Contribution and therapeutic implications of retroelement insertions in ataxia telangiectasia.

Certain classes of genetic variation still escape detection in clinical sequencing analysis. One such class is retroelement insertion, which has been reported as a cause of Mendelian diseases and may offer unique therapeutic implications. Here, we conducted retroelement profiling on whole-genome sequencing data from a cohort of 237 individuals with ataxia telangiectasia (A-T). We found 15 individuals carrying retroelement insertions in ATM, all but one of which integrated in noncoding regions. Systematic functional characterization via RNA sequencing, RT-PCR, and/or minigene splicing assays showed that 12 out of 14 intronic insertions led or contributed to ATM loss of function by exon skipping or activating cryptic splice sites. We also present proof-of-concept antisense oligonucleotides that suppress cryptic exonization caused by a deep intronic retroelement insertion. These results provide an initial systematic estimate of the contribution of retroelements to the genetic architecture of recessive Mendelian disorders as ∼2.1%-5.5%. Our study highlights the importance of retroelement insertions as causal variants and therapeutic targets in genetic diseases.

A framework for individualized splice-switching oligonucleotide therapy.

Splice-switching antisense oligonucleotides (ASOs) could be used to treat a subset of individuals with genetic diseases1, but the systematic identification of such individuals remains a challenge. Here we performed whole-genome sequencing analyses to characterize genetic variation in 235 individuals (from 209 families) with ataxia-telangiectasia, a severely debilitating and life-threatening recessive genetic disorder2,3, yielding a complete molecular diagnosis in almost all individuals. We developed a predictive taxonomy to assess the amenability of each individual to splice-switching ASO intervention; 9% and 6% of the individuals had variants that were 'probably' or 'possibly' amenable to ASO splice modulation, respectively. Most amenable variants were in deep intronic regions that are inaccessible to exon-targeted sequencing. We developed ASOs that successfully rescued mis-splicing and ATM cellular signalling in patient fibroblasts for two recurrent variants. In a pilot clinical study, one of these ASOs was used to treat a child who had been diagnosed with ataxia-telangiectasia soon after birth, and showed good tolerability without serious adverse events for three years. Our study provides a framework for the prospective identification of individuals with genetic diseases who might benefit from a therapeutic approach involving splice-switching ASOs.

Seasonal trends and retention of polycyclic aromatic compounds (PACs) in a remote sub-Arctic catchment.

Atmospheric deposition is the most dominant source of polycyclic aromatic hydrocarbons (PAHs) in remote and pristine areas. Despite low bioaccumulation potential, PAHs and their persistent transformation products (PAH-derivatives) are chemicals of concern as they can harm human and animal health through chronic low dose exposure. In this study, atmospheric deposition fluxes of polycyclic aromatic compounds (PACs) were measured on a seasonal basis (3-month periods) from 2012 to 2016 in a remote subarctic forest catchment in northern Europe. The target PACs included 19 PAHs and 15 PAH-derivatives (oxygenated, nitrogenated, and methylated PAHs). The deposition fluxes of Æ©PAHs and Æ©PAH-derivatives were in the same range and averaged 530 and 500 ng m2 day-1, respectively. The fluxes were found to be higher with a factor of 2.5 for Æ©PAHs and a factor of 3 for Æ©PAH-derivatives during cold (<0 °C) in comparison to warm (>10 °C) periods. PAHs and PAH-derivatives showed similar seasonal patterns, which suggests that these two compound classes have similar sources and deposition mechanisms, and that the source strength of the PAH-derivatives in air follows that of the PAHs. The terrestrial export of PACs via the outlet of the catchment stream was estimated to be 1.1% for Æ©PAHs and 1.7% for Æ©PAH-derivatives in relation to the annual amounts deposited to the catchment, which suggests that boreal forests are sinks for PACs derived from the atmosphere. Some individual PACs showed higher export than others (i.e. chrysene, cyclopenta(c,d)pyrene, carbazole, quinoline, and benzo(f)quinoline), with high export PACs mostly characterized by low molecular weight and low hydrophobicity (2-3 benzene rings; log Kow<6.0).

A stimulus-contingent positive feedback loop enables IFN-ß dose-dependent activation of pro-inflammatory genes.

Type I interferons (IFN) induce powerful antiviral and innate immune responses via the transcription factor, IFN-stimulated gene factor (ISGF3). However, in some pathological contexts, type I IFNs are responsible for exacerbating inflammation. Here, we show that a high dose of IFN-ß also activates an inflammatory gene expression program in contrast to IFN-λ3, a type III IFN, which elicits only the common antiviral gene program. We show that the inflammatory gene program depends on a second, potentiated phase in ISGF3 activation. Iterating between mathematical modeling and experimental analysis, we show that the ISGF3 activation network may engage a positive feedback loop with its subunits IRF9 and STAT2. This network motif mediates stimulus-specific ISGF3 dynamics that are dependent on ligand, dose, and duration of exposure, and when engaged activates the inflammatory gene expression program. Our results reveal a previously underappreciated dynamical control of the JAK-STAT/IRF signaling network that may produce distinct biological responses and suggest that studies of type I IFN dysregulation, and in turn therapeutic remedies, may focus on feedback regulators within it.

