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

The Role and Mechanism of Erythrocyte Invasion by Francisella tularensis

Francisella tularensis is an extremely virulent bacterium that can be transmitted naturally by blood sucking arthropods. During mammalian infection, F. tularensis infects numerous types of host cells, including erythrocytes. As erythrocytes do not undergo phagocytosis or endocytosis, it remains unknown how F. tularensisinvades these cells. Furthermore, the consequence of inhabiting the intracellular space of red blood cells (RBCs) has not been determined. Here, we provide evidence indicating that residing within an erythrocyte enhances the ability of F. tularensis to colonize ticks following a blood meal. Erythrocyte residence protected F. tularensis from a low pH environment similar to that of gut cells of a feeding tick. Mechanistic studies revealed that the F. tularensis type VI secretion system (T6SS) was required for erythrocyte invasion as mutation of mglA (a transcriptional regulator of T6SS genes), dotU, or iglC (two genes encoding T6SS machinery) severely diminished bacterial entry into RBCs. Invasion was also inhibited upon treatment of erythrocytes with venom from the Blue-bellied black snake (Pseudechis guttatus), which aggregates spectrin in the cytoskeleton, but not inhibitors of actin polymerization and depolymerization. These data suggest that erythrocyte invasion by F. tularensis is dependent on spectrin utilization which is likely mediated by effectors delivered through the T6SS. Our results begin to elucidate the mechanism of a unique biological process facilitated by F. tularensis to invade erythrocytes, allowing for enhanced colonization of ticks

Differential usage of transcriptional start sites and polyadenylation sites in FMR1 premutation alleles†

5′- and 3′-untranslated regions (UTRs) are important regulators of gene expression and play key roles in disease progression and susceptibility. The 5′-UTR of the fragile X mental retardation 1 (FMR1) gene contains a CGG repeat element that is expanded (>200 CGG repeats; full mutation) and methylated in fragile X syndrome (FXS), the most common form of inherited intellectual disability (ID) and known cause of autism. Significant phenotypic involvement has also emerged in some individuals with the premutation (55–200 CGG repeats), including fragile X-associated premature ovarian insufficiency (FXPOI) in females, and the neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS), in older adult carriers. Here, we show that FMR1 mRNA in human and mouse brain is expressed as a combination of multiple isoforms that use alternative transcriptional start sites and different polyadenylation sites. Furthermore, we have identified a novel human transcription start site used in brain but not in lymphoblastoid cells, and have detected FMR1 isoforms generated through the use of both canonical and non-canonical polyadenylation signals. Importantly, in both human and mouse, a specific regulation of the UTRs is observed in brain of FMR1 premutation alleles, suggesting that the transcript variants may play a role in premutation-related pathologies

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

Collagen-based silver nanoparticles for biological applications: synthesis and characterization

Abstract\ud \ud Background\ud Type I collagen is an abundant natural polymer with several applications in medicine as matrix to regenerate tissues. Silver nanoparticles is an important nanotechnology material with many utilities in some areas such as medicine, biology and chemistry. The present study focused on the synthesis of silver nanoparticles (AgNPs) stabilized with type I collagen (AgNPcol) to build a nanomaterial with biological utility. Three formulations of AgNPcol were physicochemical characterized, antibacterial activity in vitro and cell viability assays were analyzed. AgNPcol was characterized by means of the following: ultraviolet–visible spectroscopy, dynamic light scattering analysis, Fourier transform infrared spectroscopy, atomic absorption analysis, transmission electron microscopy and of X-ray diffraction analysis.\ud \ud \ud Results\ud All AgNPcol showed spherical and positive zeta potential. The AgNPcol at a molar ratio of 1:6 showed better characteristics, smaller hydrodynamic diameter (64.34 ± 16.05) and polydispersity index (0.40 ± 0.05), and higher absorbance and silver reduction efficiency (0.645 mM), when compared with the particles prepared in other mixing ratios. Furthermore, these particles showed antimicrobial activity against both Staphylococcus aureus and Escherichia coli and no toxicity to the cells at the examined concentrations.\ud \ud \ud Conclusions\ud The resulted particles exhibited favorable characteristics, including the spherical shape, diameter between 64.34 nm and 81.76 nm, positive zeta potential, antibacterial activity, and non-toxicity to the tested cells (OSCC).FAPESP (14/02282-6)CAPES (AUX-PERM-705/2009)

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 < 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

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