G-quadruplex structural variations in human genome associated with single-nucleotide variations and their impact on gene activity.

G-quadruplexes (G4s) formed by guanine-rich nucleic acids play a role in essential biological processes such as transcription and replication. Besides the >1.5 million putative G-4-forming sequences (PQSs), the human genome features >640 million single-nucleotide variations (SNVs), the most common type of genetic variation among people or populations. An SNV may alter a G4 structure when it falls within a PQS motif. To date, genome-wide PQS-SNV interactions and their impact have not been investigated. Herein, we present a study on the PQS-SNV interactions and the impact they can bring to G4 structures and, subsequently, gene expressions. Based on build 154 of the Single Nucleotide Polymorphism Database (dbSNP), we identified 5 million gains/losses or structural conversions of G4s that can be caused by the SNVs. Of these G4 variations (G4Vs), 3.4 million are within genes, resulting in an average load of >120 G4Vs per gene, preferentially enriched near the transcription start site. Moreover, >80% of the G4Vs overlap with transcription factor-binding sites and >14% with enhancers, giving an average load of 3 and 7.5 for the two regulatory elements, respectively. Our experiments show that such G4Vs can significantly influence the expression of their host genes. These results reveal genome-wide G4Vs and their impact on gene activity, emphasizing an understanding of genetic variation, from a structural perspective, of their physiological function and pathological implications. The G4Vs may also provide a unique category of drug targets for individualized therapeutics, health risk assessment, and drug development.

A pilot study on vitrectomy combined with scleral shortening for eyes with myopic macular retinoschisis-2y follow-up results.

AIM: To evaluate the effect of vitrectomy combined with scleral shortening for eyes with myopic macular retinoschisis. METHODS: Thirty-seven patients with myopic macular retinoschisis who underwent pars plana vitrectomy (PPV) combined with scleral shortening were reviewed. Axial length (AL), the height of macular retinoschisis, the height of retinal detachment if existed, the diameter of macular hole if existed and best corrected visual acuity (BCVA) were obtained. The preoperative and postoperative parameters were compared. RESULTS: At postoperative 24mo, the mean AL and height of macular retinoschisis were reduced significantly by 0.79 mm and 256.51 µm (t=8.064, P<0.0001; Z=-5.086, P<0.0001) respectively. In addition, the mean height of retinal detachment and diameter of macular hole were also reduced significantly by 365.38 µm and 183.68 µm (Z=-4.457, P=0.000008; Z=-2.983, P=0.003) respectively. Meanwhile, the postoperative BCVA was improved markedly (Z=-2.126, P=0.033). CONCLUSION: Vitrectomy combined with scleral shortening is an effective surgical method for eyes with myopic macular retinoschisis, whether or not macular hole and retinal detachment are present.

Protective Role of Coenzyme Q10 in Acute Sepsis-Induced Liver Injury in BALB/c Mice.

Sepsis increases the risk of the liver injury development. According to the research works, coenzyme Q10 exhibits hepatoprotective properties in vivo as well as in vitro. Current work aimed at investigating the protective impacts of coenzyme Q10 against liver injury in septic BALB/c mice. The male BALB/c mice were randomly segregated into 4 groups: the control group, the coenzyme Q10 treatment group, the puncture and cecal ligation group, and the coenzyme Q10+cecal ligation and puncture group. Cecal ligation and puncture was conducted after gavagaging the mice with coenzyme Q10 during two weeks. Following 48 h postcecal ligation and puncture, we estimated hepatic biochemical parameters and histopathological changes in hepatic tissue. We evaluated the expression of factors associated with autophagy, pyroptosis, and inflammation. Findings indicated that coenzyme Q10 decreased the plasma levels in alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase in the cecal ligation and puncture group. Coenzyme Q10 significantly inhibited the elevation of sequestosome-1, interleukin-1ß, oligomerization domain-like receptor 3 and nucleotide-binding, interleukin-6, and tumor necrosis factor-α expression levels; coenzyme Q10 also increased beclin 1 levels. Coenzyme Q10 might be a significant agent in the treatment of liver injury induced by sepsis.

Erratum to "Effect of Thymoquinone on Acute Kidney Injury Induced by Sepsis in BALB/c Mice".

[This corrects the article DOI: 10.1155/2020/1594726.].

One-Step High-Throughput Telomerase Activity Measurement of Cell Populations, Single Cells, and Single-Enzyme Complexes.

