Coordination of mRNA and tRNA methylations by TRMT10A.

The posttranscriptional modification of messenger RNA (mRNA) and transfer RNA (tRNA) provides an additional layer of regulatory complexity during gene expression. Here, we show that a tRNA methyltransferase, TRMT10A, interacts with an mRNA demethylase FTO (ALKBH9), both in vitro and inside cells. TRMT10A installs N1-methylguanosine (m1G) in tRNA, and FTO performs demethylation on N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) in mRNA. We show that TRMT10A ablation not only leads to decreased m1G in tRNA but also significantly increases m6A levels in mRNA. Cross-linking and immunoprecipitation, followed by high-throughput sequencing results show that TRMT10A shares a significant overlap of associated mRNAs with FTO, and these mRNAs have accelerated decay rates potentially through the regulation by a specific m6A reader, YTHDF2. Furthermore, transcripts with increased m6A upon TRMT10A ablation contain an overrepresentation of m1G9-containing tRNAs codons read by tRNAGln(TTG), tRNAArg(CCG), and tRNAThr(CGT) These findings collectively reveal the presence of coordinated mRNA and tRNA methylations and demonstrate a mechanism for regulating gene expression through the interactions between mRNA and tRNA modifying enzymes.

Functional analysis of the long-range regulatory element of BMP2 gene.

Recently, several pedigree-based studies have shown that abnormal replication of an enhancer element regulatory region in the downstream of the bone morphogenetic protein 2 (BMP2) gene is the cause of brachydactyly type A2 (BDA2). However, the exact molecular function of this regulatory region is unclear, and even conflicting results have emerged. In this study, based on bioinformatics analysis, we amplified target fragments of different lengths in this regulatory region by PCR technology, including a highly conserved 2.1 kb core sequence and 3 fragments that can completely cover the core 2.1 kb fragment. Then, the gene recombination vectors were constructed, and the biological function of these fragments was analyzed by the dual-luciferase reporter gene technology system. We found that the highly conserved 2.1 kb fragment did not have enhancer activity, while all of three truncated fragments showed strong enhancer activity. The results suggest that the expression regulation mode of the BMP2 gene is very complex. For the downstream regulatory region, selecting fragments of different lengths may have different effects on the regulation of BMP2 expression, which may due to the fragments with different lengths carrying different regulatory elements in the number of types. In summary, this study revealed the complexity of BMP2 gene regulatory elements, and provided new clues and directions for the subsequent in-depth exploration of the molecular pathogenic mechanism of BDA2.

Terminal hairpin in oligonucleotide dominantly prioritizes intramolecular cyclization by T4 ligase over intermolecular polymerization: an exclusive methodology for producing ssDNA rings.

When oligonucleotide bearing a hairpin near either its 3'- or 5'-end was treated with T4 DNA ligase, the intramolecular cyclization dominantly proceeded and its monomeric cyclic ring was obtained in extremely high selectivity. The selectivity was hardly dependent on the concentration of the oligonucleotide, and thus it could be added in one portion to the mixture at the beginning of the reaction. Without the hairpin, however, the formation of polymeric byproducts was dominant under the same conditions. Hairpin-bearing oligonucleotides primarily take the folded form, and the enzymatically reactive species (its open form) is minimal. As the result, the intermolecular reactions are efficiently suppressed due to both thermodynamic and kinetic factors. The 'terminal hairpin strategy' was applicable to large-scale preparation of a variety of DNA rings. The combination of this methodology with 'diluted buffer strategy', developed previously, is still more effective for the purpose. When large amount of l-DNA bearing a terminal hairpin (e.g. 40 µM) was treated in a diluted ligase buffer (0.1× buffer) with T4 DNA ligase, the DNA ring was prepared in 100% selectivity. Even at [l-DNA]0 = 100 µM in 0.1× buffer, the DNA ring was also obtained in pure form, simply by removing tiny quantity of linear byproducts by Exonuclease I.

Topologically Constrained Formation of Stable Z-DNA from Normal Sequence under Physiological Conditions.

Z-DNA, a left-handed duplex, has been shown to form in vivo and regulate expression of the corresponding gene. However, its biological roles have not been satisfactorily understood, mainly because Z-DNA is easily converted to the thermodynamically favorable B-DNA. Here we present a new idea to form stable Z-DNA under normal physiological conditions and achieve detailed analysis on its fundamental features. Simply by mixing two complementary minicircles of single-stranded DNA with no chemical modification, the hybridization spontaneously induces topological constraint which twines one-half of the double-stranded DNA into stable Z-DNA. The formation of Z-conformation with high stability has been proved by using circular dichroism spectroscopy, Z-DNA-specific antibody binding assay, nuclease digestion, etc. Even at a concentration of MgCl2 as low as 0.5 mM, Z-DNA was successfully obtained, avoiding the use of high salt conditions, limited sequences, ancillary additives, or chemical modifications, criteria which have hampered Z-DNA research. The resultant Z-DNA has the potential to be used as a canonical standard sample in Z-DNA research. By using this approach, further developments of Z-DNA science and its applications become highly promising.

