ALBERT

Description: SUMMARY We analyse data from seismic stations surrounding the Alboran Sea between Spain and North Africa to constrain variations of the lithosphere–asthenosphere boundary (LAB) in the region. The technique used is the receiver function technique, which uses  S -to- P  converted teleseismic waves at the LAB below the seismic stations. We confirm previous data suggesting a shallow (60–90 km) LAB beneath the Iberian Peninsula and we observe a similarly shallow LAB beneath the Alboran Sea where the lithosphere becomes progressively thinner towards the east. A deeper LAB (90–100 km) is observed beneath the Betics, the south of Portugal and Morocco. The structure of the LAB in the entire region does not seem to show any indication of subduction related features. We also observe good  P  receiver function signals from the seismic discontinuities at 410 and 660 km depth which do not indicate any upper-mantle anomaly beneath the entire region. This is in agreement with the sparse seismic activity in the mantle transition zone suggesting the presence of only weak and regionally confined anomalies.

Description: We report the evaluation of 20-, 18-, 16- and 14-mer phosphorothioate (PS)-modified tricycloDNA (tcDNA) gapmer antisense oligonucleotides (ASOs) in T m , cell culture and animal experiments and compare them to their gap-matched 20-mer 2'- O -methoxyethyl (MOE) and 14-mer 2',4'-constrained ethyl (cEt) counterparts. The sequence-matched 20-mer tcDNA and MOE ASOs showed similar T m and activity in cell culture under free-uptake and cationic lipid-mediated transfection conditions, while the 18-, 16- and 14-mer tcDNA ASOs were moderate to significantly less active. These observations were recapitulated in the animal experiments where the 20-mer tcDNA ASO formulated in saline showed excellent activity (ED 50 3.9 mg/kg) for reducing SR-B1 mRNA in liver. The tcDNA 20-mer ASO also showed better activity than the MOE 20-mer in several extra-hepatic tissues such as kidney, heart, diaphragm, lung, fat, gastrocnemius and quadriceps. Interestingly, the 14-mer cEt ASO showed the best activity in the animal experiments despite significantly lower T m and 5-fold reduced activity in cell culture relative to the 20-mer tcDNA and MOE-modified ASOs. Our experiments establish tcDNA as a useful modification for antisense therapeutics and highlight the role of chemical modifications in influencing ASO pharmacology and pharmacokinetic properties in animals.

Description: SUMMARY A molasse sequence comprising 1.5-km-thick sediments of the Lower and Middle Siwaliks Group in the sub-Himalayan North Belt along the Tinau Khola River in west-central Nepal was studied for the low-field magnetic susceptibility and anisotropy using single core specimens of siltstone/sandstone from 127 stratigraphic levels. The magnetic fabric comprises primary sedimentary-compactional and secondary tectonic components. It is characterized by (i) predominantly oblate magnetic susceptibility anisotropy ellipsoids, (ii) low anisotropy ( P ′ mostly 〈1.1) and low magnetic susceptibility (〈 ca. 10 −7 m 3 kg −1 ) contributed mainly by paramagnetic and diamagnetic minerals and (iii) WNW–ESE magnetic lineations subparallel to the fold axes/bedding strikes/thrust front, and hence normal to the direction of palaeotectonic compression. Thermal demagnetization of single core specimens from 103 levels across 1120 m of the lower part revealed a characteristic remanence of the high unblocking temperature (〉 600 °C) in hematite. Remanence ratios derived from demagnetization data allowed the first-order estimation of remanence contributions from magnetic minerals (goethite, maghemite, magnetite and hematite), and discrimination of rockmagnetic zones correlatable with distinct lithofacies, which will facilitate objective mapping. We correlated a magnetic polarity sequence, constructed from normal and reverse polarity directions from 77 levels that passed the reversal test and represented primary remanences, with the standard geomagnetic polarity timescale to constrain the depositional age between ca. 13.2 Ma (base of Chron C5AAN, 13.015–13.183 Ma) and the middle of Chron C5n.2n (9.987–11.040 Ma). We calculated the sediment accumulation rate for polarity zones from the chronologically better constrained part below Chron C5n.2n (i.e. below 11.040 Ma) to be 25–61 cm kyr −1 (average, 39 cm kyr −1 ), which is consistent with the value of 32–50 cm kyr −1 reported from Siwaik sections in Nepal. The notable increase in accumulation rate after 12.1 Ma probably reflects the peak of the earlier phase of uplift and/or unroofing of the Himalayan source region followed by rapid accumulation in the foredeep, and a link to monsoon initiation/intensification. Compared to the expected remanence from the latest APWP for the Indian Plate 10–13 Ma, the best-defined mean (351.8°/20.9°) is rotated anticlockwise by 9.2° and records an inclination shallowing of 25°. Constraining the base of the Tinau Khola north section to 13.2 Ma (i.e. older than the Tinau Khola south by 1.7 Myr) should open up new horizons for multidisciplinary and multiproxy research targeting geotectonic/climatic/environmental palaeoreconsructions of Himalaya-wide events.