Seasonal trends of per- and polyfluoroalkyl substances in river water affected by fire training sites and wastewater treatment plants.

Fire-fighting training areas and wastewater treatment plants (WWTPs) are potential sources of per- and polyfluoroalkyl substances (PFASs) to the nearby aquatic environment. This study investigated seasonal variations of PFAS levels in two river catchments in Sweden; one impacted by Stockholm Arlanda Airport (Sites 1 and 2), and the other by WWTPs and a military airport (Uppsala) (Sites 3 and 4). Æ©PFAS concentrations were up to 61 (Sites 1 and 2) and 4 (Sites 3 and 4) times higher compared to the reference site. Distinct different seasonal trends were observed in the two catchments with higher ∑PFAS concentrations during the high water flow season at Site 1 compared to the low water flow season, whereas Sites 3 and 4 showed an inverse seasonal trend. This demonstrates that the pollution is mobilized during periods of high flow in the first catchment (Stockholm Arlanda Airport), while it is diluted during high flow in the second catchment (Uppsala). Average annual loads for ∑PFASs were estimated at ∼5.2 and ∼3.7 kg yr-1 for the catchment in Uppsala and Stockholm Arlanda Airport, respectively. Thus, both catchments add PFASs to Lake Mälaren, which is Sweden's most important source area for drinking water production.

Seasonal trends of legacy and alternative flame retardants in river water in a boreal catchment.

Boreal forests store large amounts of atmospherically deposited (semi-)persistent organic pollutants (POPs). The terrestrial POPs may be exported to streams and rivers through processes that are heavily impacted by seasonality. In this screening study, concentrations of 4 legacy and 45 alternative flame retardants (FRs) were determined in the dissolved and particulate phase in streams within a relatively pristine boreal catchment in northern Europe (Krycklan Catchment Study; 3 sites) and in rivers more impacted by human activities further downstream towards the Baltic Sea (3 sites). The sampling included the main hydrological seasons (snow-free, snow-covered, and spring flood) and was conducted during two consecutive years (2014-2016). Of the 49 analyzed FRs, 11 alternative halogenated FRs (HFRs), 13 alternative organophosphorus FRs (OPFRs), and 4 legacy polybrominated diphenyl ethers (PBDEs) were detected in at least one sample. The average bulk (dissolved + particulate) concentrations of ∑FRs (including all sites) were highest for ∑HFRs (38 ±â€¯70 ng L-1), followed by the ∑OPFRs (3.9 ±â€¯4.9 ng L-1) and the ∑PBDEs (0.0040 ±â€¯0.016 ng L-1). Bulk concentrations of HFRs, OPFRs, and PBDEs were highly variable with season and sampling location, e.g., during spring flood, bulk concentrations were up to 600 times, 3.7 times, and 4.9 times higher for HFRs, OPFRs and PBDEs, respectively, than during periods of lower flow. Bulk concentrations of ∑OPFRs, were elevated at all sites ~6 days before the actual start of the spring flood in 2015, suggesting that hydrophobicity fractionation had occurred within the snowpack. Similar to previous studies of other POPs in the same headwater catchment, there was a general trend that levels of ∑FRs were higher at the mire site than at the forested site. Annual fluxes of FRs were found to be ~15 times higher downstream the city of Umeå compared to at the outlet of the pristine catchment. This study should be regarded as a screening study considering the large number of diverse FRs analyzed and variability in the results.

The Role of Spring Flood and Landscape Type in the Terrestrial Export of Polycyclic Aromatic Compounds to Streamwater.