The staining efficiency of cyanine dyes for single-stranded DNA is enormously dependent on nucleotide composition.

The staining of nucleic acids with fluorescent dyes is one of the most fundamental technologies in relevant areas of science. For reliable and quantitative analysis, the staining efficiency of the dyes should not be very dependent on the sequences of the specimens. However, this assumption has not necessarily been confirmed by experimental results, especially in the staining of ssDNA (and RNA). In this study, we found that both SYBR Green II and SYBR Gold did not stain either homopyrimidines or ssDNA composed of only adenine (A) and cytosine (C). However, these two dyes emit strong fluorescence when the ssDNA contains both guanine (G) and C (and/or both A and thymine (T)) and form potential Watson-Crick base pairs. Interestingly, SYBR Gold, but not SYBR Green II, strongly stains ssDNA consisting of G and A (or G and T). Additionally, we found that the secondary structure of ssDNA may play an important role in DNA staining. To obtain reliable results for practical applications, sufficient care must be paid to the composition and sequence of ssDNA.

Two-Holder Strategy for Efficient and Selective Synthesis of Lk 1 ssDNA Catenane.

DNA catenanes are characterized by their flexible and dynamic motions and have been regarded as one of the key players in sophisticated DNA-based molecular machines. There, the linking number (Lk) between adjacent interlocked rings is one of the most critical factors, since it governs the feasibility of dynamic motions. However, there has been no established way to synthesize catenanes in which Lk is controlled to a predetermined value. This paper reports a new methodology to selectively synthesize Lk 1 catenanes composed of single-stranded DNA rings, in which these rings can most freely rotate each other due to minimal inter-ring interactions. To the mixture for the synthesis, two holder strands (oligonucleotides of 18⁻46 nt) were added, and the structure of the quasi-catenane intermediate was interlocked through Watson⁻Crick base pairings into a favorable conformation for Lk 1 catenation. The length of the complementary part between the two quasi-rings was kept at 10 bp or shorter. Under these steric constraints, two quasi-rings were cyclized with the use of T4 DNA ligase. By this simple procedure, the formation of undesired topoisomers (Lk ≥ 2) was almost completely inhibited, and Lk 1 catenane was selectively prepared in high yield up to 70 mole%. These Lk 1 catenanes have high potentials as dynamic parts for versatile DNA architectures.

Evaluating glioblastoma tumour sphere growth and migration in interaction with astrocytes using 3D collagen-hyaluronic acid hydrogels.

Glioblastoma (GB) is an astrocytic brain tumour with a low survival rate, partly because of its highly invasive nature. The GB tumour microenvironment (TME) includes its extracellular matrix (ECM), a variety of brain cell types, unique anatomical structures, and local mechanical cues. As such, researchers have attempted to create biomaterials and culture models that mimic features of TME complexity. Hydrogel materials have been particularly popular because they enable 3D cell culture and mimic TME mechanical properites and chemical composition. Here, we used a 3D collagen I-hyaluronic acid hydrogel material to explore interactions between GB cells and astrocytes, the normal cell type from which GB likely derives. We demonstrate three different spheroid culture configurations, including GB multi-spheres (i.e., GB and astrocyte cells in spheroid co-culture), GB-only mono-spheres cultured with astrocyte-conditioned media, and GB-only mono-spheres cultured with dispersed live or fixed astrocytes. Using U87 and LN229 GB cell lines and primary human astrocytes, we investigated material and experiment variability. We then used time-lapse fluorescence microscopy to measure invasive potential by characterizing the sphere size, migration capacity, and weight-averaged migration distance in these hydrogels. Finally, we developed methods to extract RNA for gene expression analysis from cells cultured in hydrogels. U87 and LN229 cells displayed different migration behaviors. U87 migration occurred primarily as single cells and was reduced with higher numbers of astrocytes in both multi-sphere and mono-sphere plus dispersed astrocyte cultures. In contrast, LN229 migration exhibited features of collective migration and was increased in monosphere plus dispersed astrocyte cultures. Gene expression studies indicated that the most differentially expressed genes in these co-cultures were CA9, HLA-DQA1, TMPRSS2, FPR1, OAS2, and KLRD1. Most differentially expressed genes were related to immune response, inflammation, and cytokine signalling, with greater influence on U87 than LN229. These data show that 3D in vitro hydrogel co-culture models can be used to reveal cell line specific differences in migration and to study differential GB-astrocyte crosstalk.

