Strengths and limitations of reduced representation bisulfite sequencing (RRBS) in the perspective of DNA methylation analysis in fish: a case-study on rainbow trout spermatozoa.

DNA methylation in CpG dinucleotides is an important epigenetic mark in fish spermatozoa since it has been shown that some sperm methylome features are transmitted to the offspring. Reduced representation bisulfite sequencing (RRBS) is one genome-scale methods developed to assess DNA methylation at CpG sites. It allows the sequencing of a reduced fraction of the genome expected to be enriched in CpGs. The aim of this study is to characterize the extent of the CpG sites that can be identified in the RRBS-reduced sequenced fraction of rainbow trout spermatozoa, in order to evaluate the potential of RRBS for sperm DNA methylation studies. We observed that RRBS did provide a reduced amount of genomic data, the sum of the CpGs analyzed on 12 males spanning 9% of the total genomic CpGs. CpGs were only slightly enriched in the RRBS data (×1.7 times the sequenced nucleotides), the possible causes being linked to trout genome structure and sequenced fragments size. All genomic functional features were represented in our CpG dataset, with a noticeable enrichment in exons but, strikingly, not in promoters. The number of CpGs shared between biological replicates was low, but this proportion reached workable values from six biological replicates (46% of the analyzed cytosines) on. The choices that are to be made regarding fragment size selection and the options during bioinformatic data processing are discussed. In all, RRBS is a relevant first-approach method to scan the CpG DNA methylation status of spermatozoa along rainbow trout genome, although in a very reduced pattern among biological replicates.

Pembrolizumab Every 6 Weeks Versus Every 3 Weeks in Advanced Non-Small Cell Lung Cancer.

BACKGROUND: The survival benefits and adverse effects of pembrolizumab 2 mg/kg intravenously (IV) every 3 weeks (Q3W) in advanced non-small lung cancer (NSCLC) are well documented in the literature. Based on pharmacokinetic models, a pembrolizumab 4 mg/kg IV every 6 weeks (Q6W) dosing regimen is also approved in some countries. To date, there is no direct comparison in the literature between these 2 regimens in advanced NSCLC. METHODS: This retrospective study included 80 patients with advanced NSCLC who received pembrolizumab monotherapy 4 mg/kg Q6W between March 1, 2020 and December 31, 2021 and 80 patients with advanced NSCLC who received pembrolizumab monotherapy 2 mg/kg Q3W between January 1, 2017 and January 15, 2019 at Institut universitaire de cardiologie et de pneumologie de Québec (IUCPQ). The primary outcomes of this study were to compare overall survival (OS), progression-free survival (PFS) as well as the occurrence and severity of immune-mediated adverse events (AEs) in patients with advanced NSCLC who received pembrolizumab Q6W vs Q3W. Data cutoff date was December 15, 2022. RESULTS: Median follow-up was 14.5 ± 8.6 months in the Q6W group and 18.3 ± 19.6 months in the Q3W group. Median PFS was 6.9 months (CI 95% 5.0-10.7) in the Q6W group vs 8.9 months (CI 95% 5.6-14.1) in the Q3W group (adjusted HR 1.27 (CI 95% 0.85-1.89), P = .25). Median OS was not reached in the Q6W group vs 20.5 months (CI 95% 13.7-29.8) in the Q3W group (adjusted HR 0.80 (CI 95% 0.50-1.29), P = .36). Immune-mediated AEs of grade ≥ 3 occurred in 18% of patients in the Q6W group and in 19% of those in the Q3W group. CONCLUSIONS: In this unicentric retrospective study, the pembrolizumab Q6W dosing regimen was comparable to the Q3W in terms of OS, PFS, and toxicity.

Incidental findings suggestive of COVID-19 in asymptomatic cancer patients undergoing 18F-FDG PET/CT in a low prevalence region.