Telomerase, a key enzyme involved in telomere homeostasis, is a major player involved in or required for sustained cell proliferation. It is expressed in ∼90% tumor but rarely in normal somatic cells. Therefore, telomerase serves as a diagnostic marker and therapeutic target of cancers. Although many methods are available for measuring telomerase activity, a convenient, fast, sensitive, and reliable method is still lacking for routine use in both clinics and research. Here, we present a single-enzyme sensitivity telomere repeat amplification protocol for quantifying telomerase activity. With multiple optimizations, the protocol pushes the ultimate detection limit down to a single telomerase complex, enabling measurement of telomerase activity of not only multiple cancerous/normal cell samples but also single cancer cells alone or even in the presence of 8000 normal cells. Implemented in a one-step mix-and-run format, the protocol offers a most sensitive, fast, accurate, and reproducible quantification of telomerase activity with linearity ranging from 20,000 HeLa cancer cells to a single telomerase complex. It requires minimal manual operation and experimental skill and is convenient for either low or high throughput of samples. We expect that the protocol should provide practical routine analyses of telomerase in both research and clinical applications. As an example, we demonstrate how telomerase activity evolves at the single-cell level and partitions in cell division in early mouse embryo development.

Detection of genomic G-quadruplexes in living cells using a small artificial protein.

G-quadruplex (G4) structures formed by guanine-rich nucleic acids are implicated in essential physiological and pathological processes and serve as important drug targets. The genome-wide detection of G4s in living cells is important for exploring the functional role of G4s but has not yet been achieved due to the lack of a suitable G4 probe. Here we report an artificial 6.7 kDa G4 probe (G4P) protein that binds G4s with high affinity and specificity. We used it to capture G4s in living human, mouse, and chicken cells with the ChIP-Seq technique, yielding genome-wide landscape as well as details on the positions, frequencies, and sequence identities of G4 formation in these cells. Our results indicate that transcription is accompanied by a robust formation of G4s in genes. In human cells, we detected up to >123 000 G4P peaks, of which >1/3 had a fold increase of ≥5 and were present in >60% promoters and ∼70% genes. Being much smaller than a scFv antibody (27 kDa) or even a nanobody (12-15 kDa), we expect that the G4P may find diverse applications in biology, medicine, and molecular devices as a G4 affinity agent.

Effect of Thymoquinone on Acute Kidney Injury Induced by Sepsis in BALB/c Mice.

Acute kidney injury (AKI) is a common complication of sepsis and has also been observed in some patients suffering from the new coronavirus pneumonia COVID-19, which is currently a major global concern. Thymoquinone (TQ) is one of the most active ingredients in Nigella sativa seeds. It has a variety of beneficial properties including anti-inflammatory and antioxidative activities. Here, we investigated the possible protective effects of TQ against kidney damage in septic BALB/c mice. Eight-week-old male BALB/c mice were divided into four groups: control, TQ, cecal ligation and puncture (CLP), and TQ+CLP. CLP was performed after 2 weeks of TQ gavage. After 48 h, we measured the histopathological alterations in the kidney tissue and the serum levels of creatinine (CRE) and blood urea nitrogen (BUN). We also evaluated pyroptosis (NLRP3, caspase-1), apoptosis (caspase-3, caspase-8), proinflammatory (TNF-α, IL-1ß, and IL-6)-related protein and gene expression levels. Our results demonstrated that TQ inhibited CLP-induced increased serum CRE and BUN levels. It also significantly inhibited the high levels of NLRP3, caspase-1, caspase-3, caspase-8, TNF-α, IL-1ß, and IL-6 induced by CLP. Furthermore, NF-κB protein level was significantly decreased in the TQ+CLP group than in the CLP group. Together, our results indicate that TQ may be a potential therapeutic agent for sepsis-induced AKI.

Selective Targeting of Guanine-Vacancy-Bearing G-Quadruplexes by G-Quartet Complementation and Stabilization with a Guanine-Peptide Conjugate.

