Histone deacetylases suppress cgg repeat-induced neurodegeneration via transcriptional silencing in models of Fragile X Tremor Ataxia Syndrome

Fragile X Tremor Ataxia Syndrome (FXTAS) is a common inherited neurodegenerative disorder caused by expansion of a CGG trinucleotide repeat in the 59UTR of the fragile X syndrome (FXS) gene, FMR1. The expanded CGG repeat is thought to induce toxicity as RNA, and in FXTAS patients mRNA levels for FMR1 are markedly increased. Despite the critical role of FMR1 mRNA in disease pathogenesis, the basis for the increase in FMR1 mRNA expression is unknown. Here we show that overexpressing any of three histone deacetylases (HDACs 3, 6, or 11) suppresses CGG repeat-induced neurodegeneration in a Drosophila model of FXTAS. This suppression results from selective transcriptional repression of the CGG repeat-containing transgene. These findings led us to evaluate the acetylation state of histones at the human FMR1 locus. In patient-derived lymphoblasts and fibroblasts, we determined by chromatin immunoprecipitation that there is increased acetylation of histones at the FMR1 locus in pre-mutation carriers compared to control or FXS derived cell lines. These epigenetic changes correlate with elevated FMR1 mRNA expression in pre-mutation cell lines. Consistent with this finding, histone acetyltransferase (HAT) inhibitors repress FMR1 mRNA expression to control levels in pre-mutation carrier cell lines and extend lifespan in CGG repeat-expressing Drosophila. These findings support a disease model whereby the CGG repeat expansion in FXTAS promotes chromatin remodeling in cis, which in turn increases expression of the toxic FMR1 mRNA. Moreover, these results provide proof of principle that HAT inhibitors or HDAC activators might be used to selectively repress transcription at the FMR1 locus.open293

Translation of the FMR1 mRNA is not influenced by AGG interruptions

The fragile X mental retardation 1 (FMR1) gene contains a CGG-repeat element within its 5′ untranslated region (5′UTR) which, for alleles with more than ∼40 repeats, increasingly affects both transcription (up-regulation) and translation (inhibition) of the repeat-containing RNA with increasing CGG-repeat length. Translational inhibition is thought to be due to impaired ribosomal scanning through the CGG-repeat region, which is postulated to form highly stable secondary/tertiary structure. One striking difference between alleles in the premutation range (55–200 CGG repeats) and those in the normal range (<∼40 repeats) is the reduced number/absence of ‘expansion stabilizing’ AGG interruptions in the larger alleles. Such interruptions, which generally occur every 9–11 repeats in normal alleles, are thought to disrupt the extended CGG-repeat hairpin structure, thus facilitating translational initiation. To test this hypothesis, we have measured the translational efficiency of CGG-repeat mRNAs with 0–2 AGG interruptions, both in vitro (rabbit reticulocyte lysates) and in cell culture (HEK-293 cells). We demonstrate that the AGG interruptions have no detectable influence on translational efficiency in either a cell-free system or cell culture, indicating that any AGG-repeat-induced alterations in secondary/tertiary structure, if present, do not involve the rate-limiting step(s) in translational initiation

The Role of AGG Interruptions in the Transcription of FMR1 Premutation Alleles

Fragile X associated disorders are caused by a premutation allele in the fragile X mental retardation 1 gene (FMR1) and are hypothesized to result from the toxic effect of elevated levels of expanded FMR1 transcripts. Increased levels of FMR1 mRNA have indeed been reported in premutation carriers; however the mechanism by which expanded alleles lead to elevated levels of FMR1 mRNA in premutation carriers is unknown. Within the CGG repeat tract AGG interruptions are found, generally 1–3 present in normal/intermediate alleles (6–54 CGG repeats) and usually 0–1 in premutation alleles (55–200 CGG repeats). They are present at specific locations, generally occurring after 9 or 10 uninterrupted CGG repeats [(CGG)9AGG(CGG)9AGG(CGG)n]. We evaluated both the number of AGG interruptions and the resulting length of the uninterrupted 3′ CGG repeat pure tract in premutation alleles derived from two large cohorts of male and female carriers to determine whether the presence of AGG interruptions or the length of a pure stretch of CGG repeats influence the levels of FMR1 mRNA in blood. Our findings indicate that neither the number of AGG interruptions, nor their position along the CGG tract have a significant affect on mRNA levels in premutation carriers. We also, as expected based on previous findings, observed a highly significant correlation between CGG repeat number (as both total length and length of pure CGG stretch) and FMR1 mRNA expression levels, in both males and females. Importantly, we did not observe any significant difference in FMR1 mRNA levels in premutation carriers based on age

