Miracle-dz/UniFAR-FTUs huggingface dataset:
Selective nanopore sequencing of human BRCA1 by Cas9-assisted targeting of chromosome segments (CATCH)

Abstract Next generation sequencing (NGS) is challenged by structural and copy number variations larger than the typical read length of several hundred bases. Third-generation sequencing platforms such as single-molecule real-time (SMRT) and nanopore sequencing provide longer reads and are able to characterize variations that are undetected in NGS data. Nevertheless, these technologies suffer from inherent low throughput which prohibits deep sequencing at reasonable cost without target enrichment. Here, we optimized Cas9-Assisted Targeting of CHromosome segments (CATCH) for nanopore sequencing of the breast cancer gene BRCA1. A 200 kb target containing the 80 kb BRCA1 gene body and its flanking regions was isolated intact from primary human peripheral blood cells, allowing long-range amplification and long-read nanopore sequencing. The target was enriched 237-fold and sequenced at up to 70× coverage on a single flow-cell. Overall performance and single-nucleotide polymorphism (SNP) calling were directly compared to Illumina sequencing of the same enriched sample, highlighting the benefits of CATCH for targeted sequencing. The CATCH enrichment scheme only requires knowledge of the target flanking sequence for Cas9 cleavage while providing contiguous data across both coding and non-coding sequence and holds promise for characterization of complex disease-related or highly variable genomic regions.
Enzymatic synthesis of random sequences of RNA and RNA analogues by DNA polymerase theta mutants for the generation of aptamer libraries

Abstract Nucleic acid aptamers, especially RNA, exhibit valuable advantages compared to protein therapeutics in terms of size, affinity and specificity. However, the synthesis of libraries of large random RNAs is still difficult and expensive. The engineering of polymerases able to directly generate these libraries has the potential to replace the chemical synthesis approach. Here, we start with a DNA polymerase that already displays a significant template-free nucleotidyltransferase activity, human DNA polymerase theta, and we mutate it based on the knowledge of its three-dimensional structure as well as previous mutational studies on members of the same polA family. One mutant exhibited a high tolerance towards ribonucleotides (NTPs) and displayed an efficient ribonucleotidyltransferase activity that resulted in the assembly of long RNA polymers. HPLC analysis and RNA sequencing of the products were used to quantify the incorporation of the four NTPs as a function of initial NTP concentrations and established the randomness of each generated nucleic acid sequence. The same mutant revealed a propensity to accept other modified nucleotides and to extend them in long fragments. Hence, this mutant can deliver random natural and modified RNA polymers libraries ready to use for SELEX, with custom lengths and balanced or unbalanced ratios.
Substance-specific and shared transcription and epigenetic changes in the human hippocampus chronically exposed to cocaine and alcohol

The hippocampus is a key brain region involved in both short- and long-term memory processes and may play critical roles in drug-associated learning and addiction. Using whole genome sequencing of mRNA transcripts (RNA-Seq) and immunoprecipitation-enriched genomic DNA (ChIP-Seq) coupled with histone H3 lysine 4 trimethylation (H3K4me3), we found extensive hippocampal gene expression changes common to both cocaine-addicted and alcoholic individuals that may reflect neuronal adaptations common to both addictions. However, we also observed functional changes that were related only to long-term cocaine exposure, particularly the inhibition of mitochondrial inner membrane functions related to oxidative phosphorylation and energy metabolism, which has also been observed previously in neurodegenerative diseases. Cocaine- and alcohol-related histone H3K4me3 changes highly overlapped, but greater effects were detected under cocaine exposure. There was no direct correlation, however, between either cocaine- or alcohol- related histone H3k4me3 and gene expression changes at an individual gene level, indicating that transcriptional regulation as well as drug-related gene expression changes are outcomes of a complex gene-regulatory process that includes multifaceted histone modifications.
Expression, signaling proficiency, and stimulatory function of the NKG2D lymphocyte receptor in human cancer cells

