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Pharmaceuticals and Personal Care Products (PPCPs) are emerging pollutants detected in many locations of the world including Antarctica. The main objective of this review is to discuss the influence of the human population on the concentration, distribution and biological effects of PPCPs across the Antarctic coastal marine ecosystem. We carried out a review of the scientific articles published for PPCPs in Antarctic, supported by the information of the Antarctic stations reported by Council of Managers of National Antarctic Programs (CONMAP), Scientific Committee on Antarctic Research (SCAR) and Secretariat of the Antarctic Treaty (ATS). In addition, spatial data regarding the Antarctic continent was obtained from Quantarctica. Antarctic concentrations of PPCPs were more reflective of the treatment system used by research stations as opposed to the infrastructure built or the annual occupancy by station. The main problem is that most of the research stations lack tertiary treatment, resulting in elevated concentrations of PPCPs in effluents. Furthermore, the geographic distribution of Antarctic field stations in coastal areas allows for the release of PPCPs, directly into the sea, a practice that remains in compliance with the current Protocol. After their release, PPCPs can become incorporated into sea ice, which can then act as a chemical reservoir. In addition, there is no clarity on the effects on the local biota. Finally, we recommend regulating the entry and use of PPCPs in Antarctica given the difficulties of operating, and in some cases the complete absence of appropriate treatment systems. Further studies are needed on the fate, transport and biological effects of PPCPs on the Antarctic biota. It is recommended that research efforts be carried out in areas inhabited by humans to generate mitigation measures relative to potential adverse impacts. Tourism should be also considered in further studies due the temporal release of PPCPs.

CRISPR/Cas9-mediated therapeutic gene editing is a promising technology for durable treatment of incurable monogenic diseases such as myotonic dystrophies. Gene-editing approaches have been recently applied to in vitro and in vivo models of myotonic dystrophy type 1 (DM1) to delete the pathogenic CTG-repeat expansion located in the 3′ untranslated region of the DMPK gene. In DM1-patient-derived cells removal of the expanded repeats induced beneficial effects on major hallmarks of the disease with reduction in DMPK transcript-containing ribonuclear foci and reversal of aberrant splicing patterns. Here, we set out to excise the triplet expansion in a time-restricted and cell-specific fashion to minimize the potential occurrence of unintended events in off-target genomic loci and select for the target cell type. To this aim, we employed either a ubiquitous promoter-driven or a muscle-specific promoter-driven Cas9 nuclease and tetracycline repressor-based guide RNAs. A dual-vector approach was used to deliver the CRISPR/Cas9 components into DM1 patient-derived cells and in skeletal muscle of a DM1 mouse model. In this way, we obtained efficient and inducible gene editing both in proliferating cells and differentiated post-mitotic myocytes in vitro as well as in skeletal muscle tissue in vivo., Graphical abstract, This paper describes a gene-editing approach designed to remove the pathologic CTG expansion mutation that causes myotonic dystrophy type 1. By using muscle-specific and drug-inducible expression of the CRISPR/Cas9 complex, effective deletion of the CTG expansion was obtained in myogenic cells and in mouse skeletal muscle.

Background: Myotonic dystrophy type 1 (DM1) is a dominantly inherited neuromuscular disease caused by the abnormal expansion of CTG-triplets in the 3' untranslated region of the DMPK gene. While therapeutic approaches that neutralize the toxic DMPK transcript provide only short-term effects, CRISPR/Cas9-mediated gene editing strategies can eliminate permanently the pathogenic mutation. Methods and Results: Having previously applied CRISPR/Cas9-mediated gene therapy in DM1-patient-derived myogenic cells to eliminate the repeat expansion, we have recently employed a dual vector-mediated approach to transduce drug-inducible CRISPR/Cas9 complex components into DM1 patient-derived cells and DM1 mice carrying a mutated human DMPK transgene, either in skeletal muscle or systemically. These mice exhibit a pathologic neuromuscular phenotype and altered behavior similar to those observed in human disease. By using this strategy, we obtained efficient and inducible DMPK gene editing in the absence of off-target unintended events both in myogenic cells in vitro and in skeletal muscle tissues in vivo. Conclusions and Significance: CRISPR/Cas9-induced deletion of CTG expansion could potentially result in a durable therapeutic response in post-mitotic adult tissue, opening the way for future gene therapy application in humans. Importantly, the use of tissue-specific Cas9 and spatio-temporal control of gene editing minimizes unintended off-target activity.

Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by abnormally expanded stretches of CTG DNA triplets in the DMPK gene, leading to mutated-transcript RNA-toxicity. MicroRNAs (miRNAs) are short non-coding RNAs that, after maturation, are loaded onto the RISC effector complex that destabilizes target mRNAs and represses their translation. In DM1 muscle biopsies not only the expression, but also the intracellular localization of specific miRNAs is disrupted, leading to the dysregulation of the relevant mRNA targets. To investigate the functional alterations of the miRNA/target interactions in DM1, we analyzed by RNA-sequencing the RISC-associated RNAs in skeletal muscle biopsies derived from DM1 patients and matched controls. The mRNAs found deregulated in DM1 biopsies were involved in pathways and functions relevant for the disease, such as energetic metabolism, calcium signaling, muscle contraction and p53-dependent apoptosis. Bioinformatic analysis of the miRNA/mRNA interactions based on the RISC enrichment profiles, identified 24 miRNA/mRNA correlations. Following validation in 21 independent samples, we focused on the couple miR-29c/ASB2 because of the role of miR-29c in fibrosis (a feature of late-stage DM1 patients) and of ASB2 in the regulation of muscle mass. Luciferase reporter assay confirmed the direct interaction between miR-29c and ASB2. Moreover, decreased miR-29c and increased ASB2 levels were verified also in immortalized myogenic cells and primary fibroblasts, derived from biopsies of DM1 patients and controls. CRISPR/Cas9-mediated deletion of CTG expansions rescued normal miR-29c and ASB2 levels, indicating a direct link between the mutant repeats and the miRNA/target expression. In conclusion, functionally relevant miRNA/mRNA interactions were identified in skeletal muscles of DM1 patients, highlighting the dysfunction of miR-29c and ASB2.

Penguins can bioaccumulate metals, a portion of which can be deposited in the environment through organic remains such as excrement, carcasses, and eggshells. In order to determine Cu and Pb concentrations and their relationship to soil, organic matter and grain size were determined in 27 samples collected in zones without penguins, penguin transit zones, and Adelie (Pygoscelis adeliae), Chinstrap (P. antarctica), and Gentoo penguin (P. papua) colonies on the Ardley Peninsula, Maritime Antarctica. An atomic absorption spectrophotometry analysis was carried out, organic matter was determined by loss on ignition, and grain size was measured with a laser diffraction particle size analyzer. The principal component analysis shows a relationship between the variables Cu, Pb, and grain size and areas with penguin presence. Cu concentrations in soils varied among areas (χ2, 15.707; p = 0.0004), with higher concentrations in transit zones and penguin colonies (142.63 and 140.79 mg/kg, respectively) than in zones without penguins (83.33 mg/kg). Pb concentrations in soils also varied among areas (χ2, 6.5029; p = 0.0387), and were higher in transit zones (5.92 mg/kg) than in the penguin colonies (4.45 mg/kg). Grain size differed significantly among areas (χ2, 13.506; p = 0.0012), with higher values in transit zones (avg. 37.38 μm) than in penguin colonies (avg. 26.93 μm) and zones without penguins (avg. 20.72 μm). Organic matter did not differ significantly among the studied zones (χ2, 2.0882; p = 0.3520). There is a positive correlation between Cu-Pb (Rho, 0.5532; p = 0.0028), Cu-grain size (Rho, 0.4756; p = 0.0130) and Pb-grain size (Rho, 0.4879; p = 0.0098). The presence of penguins increases Cu concentrations in Antarctic soils due to its bioaccumulation and elimination through excrement; however, the presence of penguins has a minor influence on Pb concentration in soil, probably because this metal is stored efficiently in bones, feathers, and eggshells.

Circular RNAs (circRNAs) constitute a recently re-discovered class of non-coding RNAs functioning as sponge for miRNAs and proteins, affecting RNA splicing and regulating transcription. CircRNAs are generated by “back-splicing”, linking covalently 3’- and 5’-ends of exons. Thus, circRNA levels might be deregulated in conditions associated to altered RNA-splicing. Indeed, increasing evidence indicates their role in human diseases. Specifically, myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by expanded CTG-repeats in the DMPK gene, resulting in abnormal mRNA-splicing. In this investigation, circRNAs expressed in DM1 skeletal muscles were identified by analyzing RNA-sequencing data-sets followed by qPCR validation. In muscle biopsies, out of 9 tested, 4 transcripts showed an increased circular fraction: CDYL, HIPK3, RTN4_03 and ZNF609. The circular fraction values correlated positively with skeletal muscle strength and Receiver-Operating-Characteristics curves showed that these four circRNAs allow to distinguish DM1 patients from controls. The identified circRNAs were also detectable in peripheral-blood-mononuclear-cells (PBMCs) and plasma of DM1 patients, but they were not regulated significantly, indicating a tissue-selectivity of the identified modulations. Finally, increased circular fractions of RTN4_03 and ZNF609 were also observed in differentiated myogenic cell lines derived from DM1 patients. In conclusion, this proof-of-principle study identified circRNA dysregulation in DM1 patients.