PURPOSE: To assess the rate and pattern of incidental interstitial lung abnormalities suggestive of COVID-19 on 18F-FDG PET/CT in asymptomatic cancer patients during the period of active COVID-19 circulation between March and April 2020 in a geographic area of low prevalence of the virus. METHODS: 1396 18F-FDG PET/CT performed between January 1, 2020, and February 21, 2020, and between March 16, 2020, and April 17, 2020 for routine oncological indication were retrospectively analyzed. No patients had symptoms suggestive of COVID-19 at the time of the 18F-FDG PET/CT. Incidental interstitial pneumonias suggestive of COVID-19 were identified, and the 18F-FDG PET/CT patterns were described. We compared the incidence of these lesions in the pre-COVID and pandemic phases. RESULTS: We observed a 1.6% increase in interstitial lung abnormalities during the period of COVID-19 circulation. All had < 50% lung involvement. We describe a case series with typical and atypical interstitial pneumonias suggestive of COVID-19 as unilateral or bilateral with ground-glass opacity, consolidation, or crazy-paving patterns. CONCLUSION: The relatively low increase in incidental findings suggestive of COVID-19 infection on 18F-FDG PET/CT in asymptomatic cancer patients was in accordance with the low COVID-19 transmission in our geographic region. Nevertheless, nuclear medicine physicians should familiarize themselves with typical and atypical 18F-FDG PET/CT patterns suggestive of COVID-19 pneumonia and initiate appropriate intervention where necessary.

Epidemiological characteristics of the COVID-19 spring outbreak in Quebec, Canada: a population-based study.

BACKGROUND: By mid-July 2020, more than 108,000 COVID-19 cases had been diagnosed in Canada with more than half in the province of Quebec. In this context, we launched a study to analyze the epidemiological characteristics and the socio-economic impact of the spring outbreak in the population. METHOD: We conducted an online survey of the participants of the CARTaGENE population-based cohort, composed of middle-aged and older adults. We collected information on socio-demographic, lifestyle, health condition, COVID-19 related symptoms and COVID-19 testing. We studied the association between these factors and two outcomes: the status of having been tested for SARS-CoV-2 and the status of having received a positive test. These associations were measured with univariate and multivariate analyses using a hybrid tree-based regression model. RESULTS: Among the 8,129 respondents from the CARTaGENE cohort, 649 were tested for COVID-19 and 41 were positive. Medical workers and individuals having a contact with a COVID-19 patient had the highest probabilities of being tested (32% and 42.4%, respectively) and of being positive (17.2% and 13.0%, respectively) among those tested. Approximately 8% of the participants declared that they have experienced at least one of the four COVID-19 related symptoms chosen by the Public Health authorities (fever, cough, dyspnea, anosmia) but were not tested. Results from the tree-based model analyses adjusted on exposure factors showed that the combination of dyspnea, dry cough and fever was highly associated with being tested whereas anosmia, fever, and headache were the most discriminant factors for having a positive test among those tested. During the spring outbreak, more than one third of the participants have experienced a decrease in access to health services. There were gender and age differences in the socio-economic and emotional impacts of the pandemic. CONCLUSION: We have shown some discrepancies between the symptoms associated with being tested and being positive. In particular, the anosmia is a major discriminant symptom for positivity whereas ear-nose-throat symptoms seem not to be COVID-19 related. The results also emphasize the need of increasing the accessibility of testing for the general population.

DNA methylation stability in fish spermatozoa upon external constraint: Impact of fish hormonal stimulation and sperm cryopreservation.

Highly differentiated mature spermatozoa carry not only genetic but also epigenetic information that is to be transmitted to the embryo. DNA methylation is one epigenetic actor associated with sperm nucleus compaction, gene silencing, and prepatterning of embryonic gene expression. Therefore, the stability of this mark toward reproductive biotechnologies is a major issue in animal production. The present work explored the impact of hormonal induction of spermiation and sperm cryopreservation in two cyprinids, the goldfish (Carassius auratus) and the zebrafish (Danio rerio), using LUminometric Methylation Assay (LUMA). We showed that while goldfish hormonal treatment did increase sperm production, it did not alter global DNA methylation of spermatozoa. Different sperm samples repeatedly collected from the same males for 2 months also showed the same global DNA methylation level. Similarly, global DNA methylation was not affected after cryopreservation of goldfish spermatozoa with methanol, whereas less efficient cryoprotectants (dimethylsulfoxide and 1,2-propanediol) decreased DNA methylation. In contrast, cryopreservation of zebrafish spermatozoa with methanol induced a slight, but significant, increase in global DNA methylation. In the less compact nuclei, that is, goldfish fin somatic cells, cryopreservation did not change global DNA methylation regardless of the choice of cryoprotectant. To conclude, global DNA methylation is a robust parameter with respect to biotechnologies such as hormonal induction of spermiation and sperm cryopreservation, but it can be altered when the best sperm manipulation conditions are not met.