The stimulatory natural killer group 2 member D (NKG2D) lymphocyte receptor and its tumor-associated ligands are important mediators in the immune surveillance of cancer. With advanced human tumors, however, persistent NKG2D ligand expression may favor tumor progression. We have found that cancer cells themselves express NKG2D in complex with the DNAX-activating protein 10 (DAP10) signaling adaptor. Triggering of NKG2D on ex vivo cancer cells or on tumor lines which express only few receptor complexes activates the oncogenic PI3K–protein kinase B (PKB/AKT)–mammalian target of rapamycin (mTOR) signaling axis and downstream effectors, the ribosomal protein S6 kinase 1 (S6K1) and the translation initiation factor 4E-binding protein 1 (4E-BP1). In addition, as in lymphocytes, NKG2D ligand engagement stimulates phosphorylation of JNK and ERK in MAP kinase cascades. Consistent with these signaling activities, above-threshold expression of NKG2D–DAP10 in a ligand-bearing tumor line increases its bioenergetic metabolism and proliferation, thus suggesting functional similarity between this immunoreceptor and tumor growth factor receptors. This relationship is supported by significant correlations between percentages of cancer cells that are positive for surface NKG2D and criteria of tumor progression. Hence, in a conceptual twist, these results suggest that tumor co-option of NKG2D immunoreceptor expression may complement the presence of its ligands for stimulation of tumor growth.
A modest decrease in endothelial NOS in mice comparable to that associated with human NOS3 variants exacerbates diabetic nephropathy

Polymorphisms in the human endothelial nitric oxide synthase (eNOS) gene (NOS3) have been associated with advanced nephropathy in diabetic patients and with decreased expression in tissue culture. However, direct proof that modest genetic decreases in eNOS expression worsen diabetic nephropathy is lacking. To investigate this effect, we took advantage of the hybrid vigor and genetic uniformity of the F1 progeny (eNOS+/+, eNOS+/−, or eNOS−/− with or without diabetes) of a cross between heterozygous 129S6/SvEvTac eNOS+/− inbred females and heterozygous C57BL/6J eNOS+/− inbred males carrying the dominant Akita diabetogenic mutation Ins2C96Y/+. Whereas all C57BL/6J inbred eNOS−/− and eNOS+/− diabetic mice died before 5 mo, almost half of the F1 hybrid eNOS−/− and eNOS+/− diabetic mice lived until killed at 7 mo. Heterozygous eNOS+/− diabetic mice expressed ∼35% eNOS mRNA in the kidney and ∼25% glomerular eNOS protein relative to their eNOS+/+ diabetic littermates. These decreases in eNOS elevated blood pressure (BP) but not blood glucose. Urinary albumin excretion, mesangial expansion, glomerulosclerosis, mesangiolysis, and glomerular filtration rate increased in the order: eNOS+/+ Akita < eNOS+/− Akita < eNOS−/− Akita, independently of BP. Glomerular basement membrane thickening depended on increased BP. Renal expression of tissue factor and other inflammatory factors increased with the nephropathy; Nos2 also increased. Surprisingly, however, decreased eNOS expression ameliorated the increases in oxidative stress and tubulointerstitial fibrosis caused by diabetes. Our data demonstrate that a modest decrease in eNOS, comparable to that associated with human NOS3 variants, is sufficient to enhance diabetic nephropathy independently of its effects on BP.
Suppression and prevention of adjuvant arthritis in rats by a monoclonal antibody to the α/β T cell receptor

Adjuvant arthritis (AA) in rats is an experimentally induced autoimmune disease mediated by T lymphocytes specific for Mycobacterium tuberculosis. We raised the question whether T cells carrying the γ/δ Tcell receptor (TcR), reactive or not to mycobacterial antigens, are involved in the pathogenesis of AA. For this purpose, T cells bearing the TcR α/β were depleted from circulation by treatment with a monoclonal antibody against the rat TcR α/β (R73). This treatment efficiently suppressed existing disease. Even more efficient was pretreatment with R73 from birth, which prevented AA induction completely. In these α/β+ T cell‐depleted animals an elevated level of α/β− T cells (about 15% vs. 1% in normal rats) was evident, which was not significantly increased by Mycobacterium tuberculosis injection. We found no positive evidence that γ/δ+ T cells do contribute to AA induction. Moreover, treatment with an anti‐TcR α/β monoclonal antibody may be very efficient in treatment of Tcell‐mediated autoimmune diseases.
Alternative mechanisms of receptor editing in autoreactive B cells