Stabilization of G-quadruplexes (G4s) formed in guanine-rich (G-rich) nucleic acids by small-molecule ligands has been extensively explored as a therapeutic approach for diseases such as cancer. Finding ligands with sufficient affinity and specificity toward G4s remains a challenge, and many ligands reported seemed to compromise between the two features. To cope with this challenge, we focused on targeting a particular type of G4s, i.e., the G-vacancy-bearing G-quadruplexes (GVBQs), by taking a structure complementation strategy to enhance both affinity and selectivity. In this approach, a G-quadruplex-binding peptide RHAU23 is guided toward a GVBQ by a guanine moiety covalently linked to the peptide. The filling-in of the vacancy in a GVBQ by the guanine ensures an exclusive recognition of GVBQ. Moreover, the synergy between the RHAU23 and the guanine dramatically improves both the affinity toward and stabilization of the GVBQ. Targeting a GVBQ in DNA by this bifunctional peptide strongly suppresses in vitro replication. This study demonstrates a novel and promising alternative targeting strategy to a distinctive panel of G4s that are as abundant as the canonical ones in the human genome.

DNA:RNA hybrid G-quadruplex formation upstream of transcription start site.

Bioinformatic analysis reveals an enrichment of putative DNA:RNA hybrid G-quadruplex-forming sequences (PHQS) on both sides of the transcription start sites (TSSs) in the genome of warm-blooded animals, suggesting a positive selection of PHQSs in evolution and functional role of DNA:RNA hybrid G-quadruplexes (HQs) in transcription. The formation of HQs downstream of TSS in transcribed DNA has been documented under in vitro conditions; however, it is still not known if such HQs can form at the upstream side of TSSs. In this study, we report that such HQs can form in transcription in DNA with two to three guanine tracts if RNA carrying the required number of G-tracts is supplied. We also show that the formation of such HQs is dependent on the negative supercoiling generated by RNA polymerases. These results suggest that HQs may also form at the upstream side of TSSs in vivo and play a role in transcription since the two requirements are satisfied in cells.

Targeting nucleic acids with a G-triplex-to-G-quadruplex transformation and stabilization using a peptide-PNA G-tract conjugate.

A dual-functional peptide-PNA (peptide nucleic acid) conjugate consisting of a PNA G3-tract and an RHAU23 peptide is devised to target nucleic acids bearing three tandem guanine tracts (G-tracts). The PNA G3-tract joins the three G-tracts to form a stable bimolecular G-quadruplex (G4) and the resulting G4 is then bound by the RHAU23 moiety to form an extra stable G4-peptide complex. Owing to this synergistic dual structural enforcement, the conjugate accomplished extremely high selectivity and nM to sub-nM affinities towards its targets that are up to 1000 times greater than the small molecule G4 ligands. As a result, the conjugate impacts the tracking activity of motor proteins on DNA with superior selectivity and potency that are rarely seen in other G4-targeting approaches.

Kinetics, conformation, stability, and targeting of G-quadruplexes from a physiological perspective.

The particular enrichment of G-quadruplex-forming sequences near transcription start sites signifies the involvement of G-quadruplexes in the regulation of transcription. The characterization of G-quadruplex formation, which holds the key to understand the function it plays in physiological and pathological processes, is mostly performed under simplified in vitro experimental conditions. Formation of G-quadruplexes in cells, however, occurs in an environment far different from the ones in which the in vitro studies on G-quadruplexes are normally carried out. Therefore, the characteristics of G-quadruplex structures obtained under the in vitro conditions may not faithfully reveal how the G-quadruplexes would behave in a physiologically relevant situation. In this mini-review, we attempt to briefly summarize the differences in a few important characteristics, including kinetics, conformation, and stability of G-quadruplex formation observed under the two conditions to illustrate how the intracellular environment might affect the behavior of G-quadruplexes largely based on the previous work carried out in the authors' laboratory. We also propose that unstable G-quadruplex variants may be better drug target candidates to improve selectivity and potency.

Free autologous internal limiting membrane transplantation in the treatment of large macular hole.

We evaluated the clinical efficacy of free internal limiting membrane (ILM) flap transplantation for the treatment of large macular hole over 500 µm in 42 consecutive patients. Quantified evaluation of the post-operative macular anatomy restoration was performed by spectral-domain optical coherence tomography in the 12mo follow-up. The results showed 41 eyes achieved successful closure (97.6%). Postoperative best corrected visual acuity, ellipsoid layer, and external limiting membrane disruption were significantly improved at all follow-up time points. The central foveal thickness was significantly higher at 1mo. We concluded that free ILM flap transplantation proves to be effective to achieve anatomical and functional improvement for the treatment of large macular hole.

Transmission of dynamic supercoiling in linear and multi-way branched DNAs and its regulation revealed by a fluorescent G-quadruplex torsion sensor.