Description: We have identified a rare small (~450 kb unique sequence) recurrent deletion in a previously linked attention-deficit hyperactivity disorder (ADHD) locus at 2q21.1 in five unrelated families with developmental delay (DD)/intellectual disability (ID), ADHD, epilepsy and other neurobehavioral abnormalities from 17 035 samples referred for clinical chromosomal microarray analysis. Additionally, a DECIPHER ( http://decipher.sanger.ac.uk ) patient 2311 was found to have the same deletion and presented with aggressive behavior. The deletion was not found in either six control groups consisting of 13 999 healthy individuals or in the DGV database. We have also identified reciprocal duplications in five unrelated families with autism, developmental delay (DD), seizures and ADHD. This genomic region is flanked by large, complex low-copy repeats (LCRs) with directly oriented subunits of ~109 kb in size that have 97.7% DNA sequence identity. We sequenced the deletion breakpoints within the directly oriented paralogous subunits of the flanking LCR clusters, demonstrating non-allelic homologous recombination as a mechanism of formation. The rearranged segment harbors five genes: GPR148, FAM123C, ARHGEF4 , FAM168B and PLEKHB2 . Expression of ARHGEF4 (Rho guanine nucleotide exchange factor 4) is restricted to the brain and may regulate the actin cytoskeletal network, cell morphology and migration, and neuronal function. GPR148 encodes a G-protein-coupled receptor protein expressed in the brain and testes. We suggest that small rare recurrent deletion of 2q21.1 is pathogenic for DD/ID, ADHD, epilepsy and other neurobehavioral abnormalities and, because of its small size, low frequency and more severe phenotype might have been missed in other previous genome-wide screening studies using single-nucleotide polymorphism analyses.

Description: R ibosome b iogenesis G TPase A protein (RbgA) is an essential GTPase required for the biogenesis of the 50S subunit in Bacillus subtilis. Homologs of RbgA are widely distributed in bacteria and eukaryotes and are implicated in ribosome assembly in the mitochondria, chloroplast and cytoplasm. Cells depleted of RbgA accumulate an immature large subunit that is missing key ribosomal proteins. RbgA, unlike many members of the Ras superfamily of GTPases, lacks a defined catalytic residue for carrying out guanosine triphosphate (GTP) hydrolysis. To probe RbgA function in ribosome assembly, we used a combined bioinformatics, genetic and biochemical approach. We identified a RNA-binding domain within the C-terminus of RbgA that is structurally similar to AmiR–NasR Transcription Anti-termination Regulator (ANTAR) domains, which are known to bind structured RNA. Mutation of key residues in the ANTAR domain altered RbgA association with the ribosome. We identified a putative catalytic residue within a highly conserved region of RbgA, His9, which is contained within a similar PGH motif found in elongation factor Tu (EF-Tu) that is required for GTP hydrolysis on interaction with the ribosome. Finally, our results support a model in which the GTPase activity of RbgA directly participates in the maturation of the large subunit rather than solely promoting dissociation of RbgA from the 50S subunit.

