A free carboxylate oxygen in the side chain of position 674 in transmembrane domain 7 is necessary for TSH receptor activation.

A specific H-bonding network formed between the central regions of transmembrane domain 6 and transmembrane domain 7 has been proposed to be critical for stabilizing the inactive state of glycoprotein hormone receptors. Many different constitutively activating TSH receptor point mutations have been identified in hyperfunctioning thyroid adenomas in the lower portion of transmembrane domain 6. Position D633 in transmembrane domain 6 of the human TSH receptor is the only one in which four different constitutively activating amino acid exchanges have been identified. Further in vitro substitutions led to constitutive activation of the TSH receptor (D633Y, F, C) as well as to the first inactivating TSH receptor mutation in transmembrane domain 6 without changes of membrane expression or TSH binding (D633R). Molecular modeling of this inactivating TSH receptor mutation revealed potential interaction partners of R633 in transmembrane domain 3 and/or transmembrane domain 7, presumably via hydrogen bonds that could be responsible for locking the TSH receptor in a completely inactive state. To further elucidate the H-bond network that most likely maintains the inactive state of the TSH receptor, we investigated these potential interactions by generating TSH receptor double mutants designed to break up possible H bonds. We excluded S508 in transmembrane domain 3 as a possible interaction partner of R633. In contrast, a partial response to TSH stimulation was rescued in a receptor construct with the double-substitution D633R/N674D. Our results therefore confirm the H bond between position 633 in transmembrane domain 6 and 674 in transmembrane domain 7 suggested by molecular modeling of the inactivating mutation D633R. Moreover, the mutagenesis results, together with a three-dimensional structure model, indicate that for TSH receptor activation and G protein-coupled signaling, at least one free available carboxylate oxygen is required as a hydrogen acceptor atom at position 674 in transmembrane domain 7.     

Reduced levels of protein recoding by A-to-I RNA editing in Alzheimer's disease

Adenosine to inosine (A-to-I) RNA editing, catalyzed by the ADAR enzyme family, acts on dsRNA structures within pre-mRNA molecules. Editing of the coding part of the mRNA may lead to recoding, amino acid substitution in the resulting protein, possibly modifying its biochemical and biophysical properties. Altered RNA editing patterns have been observed in various neurological pathologies. Here, we present a comprehensive study of recoding by RNA editing in Alzheimer''s disease (AD), the most common cause of irreversible dementia. We have used a targeted resequencing approach supplemented by a microfluidic-based high-throughput PCR coupled with next-generation sequencing to accurately quantify A-to-I RNA editing levels in a preselected set of target sites, mostly located within the coding sequence of synaptic genes. Overall, editing levels decreased in AD patients’ brain tissues, mainly in the hippocampus and to a lesser degree in the temporal and frontal lobes. Differential RNA editing levels were observed in 35 target sites within 22 genes. These results may shed light on a possible association between the neurodegenerative processes typical for AD and deficient RNA editing.     

Optimized nickase- and nuclease-based prime editing in human and mouse cells

Precise genomic modification using prime editing (PE) holds enormous potential for research and clinical applications. In this study, we generated all-in-one prime editing (PEA1) constructs that carry all the components required for PE, along with a selection marker. We tested these constructs (with selection) in HEK293T, K562, HeLa and mouse embryonic stem (ES) cells. We discovered that PE efficiency in HEK293T cells was much higher than previously observed, reaching up to 95% (mean 67%). The efficiency in K562 and HeLa cells, however, remained low. To improve PE efficiency in K562 and HeLa, we generated a nuclease prime editor and tested this system in these cell lines as well as mouse ES cells. PE-nuclease greatly increased prime editing initiation, however, installation of the intended edits was often accompanied by extra insertions derived from the repair template. Finally, we show that zygotic injection of the nuclease prime editor can generate correct modifications in mouse fetuses with up to 100% efficiency.     

Evaluation of growth rate and semi‐refined carrageenan properties of tissue‐cultured Kappaphycus alvarezii (Rhodophyta,Gigartinales)

This study aimed to evaluate and compare the quality of κ‐carrageenan obtained from tissue‐cultured and field‐cultured Kappaphycus alvarezii. Carrageenan properties including yield, viscosity, gel strength and sulfate content were studied. After 60 days of cultivation, tissue‐cultured K. alvarezii showed a higher growth rate (6.3 ± 0.01% day^?1) than field‐cultured seedlings (3.4 ± 0.3% day^?1). The obtained carrageenan yield from tissue‐cultured (67.3 ± 16.4%) was higher than field‐cultured K. alvarezii (51.5 ± 21.0%). Gel viscosity of carrageenans from tissue‐cultured K. alvarezii (1280.0 ± 25.0 cP) was found significantly higher than field‐cultured samples (87.8 ± 20.9 cP). The 1.5% gel solution of tissue‐cultured and field‐cultured K. alvarezii exhibited gel strengths of 703.5 ± 14.1 and 288.3 ± 19.3 g cm^?2, respectively. The average sulfate content of carrageenans was found to be significantly different between tissue‐cultured and field‐cultured K. alvarezii with 34.2 ± 10.9 and 7.5 ± 6.7%, respectively. Tissue culture is recommended to produce high quality seedlings by providing optimized culture conditions to the seaweed. This approach can serve as an alternative way to solve the seedling shortage problems currently faced by the seaweed industry.     