DNA supercoiling is an important regulator of gene activity. The transmission of transcription-generated supercoiling wave along a DNA helix provides a way for a gene being transcribed to communicate with and regulate its neighboring genes. Currently, the dynamic behavior of supercoiling transmission remains unclear owing to the lack of a suitable tool for detecting the dynamics of supercoiling transmission. In this work, we established a torsion sensor that quantitatively monitors supercoiling transmission in real time in DNA. Using this sensor, we studied the transmission of transcriptionally generated negative supercoiling in linear and multi-way DNA duplexes. We found that transcription-generated dynamic supercoiling not only transmits along linear DNA duplex but also equally diverges at and proceeds through multi-way DNA junctions. We also show that such a process is regulated by DNA-protein interactions and non-canonical DNA structures in the path of supercoiling transmission. These results imply a transcription-coupled mechanism of dynamic supercoiling-mediated intra- and inter-chromosomal signal transduction pathway and their regulation in DNA.

Superhelicity Constrains a Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of Transcription.

Transcription induces formation of intramolecular G-quadruplex structures at the upstream region of a DNA duplex by an upward transmission of negative supercoiling through the DNA. Currently the regulation of such G-quadruplex formation remains unclear. Using plasmid as a model, we demonstrate that while it is the dynamic negative supercoiling generated by a moving RNA polymerase that triggers a formation of a G-quadruplex, the constitutional superhelicity determines the potential and range of the formation of a G-quadruplex by constraining the propagation of the negative supercoiling. G-quadruplex formation is maximal in negatively supercoiled and nearly abolished in relaxed plasmids while being moderate in nicked and linear ones. The formation of a G-quadruplex strongly correlates with the presence of an R-loop. Preventing R-loop formation virtually abolished G-quadruplex formation even in the negatively supercoiled plasmid. Enzymatic action and protein binding that manipulate supercoiling or its propagation all impact the formation of G-quadruplexes. Because chromosomes and plasmids in cells in their natural form are maintained in a supercoiled state, our findings reveal a physical basis that justifies the formation and regulation of G-quadruplexes in vivo. The structural features involved in G-quadruplex formation may all serve as potential targets in clinical and therapeutic applications.

Real-Time Detection Reveals Responsive Cotranscriptional Formation of Persistent Intramolecular DNA and Intermolecular DNA:RNA Hybrid G-Quadruplexes Stabilized by R-Loop.

G-quadruplex (GQ) structures are implicated in important physiological and pathological processes. Millions of GQ-forming motifs are enriched near transcription start sites (TSSs) of animal genes. Transcription can induce the formation of GQs, which in turn regulate transcription. The kinetics of the formation and persistence of GQs in transcription is crucial for the role they play but has not yet been explored. We established a method based on the fluorescence resonance energy transfer (FRET) technique to monitor in real-time the cotranscriptional formation and post-transcriptional persistence of GQs in DNA. Using a T7 transcription model, we demonstrate that a representative intramolecular DNA GQ and DNA:RNA hybrid GQ promptly form in proportion to transcription activity and, once formed, are maintained for hours or longer at physiological temperature even after transcription is stopped. Both their formation and persistence strongly depend on R-loop, a DNA:RNA hybrid duplex formed during transcription. Enzymatic removal of R-loop dramatically slows their formation and accelerates their unfolding. These results suggest that a transcription event is promptly read-out by GQ-forming motifs and the GQ formed can either perform regulation in fast response to transcription and/or memorized in DNA to mediate time-delayed regulation under the control of RNA metabolism and GQ-resolving activity. Alternatively, GQs need to be timely resolved to warrant success of translocating activities such as replication. The kinetic characteristics of GQs and its connection with the R-loop may have implications in transcription regulation, signal transduction, G-quadruplex processing, and genome stability.

Exceptionally Selective and Tunable Sensing of Guanine Derivatives and Analogues by Structural Complementation in a G-Quadruplex.

A guanine-vacancy-bearing G-quadruplex (GVBQ) interacts with guanine and derivatives by a structural complementation to form a more stable and intact G-quadruplex. Sensors using GVBQs are devised to detect guanine and other nucleobases, and their derivatives derived from structurally similar compounds. A strict requirement of Hoogsteen hydrogen bonds between the GVBQ and analyte in the structural complementation confers exceptional selectivity on the analyte. As such, subtle modifications on analytes affecting even a single hydrogen bond can preclude the recognition. In principle, the strategy may also be expanded to detect many planar cyclic compounds. Because nucleobases and derivatives/metabolites are involved in many physiological and pathological processes, this type of sensor may find applications in risk assessment of pathogenesis and therapeutics related to nucleic acid metabolism.