Bone Marrow Osteoblast Damage by Chemotherapeutic Agents

Hematopoietic reconstitution, following bone marrow or stem cell transplantation, requires a microenvironment niche capable of supporting both immature progenitors and stem cells with the capacity to differentiate and expand. Osteoblasts comprise one important component of this niche. We determined that treatment of human primary osteoblasts (HOB) with melphalan or VP-16 resulted in increased phospho-Smad2, consistent with increased TGF-β1 activity. This increase was coincident with reduced HOB capacity to support immature B lineage cell chemotaxis and adherence. The supportive deficit was not limited to committed progenitor cells, as human embryonic stem cells (hESC) or human CD34+ bone marrow cells co-cultured with HOB pre-exposed to melphalan, VP-16 or rTGF-β1 had profiles distinct from the same populations co-cultured with untreated HOB. Functional support deficits were downstream of changes in HOB gene expression profiles following chemotherapy exposure. Melphalan and VP-16 induced damage of HOB suggests vulnerability of this critical niche to therapeutic agents frequently utilized in pre-transplant regimens and suggests that dose escalated chemotherapy may contribute to post-transplantation hematopoietic deficits by damaging structural components of this supportive niche

Whole genome sequence analysis of the TALLYHO/Jng mouse

Background: The TALLYHO/Jng (TH) mouse is a polygenic model for obesity and type 2 diabetes first described in the literature in 2001. The origin of the TH strain is an outbred colony of the Theiler Original strain and mice derived from this source were selectively bred for male hyperglycemia establishing an inbred strain at The Jackson Laboratory. TH mice manifest many of the disease phenotypes observed in human obesity and type 2 diabetes. Results: We sequenced the whole genome of TH mice maintained at Marshall University to a depth of approximately 64.8X coverage using data from three next generation sequencing runs. Genome-wide, we found approximately 4.31 million homozygous single nucleotide polymorphisms (SNPs) and 1.10 million homozygous small insertions and deletions (indels) of which 98,899 SNPs and 163,720 indels were unique to the TH strain compared to 28 previously sequenced inbred mouse strains. In order to identify potentially clinically-relevant genes, we intersected our list of SNP and indel variants with human orthologous genes in which variants were associated in GWAS studies with obesity, diabetes, and metabolic syndrome, and with genes previously shown to confer a monogenic obesity phenotype in humans, and found several candidate variants that could be functionally tested using TH mice. Further, we filtered our list of variants to those occurring in an obesity quantitative trait locus, tabw2, identified in TH mice and found a missense polymorphism in the Cidec gene and characterized this variant’s effect on protein function. Conclusions: We generated a complete catalog of variants in TH mice using the data from whole genome sequencing. Our findings will facilitate the identification of causal variants that underlie metabolic diseases in TH mice and will enable identification of candidate susceptibility genes for complex human obesity and type 2 diabetes

Rapid Selection and Proliferation of CD133(+) Cells from Cancer Cell Lines: Chemotherapeutic Implications

Cancer stem cells (CSCs) are considered a subset of the bulk tumor responsible for initiating and maintaining the disease. Several surface cellular markers have been recently used to identify CSCs. Among those is CD133, which is expressed by hematopoietic progenitor cells as well as embryonic stem cells and various cancers. We have recently isolated and cultured CD133 positive [CD133(+)] cells from various cancer cell lines using a NASA developed Hydrodynamic Focusing Bioreactor (HFB) (Celdyne, Houston, TX). For comparison, another bioreactor, the rotary cell culture system (RCCS) manufactured by Synthecon (Houston, TX) was used. Both the HFB and the RCCS bioreactors simulate aspects of hypogravity. In our study, the HFB increased CD133(+) cell growth from various cell lines compared to the RCCS vessel and to normal gravity control. We observed a (+)15-fold proliferation of the CD133(+) cellular fraction with cancer cells that were cultured for 7-days at optimized conditions. The RCCS vessel instead yielded a (−)4.8-fold decrease in the CD133(+)cellular fraction respect to the HFB after 7-days of culture. Interestingly, we also found that the hypogravity environment of the HFB greatly sensitized the CD133(+) cancer cells, which are normally resistant to chemo treatment, to become susceptible to various chemotherapeutic agents, paving the way to less toxic and more effective chemotherapeutic treatment in patients. To be able to test the efficacy of cytotoxic agents in vitro prior to their use in clinical setting on cancer cells as well as on cancer stem cells may pave the way to more effective chemotherapeutic strategies in patients. This could be an important advancement in the therapeutic options of oncologic patients, allowing for more targeted and personalized chemotherapy regimens as well as for higher response rates