DNA-caged nanoparticles via electrostatic self-assembly.

DNA-modified nanoparticles enable DNA sensing and therapeutics in nanomedicine and are also crucial for nanoparticle self-assembly with DNA-based materials. However, methods to conjugate DNA to nanoparticle surfaces are limited, inefficient, and lack control. Inspired by DNA tile nanotechnology, we demonstrate a new approach to nanoparticle modification based on electrostatic attraction between negatively charged DNA tiles and positively charged nanoparticles. This approach does not disrupt nanoparticle surfaces and leverages the programmability of DNA nanotechnology to control DNA presentation. We demonstrated this approach using a vareity of nanoparticles, including polymeric micelles, polystyrene beads, gold nanoparticles, and superparamagnetic iron oxide nanoparticles with sizes ranging from 5-20 nm in diameter. DNA cage formation was confirmed through transmission electron microscopy (TEM), neutralization of zeta potential, and a series of fluorescence experiments. DNA cages present "handle" sequences that can be used for reversible target attachment or self-assembly. Handle functionality was verified in solution, at the solid-liquid interface, and inside fixed cells, corresponding to applications in biosensing, DNA microarrays, and erasable immunocytochemistry. These experiments demonstrate the versatility of the electrostatic DNA caging approach and provide a new pathway to nanoparticle modification with DNA that will empower further applications of these materials in medicine and materials science.

Extralinguistic Consultation in English-Chinese Translation: A Study Drawing on Eye-Tracking and Screen-Recording Data.

Both linguistic and extralinguistic consultations are essential in translation practice and have been commonly investigated as an integral topic in previous studies. However, since extralinguistic information is usually longer in extent and not specifically designed for a linguistic purpose, extralinguistic consultations involve different search strategies compared with linguistic consultations. Drawing on eye-tracking and screen-recording data, this study compares linguistic and extralinguistic consultations in terms of cognitive resources allocation and information processing patterns in English-Chinese translation. It also explores the differences among 17 language learners, 20 student translators, and 21 professional translators, and the effect of extralinguistic consultation on their translation quality. The findings are as follows: (1) all participants allocate more attention and lower cognitive load to extralinguistic consultations than to linguistic consultations; (2) participants' translation experience levels and their attention allocated to extralinguistic consultation show an inverted U-shaped relationship; and (3) participants who consult extralinguistic information before drafting or devote more attention to extralinguistic consultation produce target texts with significantly higher scores.

Detection of Hepatitis B Virus-Host Junction Sequences in Urine of Infected Patients.

Integrated hepatitis B virus (HBV) DNA, found in more than 85% of HBV-associated hepatocellular carcinomas (HBV-HCCs), can play a significant role in HBV-related liver disease progression. HBV-host junction sequences (HBV-JSs), created through integration events, have been used to determine HBV-HCC clonality. Here, we investigate the feasibility of analyzing HBV integration in a noninvasive urine liquid biopsy. Using an HBV-targeted next-generation sequencing (NGS) assay, we first identified HBV-JSs in eight HBV-HCC tissues and designed short-amplicon junction-specific polymerase chain reaction assays to detect HBV-JSs in matched urine. We detected and validated tissue-derived junctions in five of eight matched urine samples. Next, we screened 32 urine samples collected from 25 patients infected with HBV (5 with hepatitis, 10 with cirrhosis, 4 with HCC, and 6 post-HCC). Encouragingly, all 32 urine samples contained HBV-JSs detectable by HBV-targeted NGS. Of the 712 total HBV-JSs detected in urine, 351 were in gene-coding regions, 11 of which, including TERT (telomerase reverse transcriptase), had previously been reported as recurrent integration sites in HCC tissue and were found only in the urine patients with cirrhosis or HCC. The integration breakpoints of HBV DNA detected in urine were found predominantly (~70%) at a previously identified integration hotspot, HBV DR1-2 (down-regulator of transcription 1-2). Conclusion: HBV viral-host junction DNA can be detected in urine of patients infected with HBV. This study demonstrates the potential for a noninvasive urine liquid biopsy of integrated HBV DNA to monitor patients infected with HBV for HBV-associated liver diseases and the efficacy of antiviral therapy.

Hyaluronic acid induces ROCK-dependent amoeboid migration in glioblastoma cells.