Pathogenic anti-DNA antibodies expressed in systemic lupus erythematosis bind DNA mainly through electrostatic interactions between the positively charged Arg residues of the antibody complementarity determining region (CDR) and the negatively charged phosphate groups of DNA. The importance of Arg in CDR3 for DNA binding has been shown in mice with transgenes coding for anti-DNA VH regions; there is also a close correlation between arginines in CDR3 of antibodies and DNA binding. Codons for Arg can readily be formed by V(D)J rearrangement; thereby, antibodies that bind DNA are part of the preimmune repertoire. Anti-DNAs in healthy mice are regulated by receptor editing, a mechanism that replaces κ light (L) chains compatible with DNA binding with κ L chains that harbor aspartic residues. This negatively charged amino acid is thought to neutralize Arg sites in the VH. Editing by replacement is allowed at the κ locus, because the rearranged VJ is nested between unrearranged Vs and Js. However, neither λ nor heavy (H) chain loci are organized so as to allow such second rearrangements. In this study, we analyze regulation of anti-DNA H chains in mice that lack the κ locus, κ-/κ- mice. These mice show that the endogenous preimmune repertoire does indeed include a high frequency of antibodies with Arg in their CDR3s (putative anti-DNAs) and they are associated mainly with the editor L chain λx. The editing mechanisms in the case of λ-expressing B cells include L chain allelic inclusion and VH replacement.
Silk‐Molded Flexible, Ultrasensitive, and Highly Stable Electronic Skin for Monitoring Human Physiological Signals

Flexible and transparent E-skin devices are achieved by combining silk-molded micro-patterned polydimethylsiloxane (PDMS) with single-walled carbon nanotube (SWNT) ultrathin films. The E-skin sensing device demonstrates superior sensitivity, a very low detectable pressure limit, a fast response time, and a high stability for the detection of superslight pressures, which may broaden their potential use as cost-effective wearable electronics for healthcare applications.
γδ intraepithelial lymphocytes are essential mediators of host–microbial homeostasis at the intestinal mucosal surface

The mammalian gastrointestinal tract harbors thousands of bacterial species that include symbionts as well as potential pathogens. The immune responses that limit access of these bacteria to underlying tissue remain poorly defined. Here we show that γδ intraepithelial lymphocytes (γδ IEL) of the small intestine produce innate antimicrobial factors in response to resident bacterial “pathobionts” that penetrate the intestinal epithelium. γδ IEL activation was dependent on epithelial cell-intrinsic MyD88, suggesting that epithelial cells supply microbe-dependent cues to γδ IEL. Finally, γδ T cells protect against invasion of intestinal tissues by resident bacteria specifically during the first few hours after bacterial encounter, indicating that γδ IEL occupy a unique temporal niche among intestinal immune defenses. Thus, γδ IEL detect the presence of invading bacteria through cross-talk with neighboring epithelial cells and are an essential component of the hierarchy of immune defenses that maintain homeostasis with the intestinal microbiota.
Social evolution in multispecies biofilms

Microbial ecology is revealing the vast diversity of strains and species that coexist in many environments, ranging from free-living communities to the symbionts that compose the human microbiome. In parallel, there is growing evidence of the importance of cooperative phenotypes for the growth and behavior of microbial groups. Here we ask: How does the presence of multiple species affect the evolution of cooperative secretions? We use a computer simulation of spatially structured cellular groups that captures key features of their biology and physical environment. When nutrient competition is strong, we find that the addition of new species can inhibit cooperation by eradicating secreting strains before they can become established. When nutrients are abundant and many species mix in one environment, however, our model predicts that secretor strains of any one species will be surrounded by other species. This “social insulation” protects secretors from competition with nonsecretors of the same species and can improve the prospects of within-species cooperation. We also observe constraints on the evolution of mutualistic interactions among species, because it is difficult to find conditions that simultaneously favor both within- and among-species cooperation. Although relatively simple, our model reveals the richness of interactions between the ecology and social evolution of multispecies microbial groups, which can be critical for the evolution of cooperation.
Exopolymer alteration of physical properties of sea ice and implications for ice habitability and biogeochemistry in a warmer Arctic