Aims: Peripheral artery disease is caused by the restriction or occlusion of arteries supplying the leg. Better understanding of the molecular mechanisms underpinning tissue response to ischemia is urgently needed to improve therapeutic options. The aim of this study is to investigate hypoxia-induced miR-210 regulation and its role in a mouse model of hindlimb ischemia. Results: miR-210 expression was induced by femoral artery dissection. To study the role of miR-210, its function was inhibited by the systemic administration of a miR-210 complementary locked nucleic acid (LNA)-oligonucleotide (anti-miR-210). In the ischemic skeletal muscle, anti-miR-210 caused a marked decrease of miR-210 compared with LNA-scramble control, while miR-210 target expression increased accordingly. Histological evaluation of acute tissue damage showed that miR-210 inhibition increased both apoptosis at 1 day and necrosis at 3 days. Capillary density decrease caused by ischemia was significantly more pronounced in anti-miR-210-treated mice; residual limb perfusion decreased accordingly. To investigate the molecular mechanisms underpinning the increased damage triggered by miR-210 blockade, we tested the impact of anti-miR-210 treatment on the transcriptome. Gene expression analysis highlighted the deregulation of mitochondrial function and redox balance. Accordingly, oxidative damage was more severe in the ischemic limb of anti-miR-210-treated mice and miR-210 inhibition increased oxidative metabolism. Further, oxidative-stress resistant p66Shc-null mice displayed decreased tissue damage following ischemia. Innovation: This study identifies miR-210 as a crucial element in the adaptive mechanisms to acute peripheral ischemia. Conclusions: The physiopathological significance of miR-210 is context dependent. In the ischemic skeletal muscle it seems to be cytoprotective, regulating oxidative metabolism and oxidative stress. Antioxid. Redox Signal. 21, 1177–1188.

Long noncoding RNAs (lncRNAs) are non-protein coding RNAs regulating gene expression. Although for some lncRNAs a relevant role in hypoxic endothelium has been shown, the regulation and function of lncRNAs is still largely unknown in the vascular physio-pathology. Taking advantage of next-generation sequencing techniques, transcriptomic changes induced by endothelial cell exposure to hypoxia were investigated. Paired-end sequencing of polyadenylated RNA derived from human umbilical vein endothelial cells (HUVECs) exposed to 1% O2 or normoxia was performed. Bioinformatics analysis identified ≈2000 differentially expressed genes, including 122 lncRNAs. Extensive validation was performed by both microarray and qPCR. Among the validated lncRNAs, H19, MIR210HG, MEG9, MALAT1 and MIR22HG were also induced in a mouse model of hindlimb ischemia. To test the functional relevance of lncRNAs in endothelial cells, knockdown of H19 expression was performed. H19 inhibition decreased HUVEC growth, inducing their accumulation in G1 phase of the cell cycle; accordingly, p21 (CDKN1A) expression was increased. Additionally, H19 knockdown also diminished HUVEC ability to form capillary like structures when plated on matrigel. In conclusion, a high-confidence signature of lncRNAs modulated by hypoxia in HUVEC was identified and a significant impact of H19 lncRNA was shown.