Industrial scale isolation, structural and spectroscopic characterization of epiisopiloturine from Pilocarpus microphyllus stapf leaves: a promising alkaloid against schistosomiasis

This paper presents an industrial scale process for extraction, purification, and isolation of epiisopiloturine (EPI) (2(3H)- Furanone,dihydro-3-(hydroxyphenylmethyl)-4-[(1-methyl-1H-imidazol-4-yl)methyl]-, [3S-[3a(R*),4b]]), which is an alkaloid from jaborandi leaves (Pilocarpus microphyllus Stapf). Additionally for the first time a set of structural and spectroscopic techniques were used to characterize this alkaloid. EPI has shown schistomicidal activity against adults and young forms, as well as the reduction of the egg laying adult worms and low toxicity to mammalian cells (in vitro). At first, the extraction of EPI was done with toluene and methylene chloride to obtain a solution that was alkalinized with ammonium carbonate. The remaining solution was treated in sequence by acidification, filtration and alkalinization. These industrial procedures are necessary in order to remove impurities and subsequent application of the high performance liquid chromatography (HPLC). The HPLC was employed also to remove other alkaloids, to obtain EPI purity higher than 98%. The viability of the method was confirmed through HPLC and electrospray mass spectrometry, that yielded a pseudo molecular ion of m/z equal to 287.1 Da. EPI structure was characterized by single crystal X-ray diffraction (XRD), 1H and 13C nuclear magnetic resonance (NMR) in deuterated methanol/chloroform solution, vibrational spectroscopy and mass coupled thermal analyses. EPI molecule presents a parallel alignment of the benzene and the methyl imidazol ring separated by an interplanar spacing of 3.758 Å indicating a π-π bond interaction. The imidazole alkaloid melts at 225°C and decomposes above 230°C under air. EPI structure was used in theoretical Density Functional Theory calculations, considering the single crystal XRD data in order to simulate the NMR, infrared and Raman spectra of the molecule, and performs the signals attribution

Communication and visiting policies in Italian intensive care units during the first COVID-19 pandemic wave and lockdown: a nationwide survey

Background: During the first coronavirus disease 2019 (COVID-19) pandemic wave, an unprecedented number of patients with respiratory failure due to a new, highly contagious virus needed hospitalization and intensive care unit (ICU) admission. The aim of the present study was to describe the communication and visiting policies of Italian intensive care units (ICUs) during the first COVID-19 pandemic wave and national lockdown and compare these data with prepandemic conditions. Methods: A national web-based survey was conducted among 290 Italian hospitals. Each ICU (active between February 24 and May 31, 2020) was encouraged to complete an individual questionnaire inquiring the hospital/ICU structure/organization, communication/visiting habits and the role of clinical psychology prior to, and during the first COVID-19 pandemic wave. Results: Two hundred and nine ICUs from 154 hospitals (53% of the contacted hospitals) completed the survey (202 adult and 7 pediatric ICUs). Among adult ICUs, 60% were dedicated to COVID-19 patients, 21% were dedicated to patients without COVID-19 and 19% were dedicated to both categories (Mixed). A total of 11,102 adult patients were admitted to the participating ICUs during the study period and only approximately 6% of patients received at least one visit. Communication with family members was guaranteed daily through an increased use of electronic devices and was preferentially addressed to the same family member. Compared to the prepandemic period, clinical psychologists supported physicians more often regarding communication with family members. Fewer patients received at least one visit from family members in COVID and mixed-ICUs than in non-COVID ICUs, l (0 [0–6]%, 0 [0–4]% and 11 [2–25]%, respectively, p &lt; 0.001). Habits of pediatric ICUs were less affected by the pandemic. Conclusions: Visiting policies of Italian ICUs dedicated to adult patients were markedly altered during the first COVID-19 wave. Remote communication was widely adopted as a surrogate for family meetings. New strategies to favor a family-centered approach during the current and future pandemics are warranted