Concentrations of polycyclic aromatic compounds (PACs), including 19 polycyclic aromatic hydrocarbons (PAHs) and 15 PAH-derivatives (oxygenated and nitrogen heterocyclic PAHs), were measured in streams in a remote headwater catchment in northern Europe and in more urbanized, downstream areas. Sampling was conducted during 2014 to 2016 and included the main hydrological seasons (snow-free, snow-covered, and spring flood) at six sampling sites. Levels of the targeted PACs varied substantially over time and space and were up to 110-fold (on average 17-fold) and 7000-fold (on average 670-fold) higher for PAHs and PAH-derivatives, respectively, during spring flood compared with preceding snow-covered and snow-free seasons. Higher levels of ∑PACs were generally found in a headwater stream draining a mire than at an adjacent forested site, with up to 20 times and 150 times higher levels for ∑PAH and ∑PAH-derivatives, respectively. The particle-bound PAC levels were positively correlated to surface runoff in the mire stream (∑PAHs: p = 0.032; ∑PAH-derivatives: p = 0.040) but not in the corresponding forest stream, during snowmelt and winter base flow. In more urbanized downstream areas, particle-bound PACs were instead strongly associated with suspended particulate matter ( p < 0.05; ∑PAHs and ∑PAH-derivatives except one site). Levels of ∑PACs in the streamwater were on average 3-fold higher downstream of the most densely populated area than at the outlet of the headwater catchment. The higher PAC levels in the downstream water compared to the remote headwater were clearer when normalized to SPM amounts (instead of water volume), with a gradual downstream trend between the sites.

Design, Synthesis, and Evaluation of (2-(Pyridinyl)methylene)-1-tetralone Chalcones for Anticancer and Antimicrobial Activity.

BACKGROUND: Chalcones, natural products produced by plants as a natural defense mechanisms against various pathogens, are molecules with structures that include two aromatic rings joined by an α, ß unsaturated carbonyl system. Previous research has demonstrated that chalcones exhibit a wide variety of biological activities, including anticancer, antifungal, and antibiotic properties. OBJECTIVE: Our goal is to synthesize novel heterocyclic-containing chalcones and have their biological activities evaluated. Methods Sixteen chalcones were synthesized by the crossed aldol condensation of substituted tetralones with substituted pyridinylaldehydes. The products were purified by recrystallization in MeOH/H2O and characterized by 1H NMR, 13C NMR, and HRMS. Anticancer assays were performed by NCI (National Cancer Institute) against the NCI-60 panel of 60 different human cancer cell lines, including leukemia, non-small-cell lung cancer, colon, central nervous system, melanoma, ovarian, renal, prostate, and breast cancer. Antimicrobial assays were performed by COADD (Community for Open Antimicrobial Drug Discovery) against Escherichia coli, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Cryptococcus neoformans var. grubii, and Candida albicans. RESULT: Chalcone 3d had demonstrated growth inhibition greater than 60% against a variety of cancers: leukemia (MOLT-4, SR), non-small cell lung cancer (NCI-H522), colon cancer (HCT- 116), prostate cancer (DU-145), and breast cancer (MCF7, MDA-MB-468) and was also cytotoxic to three different cell lines (CCRF-CEM, RPMI-8226, and KM12). 5c was active against leukemia (CCRF-CEM, RPMI-8226, SR) and breast cancer (MCF7) and 5e was active only against leukemia (RPMI-8226, SR). 5h was partially active and the best compound with growth inhibition of MRSA by 75%. 3b was the best compound against EC, KP, and PA and 3f had the greatest activity against AB. For fungi, 3f and 3e demonstrated the best growth inhibition. CONCLUSION: A small library of heterocyclic-containing chalcones was developed and initial screening demonstrates modest activity against cancers, bacteria, and fungi.

Influence of natural organic matter on the extraction efficiency of flame retardants from surface waters.

The influence of natural organic matter (NOM) on the solid-phase extraction (SPE) efficiency was investigated for legacy and emerging flame retardants (FRs; n=26) in surface water. Three different groups of FRs were analyzed: polybrominated diphenyl ethers (PBDEs), halogenated flame retardants (HFRs), and organophosphorus flame retardants (OPFRs). In addition, five sorbents (Amberlite XAD-2, Amberlite IRA-743, Oasis HLB, Chromabond HR-P, and Chromabond HR-X) were evaluated for the extraction of FRs (n=33) in water, of which Oasis HLB eluted with dichloromethane and acetone:n-hexane (1:1, v/v) provided the highest overall recoveries. In subsequent NOM experiments, where FRs were extracted from water containing different NOM concentrations, both increased and decreased extraction efficiency with increasing NOM level were observed. Physicochemical and semi-empirical quantum chemistry properties were calculated for the FRs and used for analyzing relations between FRs. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) showed that the FRs separated into four different groups based on their properties. The FRs within each group responded similarly to increasing NOM, while differences in behavior were observed between the groups. This suggests that the structural properties of micropollutants highly influence NOM-FR interaction mechanisms. For instance, at high NOM levels, recoveries decreased substantially for FRs containing a moiety that can form strong hydrogen bonds (such as the double-bonded oxygen in e.g., OPFRs). Many of the compounds showed maximum extraction efficiency at higher levels of NOM. This suggests that binding of NOM to the sorbent and subsequent interaction between sorbent-bound NOM and FRs is an important mechanism for extraction of micropollutants from surface waters.

Spatial distribution and source tracing of per- and polyfluoroalkyl substances (PFASs) in surface water in Northern Europe.