Background Long noncoding RNAs (lncRNAs) are non-protein coding transcripts regulating a variety of physiological and pathological functions. However, their implication in heart failure is still largely unknown. The aim of this study is to identify and characterize lncRNAs deregulated in patients affected by ischemic heart failure. Methods LncRNAs were profiled and validated in left ventricle biopsies of 18 patients affected by non end-stage dilated ischemic cardiomyopathy and 17 matched controls. Further validations were performed in left ventricle samples derived from explanted hearts of end-stage heart failure patients and in a mouse model of cardiac hypertrophy, obtained by transverse aortic constriction. Peripheral blood mononuclear cells of heart failure patients were also analyzed. LncRNA distribution in the heart was assessed by in situ hybridization. Function of the deregulated lncRNA was explored analyzing the expression of the neighbor mRNAs and by gene ontology analysis of the correlating coding transcripts. Results Fourteen lncRNAs were significantly modulated in non end-stage heart failure patients, identifying a heart failure lncRNA signature. Nine of these lncRNAs (CDKN2B-AS1/ANRIL, EGOT, H19, HOTAIR, LOC285194/TUSC7, RMRP, RNY5, SOX2-OT and SRA1) were also confirmed in end-stage failing hearts. Intriguingly, among the conserved lncRNAs, h19, rmrp and hotair were also induced in a mouse model of heart hypertrophy. CDKN2B-AS1/ANRIL, HOTAIR and LOC285194/TUSC7 showed similar modulation in peripheral blood mononuclear cells and heart tissue, suggesting a potential role as disease biomarkers. Interestingly, RMRP displayed a ubiquitous nuclear distribution, while H19 RNA was more abundant in blood vessels and was both cytoplasmic and nuclear. Gene ontology analysis of the mRNAs displaying a significant correlation in expression with heart failure lncRNAs identified numerous pathways and functions involved in heart failure progression. Conclusions These data strongly suggest lncRNA implication in the molecular mechanisms underpinning HF. Electronic supplementary material The online version of this article (doi:10.1186/s12967-016-0926-5) contains supplementary material, which is available to authorized users.

Non-invasive and simple to measure biomarkers are still an unmet need for myotonic dystrophy type 1 (DM1). Indeed, muscle biopsies can be extremely informative, but their invasive nature limits their application. Extracellular microRNAs are emerging humoral biomarkers and preliminary studies identified a group of miRNAs that are deregulated in the plasma or serum of small groups of DM1 patients. Here we adopted very stringent selection and normalization criteria to validate or disprove these miRNAs in 103 DM1 patients and 111 matched controls. We confirmed that 8 miRNAs out of 12 were significantly deregulated in DM1 patients: miR-1, miR-27b, miR-133a, miR-133b, miR-206, miR-140-3p, miR-454 and miR-574. The levels of these miRNAs, alone or in combination, discriminated DM1 from controls significantly, and correlated with both skeletal muscle strength and creatine kinase values. Interestingly, miR-133b levels were significantly higher in DM1 female patients. Finally, the identified miRNAs were also deregulated in the plasma of a small group (n = 30) of DM2 patients. In conclusion, this study proposes that miRNAs might be useful as DM1 humoral biomarkers.

Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by a CTG repeat expansion in 3’UTR of DMPK gene. This mutation causes accumulation of toxic RNA in nuclear foci leading to splicing misregulation of specific genes. In view of future clinical trials with antisense oligonucleotides in DM1 patients, it is important to set up sensitive and minimally-invasive tools to monitor the efficacy of treatments on skeletal muscle. A tibialis anterior (TA) muscle sample of about 60 mg was obtained from 5 DM1 patients and 5 healthy subjects through a needle biopsy. A fragment of about 40 mg was used for histological examination and a fragment of about 20 mg was used for biomolecular analysis. The TA fragments obtained with the minimally-invasive needle biopsy technique is enough to perform all the histopathological and biomolecular evaluations useful to monitor a clinical trial on DM1 patients.

The fascinating world of noncoding RNAs has recently come to light, thanks to the development of powerful sequencing technologies, revealing a variety of RNA molecules playing important regulatory functions in most, if not all, cellular processes. Many noncoding RNAs have been implicated in regulatory networks that are determinant for skeletal muscle differentiation and disease. In this review, we outline the noncoding RNAs involved in physiological mechanisms of myogenesis and those that appear dysregulated in muscle dystrophies, also discussing their potential use as disease biomarkers and therapeutic targets.

A 64-year-old man with idiopathic CD4(+) lymphocytopenia developed cognitive impairment and gait ataxia with isolated obstructive hydrocephalus, which was fatal. Cerebrospinal fluid showed mild pleocytosis, but the etiology was not revealed by extensive analysis. At autopsy, inflammatory cells, CD8(+) lymphocytes and abundant macrophages but not CD4(+) lymphocytes were infiltrating the meninges at the base of the brain. Electron microscopy demonstrated that inflammation was caused by Cryptococcus neoformans, which was localized exclusively within macrophages, where it grew with budding. Our study suggests that, in idiopathic CD4(+) lymphocytopenia, macrophages can efficiently phagocytize but inefficiently digest C. neoformans, thus representing a vehicle of chronic intracellular infection.