Description: Triantennary N -acetyl galactosamine (GalNAc, GN3 ), a high-affinity ligand for the hepatocyte-specific asialoglycoprotein receptor (ASGPR), enhances the potency of second-generation gapmer antisense oligonucleotides (ASOs) 6–10-fold in mouse liver. When combined with next-generation ASO designs comprised of short S -cEt ( S -2'- O -Et-2',4'-bridged nucleic acid) gapmer ASOs, ~60-fold enhancement in potency relative to the parent MOE (2'- O -methoxyethyl RNA) ASO was observed. GN3 -conjugated ASOs showed high affinity for mouse ASGPR, which results in enhanced ASO delivery to hepatocytes versus non-parenchymal cells. After internalization into cells, the GN3 -ASO conjugate is metabolized to liberate the parent ASO in the liver. No metabolism of the GN3 -ASO conjugate was detected in plasma suggesting that GN3 acts as a hepatocyte targeting prodrug that is detached from the ASO by metabolism after internalization into the liver. GalNAc conjugation also enhanced potency and duration of the effect of two ASOs targeting human apolipoprotein C-III and human transthyretin (TTR) in transgenic mice. The unconjugated ASOs are currently in late stage clinical trials for the treatment of familial chylomicronemia and TTR-mediated polyneuropathy. The ability to translate these observations in humans offers the potential to improve therapeutic index, reduce cost of therapy and support a monthly dosing schedule for therapeutic suppression of gene expression in the liver using ASOs.

Description: To evaluate the contribution of non-synonymous-coding variants of known familial and genome-wide association studies (GWAS)-linked genes for Parkinson's disease (PD) to PD risk in the East Asian population, we sequenced all the coding exons of 39 PD-related disease genes and evaluated the accumulation of rare non-synonymous-coding variants in 375 early-onset PD cases and 399 controls. We also genotyped 782 non-synonymous-coding variants of these genes in 710 late-onset PD cases and 9046 population controls. Significant enrichment of LRRK2 variants was observed in both early- and late-onset PD (odds ratio = 1.58; 95% confidence interval = 1.29–1.93; P = 8.05 x 10 –6 ). Moderate enrichment was also observed in FGF20 , MCCC1 , GBA and ITGA8 . Half of the rare variants anticipated to cause loss of function of these genes were present in healthy controls. Overall, non-synonymous-coding variants of known familial and GWAS-linked genes appear to make a limited contribution to PD risk, suggesting that clinical sequencing of these genes will provide limited information for risk prediction and molecular diagnosis.

Description: Phosphorothioate (PS) antisense oligonucleotides (ASOs) have been successfully developed as drugs to reduce the expression of disease-causing genes. PS-ASOs can be designed to induce degradation of complementary RNAs via the RNase H pathway and much is understood about that process. However, interactions of PS-ASOs with other cellular proteins are not well characterized. Here we report that in cells transfected with PS-ASOs, the chaperonin T-complex 1 (TCP1) proteins interact with PS-ASOs and enhance antisense activity. The TCP1-β subunit co-localizes with PS-ASOs in distinct nuclear structures, termed phosphorothioate bodies or PS-bodies. Upon Ras-related nuclear protein (RAN) depletion, cytoplasmic PS-body-like structures were observed and nuclear concentrations of PS-ASOs were reduced, suggesting that TCP1-β can interact with PS-ASOs in the cytoplasm and that the nuclear import of PS-ASOs is at least partially through the RAN-mediated pathway. Upon free uptake, PS-ASOs co-localize with TCP1 proteins in cytoplasmic foci related to endosomes/lysosomes. Together, our results indicate that the TCP1 complex binds oligonucleotides with TCP1-β subunit being a nuclear PS-body component and suggest that the TCP1 complex may facilitate PS-ASO uptake and/or release from the endocytosis pathway.

Description: Metagenomic sequencing provides a unique opportunity to explore earth’s limitless environments harboring scores of yet unknown and mostly unculturable microbes and other organisms. Functional analysis of the metagenomic data plays a central role in projects aiming to explore the most essential questions in microbiology, namely ‘In a given environment, among the microbes present, what are they doing, and how are they doing it?’ Toward this goal, several large-scale metagenomic projects have recently been conducted or are currently underway. Functional analysis of metagenomic data mainly suffers from the vast amount of data generated in these projects. The shear amount of data requires much computational time and storage space. These problems are compounded by other factors potentially affecting the functional analysis, including, sample preparation, sequencing method and average genome size of the metagenomic samples. In addition, the read-lengths generated during sequencing influence sequence assembly, gene prediction and subsequently the functional analysis. The level of confidence for functional predictions increases with increasing read-length. Usually, the most reliable functional annotations for metagenomic sequences are achieved using homology-based approaches against publicly available reference sequence databases. Here, we present an overview of the current state of functional analysis of metagenomic sequence data, bottlenecks frequently encountered and possible solutions in light of currently available resources and tools. Finally, we provide some examples of applications from recent metagenomic studies which have been successfully conducted in spite of the known difficulties.