Anaplastic lymphoma kinase 5A4 immunohistochemistry as a diagnostic assay in lung cancer: A Canadian reference testing center's results in population-based reflex testing.

BACKGROUND: The presence of anaplastic lymphoma kinase (ALK) rearrangement predicts response to ALK tyrosine kinase inhibitor (TKI) therapy. Fluorescence in situ hybridization (FISH) was the initial reference standard to detect ALK rearrangement, but immunohistochemistry (IHC) using D5F3 has gained acceptance as an alternative diagnostic method. ALK IHC assays using other ALK antibodies have also been used as screening methods, but data supporting their utility as diagnostic tests have not been widely reported. METHODS: Data from reflexive clinical ALK IHC test using the 5A4 clone concurrent with epidermal growth factor receptor (EGFR) mutation testing were analyzed. ALK IHC results were reported as negative (-), equivocal, or positive (+), with equivocal or positive staining validated by FISH break-apart probe testing. Treatment outcomes were reviewed for ALK IHC+ patients. RESULTS: Between 2012 and 2015, 146 (2.5%) cases were reported as ALK IHC+, 188 (3.2%) were reported as equivocal, and 5624 (94.4%) were reported as ALK IHC-. Of the ALK IHC+ cases, 131/143(91.6%) were ALK FISH+. Excluding 6 cases in which FISH was inconclusive or not performed, the positive predictive value was 95.6%, and the negative predictive value was 100%. Most specimens (n = 5352 [89.6%]) were also successfully tested for EGFR. Clinical responses to ALK TKIs were noted in 49 ALK IHC+ patients, with a median progression-free survival of 9.9 months. CONCLUSIONS: ALK 5A4 IHC can serve as a robust diagnostic test for ALK-rearranged lung cancer and is associated with treatment response and survival. Optimized tissue allocation resulted in high success rates of combined reflex EGFR and ALK testing.

The Impact of Brain Metastases and Associated Neurocognitive Aspects on Health Utility Scores in EGFR Mutated and ALK Rearranged NSCLC: A Real World Evidence Analysis.

BACKGROUND: In lung cancer, brain metastases (BM) and their treatment are associated with high economic burden and inferior health-related quality of life. In the era of targeted therapy, real world evidence through health utility scores (HUS) is critical for economic analyses. MATERIALS AND METHODS: In a prospective observational cohort study (2014-2016), outpatients with stage IV lung cancer completed demographic and EQ-5D-3L surveys (to derive HUS). Health states and clinicopathologic variables were obtained from chart abstraction. Patients were categorized by the presence or absence of BM; regression analyses identified factors that were associated with HUS. A subset of patients prospectively completed neurocognitive function (NCF) tests and/or the FACT-brain (FACT-Br) questionnaire, which were then correlated with HUS (Spearman coefficients; regression analyses). RESULTS: Of 519 patients with 1,686 EQ-5D-3L-derived HUS, 94 (18%) completed NCF tests and 107 (21%) completed FACT-Br; 301 (58%) never developed BM, 24 (5%) developed first BM during study period, and 194 (37%) had BM at study entry. The sample was enriched (46%) for EGFR mutations (EGFRm) and ALK-rearrangements (ALKr). There were no HUS differences by BM status overall and in subsets by demographics. In multivariable analyses, superior HUS was associated with having EGFRm/ALKr (p < .0001), no prior radiation for extracranial disease (p < .001), and both intracranial (p = .002) and extracranial disease control (p < .01). HUS correlated with multiple elements of the FACT-Br and tests of NCF. CONCLUSION: Having BM in lung cancer is not associated with inferior HUS in a population enriched for EGFRm and ALKr. Patients exhibiting disease control and those with oncogene-addicted tumors have superior HUS. IMPLICATIONS FOR PRACTICE: In the setting of EGFR mutations or ALK rearrangement non-small cell lung cancer (NSCLC), a diagnosis of brain metastases no longer consigns the patient to an inferior health state suggesting that new economic analyses in NSCLC are needed in the era of targeted therapies. Additionally, the EQ-5D questionnaire is associated with measures of health-related quality of life and neurocognitive scores suggesting this tool should be further explored in prospective clinical studies.

