolateral thoracotomy) at 52.0 +/- 7.5% (p less than 0.05) than in the control group (38.4 +/- 8.8%) five days postoperatively. It is concluded that bedside spirometry is a simple and reliable technique to assess postoperative changes in ventilatory mechanics due to pain. The pain that follows posterolateral thoracotomy can be substantially decreased with a continuous intercostal nerve block. Anterolateral thoracotomy is notably less painful than posterolateral thoracotomy and should be considered the approach of choice for patients with decreased pulmonary reserve who undergo uncomplicated pulmonary resection.

Two patients who had oropharyngeal infection with extension into the thoracic cavity through the fascial planes were recently seen at the Health Sciences Centre in Winnipeg. In both instances the infection was odontogenic. A 5-year review of the literature yielded 14 other cases with a similar presentation. In most cases the infection was odontogenic. The overall mortality was 25% (four deaths in 16 patients). Transient deficiency of the swallowing mechanism led to aspiration pneumonia in 44% of the patients. An aggressive surgical approach and appropriate antibiotic therapy are essential in managing these patients. Oral intake should be restricted and nasogastric tube feedings should be considered to diminish the risk of aspiration pneumonia.

Thrombotic malfunction of a Björk-Shiley aortic valve prosthesis occurred in three patients 6 to 16 months postoperatively. None of the patients had been taking anticoagulants. Although the presentation was acute, prodromal symptoms could be identified retrospectively in two of the patients. Two patients survived thrombectomy. Postoperative anticoagulant therapy is recommended in patients with these prostheses despite factors that may make such therapy riskier in specific patients. Attention to the character of murmurs and of the closure sound of the prosthetic valve must be part of the routine follow-up. In the emergency situation, when delay must be avoided, catheterization and angiography are unnecessary. The operative approach consists of complete thrombectomy without replacement of the valve or any of its components unless there is obvious periprosthetic leak or prosthetic wear.

Rupture of the trachea and major bronchi usually results from blunt trauma to the chest. To define the characteristics of the condition, the mechanism of injury, presentation and hospital course, the authors reviewed seven cases of tracheobronchial rupture after blunt trauma. Four were injured in a motor vehicle accident, two had a crush injury and one child had a fall from a playground swing. The site of injury was the left main bronchus in three, membranous trachea in two and bilateral bronchi and bronchus intermedius in one patient each. Bronchoplastic repair was performed in four patients with salvage of lung tissue in three. One patient required pulmonary resection. Two patients with a laceration of the membranous trachea did not undergo operation. Two patients died secondary to associated head injuries; one patient underwent repair of the bronchus intermedius and one patient had a lacerated membranous trachea. The possible mechanisms of injury are: rapid deceleration with forward swing of the trachea, widening of the transverse diameter of the chest, exerting traction on the carina, and a rapid rise of airway pressure on impact. Surgical intervention is recommended at an early stage to avoid loss of lung tissue.

Acute renal failure secondary to renal artery occlusion is rare but can be reversed and is therefore important to cases to draw attention to the diagnosis and management of the condition. In one case, occlusion of the artery to a single functioning kidney was responsible for the clinical presentation; in the other, bilateral renal artery emboli were responsible. The authors present a detailed review of the English literature to determine the prognostic value of the duration of anuria preceding operation in patients with renal artery occlusion to a solitary kidney. This factor showed no correction with viability of renal tissue, functional recovery or patient survival. Surgical management offers the best prospect of success in these patients.

The relation between mixed venous oxygen saturation and cardiac index was determined in 11 children who underwent surgical treatment for congenital heart disease. The correlation between these two variables was found to be reliable (r = 0.78, P = 0.001). The simple determination of mixed venous oxygen saturation performance, particularly when sophisticated equipment for measuring cardiac output is not available.