Defects in Cu(In,Ga)Se2 Chalcopyrite Semiconductors: A Comparative Study of Material Properties,Defect States,and Photovoltaic Performance

Understanding defects in Cu(In,Ga)(Se,S)₂ (CIGS), especially correlating changes in the film formation process with differences in material properties, photovoltaic (PV) device performance, and defect levels extracted from admittance spectroscopy, is a critical but challenging undertaking due to the complex nature of this polycrystalline compound semiconductor. Here we present a systematic comparative study wherein varying defect density levels in CIGS films were intentionally induced by growing CIGS grains using different selenium activity levels. Material characterization results by techniques including X‐ray diffraction, scanning electron microscopy, transmission electron microscopy, secondary ion mass spectrometry, X‐ray photoelectron spectroscopy, and medium energy ion scattering indicate that this process variation, although not significantly affecting CIGS grain structure, crystal orientation, or bulk composition, leads to enhanced formation of a defective chalcopyrite layer with high density of indium or gallium at copper antisite defects ((In, Ga)_Cu) near the CIGS surface, for CIGS films grown with insufficient selenium supply. This defective layer or the film growth conditions associated with it is further linked with observed current‐voltage characteristics, including rollover and crossover behavior, and a defect state at around 110 meV (generally denoted as the N1 defect) commonly observed in admittance spectroscopy. The impact of the (In, Ga)_Cu defects on device PV performance is also established.     

Improved method for simultaneous isolation of proteins and nucleic acids

Here we combine the use of fluorescence-enhancing silicon substrates coated by copoly(DMA–NAS–MAPS), a ter-copolymer based on N,N-dimethylacrylamide (DMA), N-acryloyloxysuccinimide (NAS), and 3-(trimethoxysilyl)propyl-methacrylate (MAPS), with an efficient dynamic incubation to overcome mass transport limitations and obtain femtomolar limits of detection. The high sensitivity was obtained with a conventional microarray scanner without the use of any sophisticated detection strategy or protocol. When the method was applied, an improvement of the analytical sensitivity of approximately three orders of magnitude was achieved for antibody detection when compared with the same assay performed on regular glass slides and static conditions. Moreover, limits of detection of 45 and 54 pg/ml were obtained for hepatitis B superficial antigen and HIV p24 antigen, respectively.     

Biodiversity of leaf-litter ants in fragmented tropical rainforests of Borneo: the value of publically and privately managed forest fragments

In view of the rapid rate of expansion of agriculture in tropical regions, attention has focused on the potential for privately-managed rainforest patches within agricultural land to contribute to biodiversity conservation. However, these sites generally differ in their history of forest disturbance and management compared with other forest fragments, and more information is required on the biodiversity value of these privately-managed sites, particularly in oil-palm dominated landscapes of SE Asia. Here we address this issue, using tropical leaf-litter ants in rainforest fragments surrounded by mature oil palm plantations in Sabah, Borneo as a model system. We compare the species richness and composition of ant assemblages in privately-managed forest fragments (‘high conservation value’ fragments; HCVs) with those in publically-managed fragments of forest (virgin jungle reserves; VJRs) and control sites in extensive tracts of primary forest. In this way, we test the hypothesis that privately-managed and publically-managed forest fragments differ in their species richness and composition as a result of differences in history and management and hence in habitat quality. In support of this hypothesis, we found that HCVs had much poorer habitat quality than VJRs, including lower sizes and densities of trees, less canopy cover, fewer dipterocarp trees and shallower leaf litter. Consequently, HCVs supported only half the species richness of ants in VJRs, which in turn supported 70 % of the species richness of control sites, with vegetation structure and composition explaining 77 % of the variation among forest fragments in ant species richness. HCVs were also much smaller than VJRs but there was only a weak relationship between fragment size and habitat quality, and species richness was not related to fragment size. VJRs supported 78 % of the 156 species found in extensive tracts of forest whereas HCVs supported only 22 %, which was only slightly higher than the proportion previously recorded in oil palm (19 %). These data support previous findings that publically-managed VJR fragments can make an important contribution to biodiversity conservation within agricultural landscapes. However, we suggest that for these HCVs to be effective as reservoirs of biodiversity, management is required to restore vegetation structure and habitat quality, for instance through enrichment planting with native tree species.     

Phylogenetic relationships of Malaysia’s long-tailed macaques,Macaca fascicularis,based on cytochrome b sequences

Phylogenetic relationships among Malaysia’s long-tailed macaques have yet to be established, despite abundant genetic studies of the species worldwide. The aims of this study are to examine the phylogenetic relationships of Macaca fascicularis in Malaysia and to test its classification as a morphological subspecies. A total of 25 genetic samples of M. fascicularis yielding 383 bp of Cytochrome b (Cyt b) sequences were used in phylogenetic analysis along with one sample each of M. nemestrina and M. arctoides used as outgroups. Sequence character analysis reveals that Cyt b locus is a highly conserved region with only 23% parsimony informative character detected among ingroups. Further analysis indicates a clear separation between populations originating from different regions; the Malay Peninsula versus Borneo Insular, the East Coast versus West Coast of the Malay Peninsula, and the island versus mainland Malay Peninsula populations. Phylogenetic trees (NJ, MP and Bayesian) portray a consistent clustering paradigm as Borneo’s population was distinguished from Peninsula’s population (99% and 100% bootstrap value in NJ and MP respectively and 1.00 posterior probability in Bayesian trees). The East coast population was separated from other Peninsula populations (64% in NJ, 66% in MP and 0.53 posterior probability in Bayesian). West coast populations were divided into 2 clades: the North-South (47%/54% in NJ, 26/26% in MP and 1.00/0.80 posterior probability in Bayesian) and Island-Mainland (93% in NJ, 90% in MP and 1.00 posterior probability in Bayesian). The results confirm the previous morphological assignment of 2 subspecies, M. f. fascicularis and M. f. argentimembris, in the Malay Peninsula. These populations should be treated as separate genetic entities in order to conserve the genetic diversity of Malaysia’s M. fascicularis. These findings are crucial in aiding the conservation management and translocation process of M. fascicularis populations in Malaysia.