Association of MAPT H1 subhaplotypes with neuropathology of lewy body disease.

BACKGROUND: Genetic variation at the microtubule-associated protein tau locus is associated with clinical parkinsonism. However, it is unclear as to whether microtubule-associated protein tau H1 subhaplotypes are associated with the burden of neuropathological features of Lewy body disease. OBJECTIVES: To evaluate associations of microtubule-associated protein tau haplotypes with severity of Lewy body pathology and markers of SN neuronal loss in Lewy body disease cases. METHODS: Five hundred eighty-five autopsy-confirmed Lewy body disease cases were included. Six microtubule-associated protein tau variants (rs1467967, rs242557, rs3785883, rs2471738, rs8070723, and rs7521) were genotyped to define common microtubule-associated protein tau haplotypes. Lewy body counts were measured in five cortical regions. Ventrolateral and medial SN neuronal loss were assessed semiquantitatively. Nigrostriatal dopaminergic degeneration was quantified by image analysis of tyrosine hydroxylase immunoreactivity in the dorsolateral and ventromedial putamen. RESULTS: The common microtubule-associated protein tau H2 haplotype did not show a strong effect on pathological burden in Lewy body disease. The rare H1j haplotype (1.3%) was significantly associated with a lower dorsolateral putaminal tyrosine hydroxylase immunoreactivity (and therefore greater dopaminergic degeneration) compared to other microtubule-associated protein tau haplotypes (P = 0.0016). Microtubule-associated protein tau H1j was also nominally (P ≤ 0.05) associated with a lower ventromedial putaminal tyrosine hydroxylase immunoreactivity (P = 0.010), but this did not survive multiple testing correction. Other nominally significant associations between microtubule-associated protein tau H1 subhaplotypes and neuropathological outcomes were observed. CONCLUSIONS: A rare microtubule-associated protein tau H1 subhaplotype (H1j) may be associated with more severe putaminal dopaminergic degeneration in Lewy body disease cases. Microtubule-associated protein tau H1j has been associated previously with an increased risk of PD, and therefore our exploratory findings provide insight into the mechanism by which H1j modulates PD risk. © 2019 International Parkinson and Movement Disorder Society.

Multiple system atrophy and apolipoprotein E.

BACKGROUND: Dysregulation of the specialized lipid metabolism involved in myelin synthesis and maintenance by oligodendrocytes has been associated with the unique neuropathology of MSA. We hypothesized that apolipoprotein E, which is associated with neurodegeneration, may also play a role in the pathogenesis of MSA. OBJECTIVE: This study evaluated genetic associations of Apolipoprotein E alleles with risk of MSA and α-synuclein pathology, and also examined whether apolipoprotein E isoforms differentially affect α-synuclein uptake in a oligodendrocyte cell. METHODS: One hundred sixty-eight pathologically confirmed MSA patients, 89 clinically diagnosed MSA patients, and 1,277 control subjects were genotyped for Apolipoprotein E. Human oligodendrocyte cell lines were incubated with α-synuclein and recombinant human apolipoprotein E, with internalized α-synuclein imaged by confocal microscopy and cells analyzed by flow cytometry. RESULTS: No significant association with risk of MSA or was observed for either Apolipoprotein E ɛ2 or ɛ4. α-Synuclein burden was also not associated with Apolipoprotein E alleles in the pathologically confirmed patients. Interestingly, in our cell assays, apolipoprotein E ɛ4 significantly reduced α-synuclein uptake in the oligodendrocytic cell line. CONCLUSIONS: Despite differential effects of apolipoprotein E isoforms on α-synuclein uptake in a human oligodendrocytic cell, we did not observe a significant association at the Apolipoprotein E locus with risk of MSA or α-synuclein pathology. © 2018 International Parkinson and Movement Disorder Society.