The physical properties of Arctic sea ice determine its habitability. Whether ice-dwelling organisms can change those properties has rarely been addressed. Following discovery that sea ice contains an abundance of gelatinous extracellular polymeric substances (EPS), we examined the effects of algal EPS on the microstructure and salt retention of ice grown from saline solutions containing EPS from a culture of the sea-ice diatom, Melosira arctica. We also experimented with xanthan gum and with EPS from a culture of the cold-adapted bacterium Colwellia psychrerythraea strain 34H. Quantitative microscopic analyses of the artificial ice containing Melosira EPS revealed convoluted ice-pore morphologies of high fractal dimension, mimicking features found in EPS-rich coastal sea ice, whereas EPS-free (control) ice featured much simpler pore geometries. A heat-sensitive glycoprotein fraction of Melosira EPS accounted for complex pore morphologies. Although all tested forms of EPS increased bulk ice salinity (by 11–59%) above the controls, ice containing native Melosira EPS retained the most salt. EPS effects on ice and pore microstructure improve sea ice habitability, survivability, and potential for increased primary productivity, even as they may alter the persistence and biogeochemical imprint of sea ice on the surface ocean in a warming climate.
Mapping quantitative trait loci for egg production traits in an F2 intercross of Oh-Shamo and White Leghorn chickens.

We performed quantitative trait locus (QTL) analyses for egg production traits, including age at first egg (AFE) and egg production rates (EPR) measured every 4 weeks from 22 to 62 weeks of hen age, in a population of 421 F(2) hens derived from an intercross between the Oh-Shamo (Japanese Large Game) and White Leghorn breeds of chickens. Simple interval mapping revealed a main-effect QTL for AFE on chromosome 1 and four main-effect QTL for EPR on chromosomes 1 and 11 (three on chromosome 1 and one on chromosome 11) at the genome-wide 5% levels. Among the three EPR QTL on chromosome 1, two were identified at the early stage of egg laying (26-34 weeks of hen age) and the remaining one was discovered at the late stage (54-58 weeks). The alleles at the two EPR QTL derived from the Oh-Shamo breed unexpectedly increased the trait values, irrespective of the Oh-Shamo being inferior to the White Leghorn in the trait. This suggests that the Oh-Shamo, one of the indigenous Japanese breeds, is an untapped resource that is important for further improvement of current elite commercial laying chickens. In addition, six epistatic QTL were identified on chromosomes 2, 4, 7, 8, 17 and 19, where none of the above main-effect QTL were located. This is the first example of detection of epistatic QTL affecting egg production traits. The main and epistatic QTL identified accounted for 4-8% of the phenotypic variance. The total contribution of all QTL detected for each trait to the phenotypic and genetic variances ranged from 4.1% to 16.9% and from 11.5% to 58.5%, respectively.
Perinatal dengue infection: Two new cases

Dengue infection is commonly observed in tropical countries, but its transmission from mother to baby has not been frequently described. We report two cases with neonatal dengue infection due to perinatal transmission. The first signs of dengue appeared on day 4 of life in both the infants. Initially a bacterial infection was suspected. Clinical diagnosis of dengue fever was confirmed by appropriate laboratory testing. In areas where dengue is endemic, the diagnosis of dengue should be considered in a neonate with signs of sepsis. When dengue is suspected in a parturient woman, laboratory investigation and extended observation of the newborn are advised.
Trichome-derived O-acyl sugars are a first meal for caterpillars that tags them for predation

Plant glandular trichomes exude secondary metabolites with defensive functions, but these epidermal protuberances are surprisingly the first meal of Lepidopteran herbivores on Nicotiana attenuata. O-acyl sugars, the most abundant metabolite of glandular trichomes, impart a distinct volatile profile to the body and frass of larvae that feed on them. The headspace composition of Manduca sexta larvae is dominated by the branched chain aliphatic acids hydrolyzed from ingested O-acyl sugars, which waxes and wanes rapidly with trichome ingestion. In native habitats a ground-hunting predator, the omnivorous ant Pogonomyrmex rugosus, but not the big-eyed bug Geocoris spp. , use these volatile aliphatic acids to locate their prey.
Ribosome elongating footprints denoised by wavelet transform comprehensively characterize dynamic cellular translation events