Glioblastoma (GBM) is the most aggressive and deadly adult brain tumor, primarily because of its high infiltrative capacity and development of resistance to therapy. Although GBM cells are typically believed to migrate via mesenchymal (e.g., fibroblast-like) migration modes, amoeboid (e.g., leucocyte-like) migration modes have been identified and may constitute a salvage pathway. However, the mesenchymal to amoeboid transition (MAT) process in GB is not well characterized, most likely because most culture models induce MAT via pharmacological or genetic inhibition conditions that are far from physiological. In this study, we examined the ability of hyaluronic acid (HA) content in three-dimensional collagen (Col) hydrogels to induce MAT in U87 GBM cells. HA and Col are naturally-occurring components of the brain extracellular matrix (ECM). In pure Col gels, U87 cells displayed primarily mesenchymal behaviors, including elongated cell morphology, clustered actin and integrin expression, and crawling migration behaviors. Whereas an increasing population of cells displaying amoeboid behaviors, including rounded morphology, cortical actin expression, low/no integrin expression, and squeezing or gliding motility, were observed with increasing HA content (0.1-0.2 wt% in Col). Consistent with amoeboid migration, these behaviors were abrogated by ROCK inhibition with the non-specific small molecule inhibitor Y27632. Toward identification of histological MAT classification criteria, we also examined the correlation between cell and nuclear aspect ratio (AR) in Col and Col-HA gels, finding that nuclear AR has a small variance and is not correlated to cell AR in HA-rich gels. These results suggest that HA may regulate GBM cell motility in a ROCK-dependent manner.

Moisture retention extended enhanced bioelectrochemical remediation of unsaturated soil.

Bioelectrochemical systems (BESs) have demonstrated great promise in augmented biodegradation of petroleum hydrocarbons in water-saturated soils. However, bioremediation of unsaturated soil in vadose zone has been a challenge due to poor mass transfer and low conductivity. This study proposed a moisture retention layer (2 cm thickness) around the BES anodes to enhance soil remediation under unsaturated conditions. The active soil BESs (closed circuit) includes two reactors with anodic moisture-retaining layers of soil-polyacrylamide hydrogel (SHB) and graphite granule-polyacrylamide hydrogel (GHB) mixtures, and another reactor filled with only soil (SB) without moisture-retaining layer. An open circuit SB was served as a control to simulate natural attenuation. This study demonstrated for the first time that moisture retention layers around the BES anodes could significantly extend and enhance hydrocarbon degradation in vadose zone soil. Results showed that SHB reactor could maintain 43-100% longer duration for electricity generation than other reactors. Correspondingly, SHB showed the best removal (average 21-37%) of total petroleum hydrocarbon (TPH) in spatial distribution, which was ~91% and ~164% higher than other BESs and control, respectively. This study demonstrated that by using low-cost and environmentally friendly hydrogel, BESs could become a viable remediation method for vadose zone soil.

MicroRNA-mRNA Interactions at Low Levels of Compressive Solid Stress Implicate mir-548 in Increased Glioblastoma Cell Motility.

Glioblastoma (GBM) is an astrocytic brain tumor with median survival times of <15 months, primarily as a result of high infiltrative potential and development of resistance to therapy (i.e., surgical resection, chemoradiotherapy). A prominent feature of the GBM microenvironment is compressive solid stress (CSS) caused by uninhibited tumor growth within the confined skull. Here, we utilized a mechanical compression model to apply CSS (<115 Pa) to well-characterized LN229 and U251 GBM cell lines and measured their motility, morphology, and transcriptomic response. Whereas both cell lines displayed a peak in migration at 23 Pa, cells displayed differential response to CSS with either minimal (i.e., U251) or large changes in motility (i.e., LN229). Increased migration of LN229 cells was also correlated to increased cell elongation. These changes were tied to epigenetic signaling associated with increased migration and decreases in proliferation predicted via Ingenuity® Pathway Analysis (IPA), characteristics associated with tumor aggressiveness. miRNA-mRNA interaction analysis revealed strong influence of the miR548 family (i.e., mir-548aj, mir-548az, mir-548t) on differential signaling induced by CSS, suggesting potential targets for pharmaceutical intervention that may improve patient outcomes.

RNA ligation of very small pseudo nick structures by T4 RNA ligase 2, leading to efficient production of versatile RNA rings.