Full sequencing and haplotype analysis of MAPT in Parkinson's disease and rapid eye movement sleep behavior disorder.

BACKGROUND: MAPT haplotypes are associated with PD, but their association with rapid eye movement sleep behavior disorder is unclear. OBJECTIVE: To study the role of MAPT variants in rapid eye movement sleep behavior disorder. METHODS: Two cohorts were included: (A) PD (n = 600), rapid eye movement sleep behavior disorder (n = 613) patients, and controls (n = 981); (B) dementia with Lewy bodies patients with rapid eye movement sleep behavior disorder (n = 271) and controls (n = 950). MAPT-associated variants and the entire coding sequence of MAPT were analyzed. Age-, sex-, and ethnicity-adjusted analyses were performed to examine the association between MAPT, PD, and rapid eye movement sleep behavior disorder. RESULTS: MAPT-H2 variants were associated with PD (odds ratios: 0.62-0.65; P = 0.010-0.019), but not with rapid eye movement sleep behavior disorder. In PD, the H1 haplotype odds ratio was 1.60 (95% confidence interval: 1.12-2.28; P = 0.009), and the H2 odds ratio was 0.68 (95% confidence interval: 0.48-0.96; P = 0.03). The H2/H1 haplotypes were not associated with rapid eye movement sleep behavior disorder. CONCLUSIONS: Our results confirm the protective effect of the MAPT-H2 haplotype in PD, and define its components. Furthermore, our results suggest that MAPT does not play a major role in rapid eye movement sleep behavior disorder, emphasizing different genetic background than in PD in this locus. © 2018 International Parkinson and Movement Disorder Society.

Monitoring and Management of Immune-Related Adverse Events Associated With Programmed Cell Death Protein-1 Axis Inhibitors in Lung Cancer.

Monoclonal antibodies targeting programmed cell death protein-1 (PD-1) represent a new treatment paradigm in non-small cell lung cancer. Three phase III trials have demonstrated a survival benefit and improved tolerability of nivolumab and pembrolizumab when compared with standard second-line chemotherapy. Nevertheless, the adverse events associated with PD-1 inhibitors are unique; early recognition and treatment are essential. This review summarizes the required monitoring and appropriate management of immune-related adverse events in lung cancer patients receiving these agents. THE ONCOLOGIST: 2017;22:70-80 IMPLICATIONS FOR PRACTICE: : The potential adverse events of immune checkpoint inhibitors differ from conventional chemotherapy and can require a multidisciplinary approach. Continued education is important for all physicians to ensure optimal care for patients.

The effect of cryoprotectant agents on DNA methylation patterns and progeny development in the spermatozoa of Colossoma macropomum.

DNA methylation patterns are inherited from parents and are imperative for proper embryonic development; however, alterations in these patterns can compromise fertilization and development into a fully functioning adult animal because DNA methylation is part of a complex program of gene transcription. In this study, we investigated the impact of cryoprotectant agents (CPAs) on DNA methylation patterns in spermatozoa and the consequences on embryonic development and the survival rate of progeny. Global methylation was assessed by enzymatic reactions in Colossoma macropomum spermatozoa that were cryopreserved using dimethylsulfoxide, dimethylformamide, methanol, ethyl glycol and glycerol as CPAs. Fertilization was carried out to evaluate survival rates and abnormalities in embryonic development upon treatment with each of the CPAs. Fresh semen served as the control. Our results indicated that, compared to the control group, spermatozoa cryopreservation decreased the fertilization rate and delayed embryonic development from the midblastula stage. Furthermore, spermatozoa cryopreserved in all CPAs had lower methylation levels and exhibited more delays and abnormalities during embryonic development than did fresh semen. Methanol resulted in fertilization, hatching rates and embryonic development that were closer to the control but had lower methylation levels. In conclusion, ours results show significant alterations on spermatozoa DNA methylation patterns caused by CPAs that are used in the semen cryopreservation process. DNA methylation pattern alterations affected the viability of progeny (r=0.48); however, these effects can be minimized by choosing the CPA that will compose the freezing solution.