The impact of point and diffuse sources for 26 per- and polyfluoroalkyl substances (PFASs) in northern Europe were investigated by studying Swedish rivers (n = 40) and recipient seawater (Baltic Sea and Kattegat; n = 18). Different composition profiles were observed in the rivers, with ten rivers having a remarkably high fraction of perfluoroalkane sulfonic acids (PFSAs; 65% of the Æ©PFASs) as compared to other rivers (19%) suggesting major impact of one or several source types dominated by PFSAs. Population density and low latitude (south) were strongly correlated to the widely used perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA) as well as to perfluorohexanesulfonic acid (PFHxS). Significant relationships between several PFCAs and PFSAs (i.e. perfluorobutanoic acid (PFBA), perfluoroheptanoic acid (PFHpA), PFOA, perfluorobutanesulfonic acid (PFBS), and PFHxS) and dissolved organic carbon (DOC) were detected (p < 0.05), indicating chemical binding and co-transport with DOC in fresh water and seawater. Partial least squares regression analysis showed that perfluoroalkyl carboxylic acids (PFCAs) were related to latitude according to their perfluorocarbon chain length (C3, C7, C8, C9, C10 and C11), with longer chains associated with higher latitudes. This suggests the presence of mechanisms promoting higher prevalence of longer chained PFCAs in the north, e.g. precursor degradation, and/or aerosol associated stabilization of PFCAs and their precursors.

Development and comparison of gas chromatography-mass spectrometry techniques for analysis of flame retardants.

The restrictions on the use of legacy flame retardants (FRs) have increased the need of alternative FRs to comply with fire safety legislations. In this study, the feasibility of three different gas chromatography-mass spectrometry (GC-MS) techniques were investigated for the analysis of 102 legacy and alternative FRs including polybrominated diphenyl ethers (PBDEs, n=27), halogenated FRs (HFRs, containing bromine and/or chlorine, n=46), and organophosphorous FRs (OPFRs, n=29). The tested techniques included GC-single MS with (i) electon impact (EI) ionization and (ii) negative chemical ionization (NCI), and (iii) GC-tandem MS (MS/MS) with EI ionization. Out of the tested FRs, 90 could be detected under the used conditions on at least one of the three instrument setups. Later experiments included a selection of these FRs. For the majority of tested PBDEs (5 out of 6) and HFRs (24 out of 26), EI-MS/MS provided the highest detectability (i.e. the lowest detection limits), while for most tested OPFRs (8 out of 13), EI-MS performed better. The influence of matrix components on the analysis of FRs (n=45) was investigated by analyzing a fortified surface water sample with the technique with the lowest selectivity, EI-MS. Both peak enhancement and suppression were observed, and significant correlations between matrix effects and several physico-chemical properties (e.g., retention time and boiling point) were found for PBDEs. In a separate clean-up experiment using natural water spiked with legacy and alternative FRs (n=30), alumina provided the highest mean recovery (90%) in comparison to acidified silica (67%) and Florisil (78%). This study provides new knowledge on analysis of FRs including a wide range of alternative FRs, and it will aid in the efforts of FR monitoring in the environment.

Spectral shifts of mammalian ultraviolet-sensitive pigments (short wavelength-sensitive opsin 1) are associated with eye length and photic niche evolution.

Retinal opsin photopigments initiate mammalian vision when stimulated by light. Most mammals possess a short wavelength-sensitive opsin 1 (SWS1) pigment that is primarily sensitive to either ultraviolet or violet light, leading to variation in colour perception across species. Despite knowledge of both ultraviolet- and violet-sensitive SWS1 classes in mammals for 25 years, the adaptive significance of this variation has not been subjected to hypothesis testing, resulting in minimal understanding of the basis for mammalian SWS1 spectral tuning evolution. Here, we gathered data on SWS1 for 403 mammal species, including novel SWS1 sequences for 97 species. Ancestral sequence reconstructions suggest that the most recent common ancestor of Theria possessed an ultraviolet SWS1 pigment, and that violet-sensitive pigments evolved at least 12 times in mammalian history. We also observed that ultraviolet pigments, previously considered to be a rarity, are common in mammals. We then used phylogenetic comparative methods to test the hypotheses that the evolution of violet-sensitive SWS1 is associated with increased light exposure, extended longevity and longer eye length. We discovered that diurnal mammals and species with longer eyes are more likely to have violet-sensitive pigments and less likely to possess UV-sensitive pigments. We hypothesize that (i) as mammals evolved larger body sizes, they evolved longer eyes, which limited transmittance of ultraviolet light to the retina due to an increase in Rayleigh scattering, and (ii) as mammals began to invade diurnal temporal niches, they evolved lenses with low UV transmittance to reduce chromatic aberration and/or photo-oxidative damage.