T4 RNA ligase 2 catalyses two types of reactions: (i) sealing of a nick structure in double-stranded RNA and (ii) connection of two single-stranded RNA strands. In order to obtain comprehensive views on these two types of reactions and widen the application scope of this RNA ligase, we here systematically analysed the connection of single-stranded RNA strands having different secondary structures. It has been found that the ligation is enormously promoted when a stem of only 4-bp or longer is formed in the 3'-OH side of the joining site. Additional placement of a stem in the 5'-phosphate side further facilitates the ligation. In contrast, perturbation of the stem structures in RNA substrates suppresses the ligation. These results indicate that ligation of two single-stranded RNA strands by T4 RNA ligase 2 is greatly promoted by forming a "nick-like intermediate". Even the unstable intermediate, formed only temporarily in the solution, is sufficiently effective. By designing the synthetic systems in terms of this finding, short single-stranded RNA rings of versatile sizes, which are otherwise hard to be obtained, are efficiently prepared in high selectivity and yield.

Dominance of electroactive microbiomes in bioelectrochemical remediation of hydrocarbon-contaminated soils with different textures.

Bioelectrochemical systems (BESs) are known to enhance the remediation of hydrocarbon-contaminated soil and sediments compared with natural attenuation, and the primary mechanism has been assumed as anaerobic degradation facilitated by electroactive bacteria (EAB) using the electrode as electron acceptor. However, known EAB were rarely found on the anodes of reported BESs, which challenged the fundamental mechanism of BESs although significant current generation was always observed during degradation of these recalcitrant substrates. This study however found the abundant EAB Geobacter (∼27.3%) in the anodic biofilms, which confirmed the role of electroactive bio-anode on the conversion of hydrocarbons into the current for the enhancement of remediation. Widespread occurrence of aerobic hydrocarbon-degrading bacteria (HDB) (e.g. ∼24.0% Parvibaculum and ∼30.6% Pseudomonas) was observed in soils with limited dissolved oxygen (∼0.4 mg/L). The higher abundance of dehydrogenase genes was found in the anode biofilms than that in soils, indicating anodic microorganisms may be mainly responsible for the removal of intermediates of aerobic hydrocarbons degradation in soils. High water saturation level and sandy soil texture showed positive impacts on bioelectrochemical remediation, while clay soil and unsaturation condition pose challenges in mass transfers in the matrix. The reactor performance was consistent with the phylogenetic molecular ecological network (pMENs) analysis, which showed that sandy soil BESs had tighter microbial network interactions than clay soil reactors.

Cyclization of secondarily structured oligonucleotides to single-stranded rings by using Taq DNA ligase at high temperatures.

Single-stranded DNA rings play important roles in nanoarchitectures, molecular machines, DNA detection, etc. Although T4 DNA ligase has been widely employed to cyclize single-stranded oligonucleotides into rings, the cyclization efficiency is very low when the oligonucleotides (l-DNAs) take complicated secondary structures at ambient temperatures. In the present study, this problem has been solved by using Thermus aquaticus DNA ligase (Taq DNA ligase) at higher temperatures (65 and 70 °C) where the secondary structures are less stable or completely destroyed. This method is based on our new finding that this ligase successfully functions even when the splint strand is short and forms no stable duplex with l-DNA (at least in the absence of the enzyme). In order to increase the efficiency of cyclization, various operation factors (lengths and sequences of splint, as well as the size of the DNA ring) have been investigated. Based on these results, DNA rings have been successfully synthesized from secondarily structured oligonucleotides in high yields and high selectivity. The present methodology is applicable to the preparation of versatile DNA rings involving complicated secondary structures, which should show novel properties and greatly widen the scope of DNA-based nanotechnology.

Magnetic Quantum Dots Steer and Detach Microtubules From Kinesin-Coated Surfaces.

The microtubule (MT)-kinesin system has been extensively studied because of its role in cellular processes, as well as its potential use for controllably transporting objects at the nanoscale. Thus, there is substantial interest in methods to evaluate MT properties, including bending radius and the binding energy of kinesin motor proteins to MT tracks. Current methods to identify these properties include optical tweezers, microfluidic devices, and magnetic fields. Here, the use of magnetic quantum dots (i.e., MagDots) is evaluated as a method to study MT-kinesin interactions via applied magnetic forces. Magnetic fields are generated using a magnetic needle whose field gradient is quantified by finite element modeling (FEM). Magnetic force is applied to MagDot-labeled MTs and demonstrated sufficient to steer and detach MTs from kinesin-coated surfaces. Taking advantage of the dual-functionality of MagDots, the magnetic force experienced by a single MagDot and the number of MagDots on MTs are determined. The total force exerted on MTs by MagDots is estimated to be ≈0.94-2.47 pN. This approach could potentially be used to interrogate MT properties and MT-kinesin interactions, enhancing our biological understanding of this system and enabling further development of MT shuttles for nanotransport.