Molecular basis of spermatogenesis and sperm quality.

Spermatozoan quality can be evaluated in different ways, here we focus on the analysis of DNA, RNA and epigenetic status of germ cells. These characterizations also can be the bases for explaining sperm quality at other levels, so we will see how some of these molecules could affect other sperm quality markers. Moreover, we consider the possibility of using some of these molecules as predictors of sperm quality in terms of the ability to produce healthy offspring. The relevant effect of different types of RNA molecules in germ line specification and spermatogenesis and the importance of germ cell DNA integrity and a proper epigenetic pattern will be also discussed. Although most studies at this level have been performed in mammals, some information is available for fish; these recent discoveries in fish models are included. We provide a general overview on how these molecules could have a deep influence in the final sperm quality.

Cryobanking of aquatic species.

This review is focused on the applications of genome cryobanking of aquatic species including freshwater and marine fish, as well as invertebrates. It also reviews the latest advances in cryobanking of model species, widely used by the scientific community worldwide, because of their applications in several fields. The state of the art of cryopreservation of different cellular types (sperm, oocytes, embryos, somatic cells and primordial germ cells or early spermatogonia) is discussed focusing on the advantages and disadvantages of each procedure according to different applications. A special review on the need of standardization of protocols has also been carried out. In summary, this comprehensive review provides information on the practical details of applications of genome cryobanking in a range of aquatic species worldwide, including the cryobanks established in Europe, USA, Brazil, Australia and New Zealand, the species and type of cells that constitute these banks and the utilization of the samples preserved. STATEMENT OF RELEVANCE: This review compiles the last advances on germplasm cryobanking of freshwater and marine fish species and invertebrates, with high value for commercial aquaculture or conservation. It is reviewed the most promising cryopreservation protocols for different cell types, embryos and larvae that could be applied in programs for genetic improvement, broodstock management or conservation of stocks to guarantee culture production.

Targeting the KRAS Pathway in Non-Small Cell Lung Cancer.

: Lung cancer remains the leading cause of cancer-related deaths worldwide. However, significant progress has been made individualizing therapy based on molecular aberrations (e.g., EGFR, ALK) and pathologic subtype. KRAS is one of the most frequently mutated genes in non-small cell lung cancer (NSCLC), found in approximately 30% of lung adenocarcinomas, and is thus an appealing target for new therapies. Although no targeted therapy has yet been approved for the treatment of KRAS-mutant NSCLC, there are multiple potential therapeutic approaches. These may include direct inhibition of KRAS protein, inhibition of KRAS regulators, alteration of KRAS membrane localization, and inhibition of effector molecules downstream of mutant KRAS. This article provides an overview of the KRAS pathway in lung cancer and related therapeutic strategies under investigation. IMPLICATIONS FOR PRACTICE: The identification of oncogene-addicted cancers and specific inhibitors has revolutionized non-small cell lung cancer (NSCLC) treatment and outcomes. One of the most commonly mutated genes in adenocarcinoma is KRAS, found in approximately 30% of lung adenocarcinomas, and thus it is an appealing target for new therapies. This review provides an overview of the KRAS pathway and related targeted therapies under investigation in NSCLC. Some of these agents may play a key role in KRAS-mutant NSCLC treatment in the future.

Epigenetic regulation in Parkinson's disease.