Abstract Translation is dynamically regulated during cell development and stress response. In order to detect actively translated open reading frames (ORFs) and dynamic cellular translation events, we have developed a computational method, RiboWave, to process ribosome profiling data. RiboWave utilizes wavelet transform to denoise the original signal by extracting 3-nt periodicity of ribosomes and precisely locate their footprint denoted as Periodic Footprint P-site (PF P-site). Such high-resolution footprint is found to capture the full track of actively elongating ribosomes, from which translational landscape can be explicitly characterized. We compare RiboWave with several published methods, like RiboTaper, ORFscore and RibORF, and found that RiboWave outperforms them in both accuracy and usage when defining actively translated ORFs. Moreover, we show that PF P-site derived by RiboWave shows superior performance in characterizing the dynamics and complexity of cellular translatome by accurately estimating the abundance of protein levels, assessing differential translation and identifying dynamic translation frameshift.
Room Temperature Formation of High‐Mobility Two‐Dimensional Electron Gases at Crystalline Complex Oxide Interfaces

Well-controlled sub-unit-cell layer-bylayer epitaxial growth of spinel alumina is achieved at room temperature on a TiO2 -terminated SrTiO3 single-crystalline substrate. By tailoring the interface redox reaction, 2D electron gases with mobilities exceeding 3000 cm 2 V(-1) s(-1) are achieved at this novel oxide interface.
Tautomeric G•U pairs within the molecular ribosomal grip and fidelity of decoding in bacteria

Abstract We report new crystallographic structures of Thermus thermophilus ribosomes complexed with long mRNAs and native Escherichia coli tRNAs. They complete the full set of combinations of Watson–Crick G•C and miscoding G•U pairs at the first two positions of the codon–anticodon duplex in ribosome functional complexes. Within the tight decoding center, miscoding G•U pairs occur, in all combinations, with a non-wobble geometry structurally indistinguishable from classical coding Watson–Crick pairs at the same first two positions. The contacts with the ribosomal grip surrounding the decoding center are all quasi-identical, except in the crowded environment of the amino group of a guanosine at the second position; in which case a G in the codons may be preferred. In vivo experimental data show that the translational errors due to miscoding by G•U pairs at the first two positions are the most frequently encountered ones, especially at the second position and with a G on the codon. Such preferred miscodings involve a switch from an A-U to a G•U pair in the tRNA/mRNA complex and very rarely from a G = C to a G•U pair. It is concluded that the frequencies of such occurrences are only weakly affected by the codon/anticodon structures but depend mainly on the stability and lifetime of the complex, the modifications present in the anticodon loop, especially those at positions 34 and 37, in addition to the relative concentration of cognate/near-cognate tRNA species present in the cellular tRNA pool.
Integrated Anatomy E-Tutorial Designed By Medical Students, Combining Articulate Storyline 2 with Images from Anatomy.TV.

Increasing accessibility to a wide variety of anatomy teaching resources online has made learning complicated for students, due to the large quantity, mixed quality and lack of review by professionals. Therefore, we set out to develop a one-stop E-tutorial, which encompasses gross and surface anatomy with clinical applications and relevance highlighted, with content verified by medical and digital professionals. The University of Glasgow’s medical curriculum was reviewed to identify areas of teaching that required extra tailored resources to aid in areas of complex anatomy. An Anatomy E-tutorial on ‘Handand-Wrist’ was created using Articulate Storyline 2 software. The tutorial we created centered on the use of Anatomy.TV gross anatomical images. In addition, self-captured surface anatomy images and videos using digital-photography were incorporated into the package. Unique features of Articulate Storyline were utilized to ensure the users remained engaged throughout the E-Tutorial. We successfully created a fully interactive anatomy tutorial on hand and wrist anatomy using a novel software package. This was tailored to students undertaking the Glasgow Medical School curriculum, but has relevance to all those who need to obtain an in-depth knowledge of hand and wrist anatomy. Student evaluation will be presented on the effectiveness of this type of tool for learning anatomy of this region. This innovative use of Articulate Storyline 2, developed by students, for students, has created an excellent revision tool bringing together key elements of the medical curriculum in a single, easily accessible format. This will benefit student engagement with a complex area of anatomy. Produced by students and tailored to the integrated anatomy teaching, this product will prove beneficial for our colleagues and other health professionals alike.