Recent efforts have shed new light on the epigenetic mechanisms driving gene expression alterations associated with Parkinson's disease (PD) pathogenesis. Changes in gene expression are a well-established cause of PD, and epigenetic mechanisms likely play a pivotal role in regulation. Studies in families with PD harboring duplications and triplications of the SNCA gene have demonstrated that gene dosage is associated with increased expression of both SNCA mRNA and protein, and correlates with a fulminant disease course. Furthermore, it is postulated that even subtle changes in SNCA expression caused by common variation is associated with disease risk. Of note, genome-wide association studies have identified over 30 loci associated with PD with most signals located in non-coding regions of the genome, thus likely influencing transcript expression levels. In health, epigenetic mechanisms tightly regulate gene expression, turning genes on and off to balance homeostasis and this, in part, explains why two cells with the same DNA sequence will have different RNA expression profiles. Understanding this phenomenon will be crucial to our interpretation of the selective vulnerability observed in neurodegeneration and specifically dopaminergic neurons in the PD brain. In this review, we discuss epigenetic mechanisms, such as DNA methylation and histone modifications, involved in regulating the expression of genes relevant to PD, RNA-based mechanisms, as well as the effect of toxins and potential epigenetic-based treatments for PD.

Cerebellar ataxia in progressive supranuclear palsy: An autopsy study of PSP-C.

BACKGROUND: Cerebellar ataxia is an exclusion criterion for the clinical diagnosis of progressive supranuclear palsy, but a variant with predominant cerebellar ataxia has been reported. The aims of this study were to estimate the frequency of progressive supranuclear palsy with predominant cerebellar ataxia in an autopsy series from the United States and to compare clinical, pathologic, and genetic differences between progressive supranuclear palsy with and without predominant cerebellar ataxia. METHOD: We selected 100 consecutive patients with pathologically confirmed progressive supranuclear palsy who had been evaluated at the Mayo Clinic (referred to as the Mayo Clinic patient series) from our brain bank database (N = 1085). We next enriched in cases likely to have cerebellar ataxia by searching the remaining 985 cases for (1) an antemortem diagnosis of multiple system atrophy or (2) neuropathologic evidence of prominent degeneration of the cerebellum or cerebellar afferent nuclei. Subsequently, clinical, pathologic, and genetic features were compared between the two groups. RESULTS: One patient in the Mayo Clinic patient series (1%) met criteria for progressive supranuclear palsy with predominant cerebellar ataxia and had both cerebellar and mild midbrain atrophy on MRI. Four patients were identified with the targeted search. Four of the five patients were clinically misdiagnosed as multiple system atrophy. The severity of tau-related pathology and cerebellar degeneration were not different between the two groups. No differences were detected in tau genotypes. CONCLUSION: Although our data cannot provide definitive information about how to make an accurate clinical diagnosis, they should serve to raise awareness of progressive supranuclear palsy with predominant cerebellar ataxia in the differential diagnosis of multiple system atrophy. © 2016 Movement Disorder Society.

Stabilization of gene expression and cell morphology after explant recycling during fin explant culture in goldfish.

The development of fin primary cell cultures for in vitro cellular and physiological studies is hampered by slow cell outgrowth, low proliferation rate, poor viability, and sparse cell characterization. Here, we investigated whether the recycling of fresh explants after a first conventional culture could improve physiological stability and sustainability of the culture. The recycled explants were able to give a supplementary cell culture showing faster outgrowth, cleaner cell layers and higher net cell production. The cells exhibited a highly stabilized profile for marker gene expression including a low cytokeratin 49 (epithelial marker) and a high collagen 1a1 (mesenchymal marker) expression. Added to the cell spindle-shaped morphology, motility behavior, and actin organization, this suggests that the cells bore stable mesenchymal characteristics. This contrast with the time-evolving expression pattern observed in the control fresh explants during the first 2 weeks of culture: a sharp decrease in cytokeratin 49 expression was concomitant with a gradual increase in col1a1. We surmise that such loss of epithelial features for the benefit of mesenchymal ones was triggered by an epithelial to mesenchymal transition (EMT) process or by way of a progressive population replacement process. Overall, our findings provide a comprehensive characterization of this new primary culture model bearing mesenchymal features and whose stability over culture time makes those cells good candidates for cell reprogramming prior to nuclear transfer, in a context of fish genome preservation.