RNA G-quadruplex formation in defined sequence in living cells detected by bimolecular fluorescence complementation.

G-quadruplexes are implicated in many essential cellular processes and sequences with potential to form a G-quadruplex are widely present in DNA and RNA. However, it is difficult to know whether a sequence of interest naturally forms a G-quadruplex in living cells. Here we report the detection of a G-quadruplex in defined RNA sequences in living cells in a natural intracellular environment. A G-quadruplex forming sequence in a RNA transcript is tagged at proximity with an aptamer. The two structures are recognized respectively by two probe proteins each of which is fused with a split half of enhanced green fluorescent protein (eGFP). Simultaneous binding of the two proteins to RNA brings the two halves of eGFP into proximity, permitting them to reconstitute into a functional eGFP that emits fluorescence to signal the formation of a G-quadruplex in RNA. We show that a G-quadruplex can form in RNA and can be detected with sequence and structure specificity under both in vitro and in vivo conditions. The results, therefore, provide direct evidence for the formation of RNA G-quadruplexes in live cells and the method provides a useful tool to validate G-quadruplex formation in a specific sequence under a natural cellular condition.

Guanine-vacancy-bearing G-quadruplexes responsive to guanine derivatives.

G-quadruplex structures formed by guanine-rich nucleic acids are implicated in essential physiological and pathological processes and nanodevices. G-quadruplexes are normally composed of four Gn (n ≥ 3) tracts assembled into a core of multiple stacked G-quartet layers. By dimethyl sulfate footprinting, circular dichroism spectroscopy, thermal melting, and photo-cross-linking, here we describe a unique type of intramolecular G-quadruplex that forms with one G2 and three G3 tracts and bears a guanine vacancy (G-vacancy) in one of the G-quartet layers. The G-vacancy can be filled up by a guanine base from GTP or GMP to complete an intact G-quartet by Hoogsteen hydrogen bonding, resulting in significant G-quadruplex stabilization that can effectively alter DNA replication in vitro at physiological concentration of GTP and Mg(2+). A bioinformatic survey shows motifs of such G-quadruplexes are evolutionally selected in genes with unique distribution pattern in both eukaryotic and prokaryotic organisms, implying such G-vacancy-bearing G-quadruplexes are present and play a role in gene regulation. Because guanine derivatives are natural metabolites in cells, the formation of such G-quadruplexes and guanine fill-in (G-fill-in) may grant an environment-responsive regulation in cellular processes. Our findings thus not only expand the sequence definition of G-quadruplex formation, but more importantly, reveal a structural and functional property not seen in the standard canonical G-quadruplexes.

Strand-Biased Formation of G-Quadruplexes in DNA Duplexes Transcribed with T7 RNA Polymerase.

G-quadruplex-forming sequences are enriched near transcription start sites (TSSs) in animal genes. They readily form G-quadruplexes in transcription, which in turn regulate transcription. Therefore, the control of G-quadruplex formation is important for their functionality. It is now shown that G-quadruplexes form efficiently on the non-template, but hardly on the template DNA strand in the downstream vicinity of TSSs in DNA duplexes when they are transcribed by the T7 RNA polymerase (RNAP). Structural analysis reveals that the T7 RNAP causes distortion in a DNA duplex both inside and in front of the enzyme. This structural distortion leads to strand-biased G-quadruplex formation when a G-quadruplex-forming sequence is partially fed into the T7 RNAP to a position about seven nucleotides away from the front of RNA synthesis. Based on these facts, we propose a model for the strand-biased formation of G-quadruplexes in transcribed DNA duplexes.

Telomere G-Quadruplex as a Potential Target to Accelerate Telomere Shortening by Expanding the Incomplete End-Replication of Telomere DNA.

Chromosomes in human cells are protected by telomeres. Telomere shortens during each round of cell division because of the DNA end-replication problem. Cancer cells maintain telomere length homeostasis by either telomerase or/and the alternative lengthening of telomere (ALT) mechanism to sustain their division potential. Telomeric DNA tends to form G-quadruplex preferentially at the extreme 3' end. This unique feature prevents the 3' end from being used as a substrate of telomerase and as a primer in the ALT. Therefore, stabilizing telomere G-quadruplex is expected to inhibit both pathways and limit the proliferation of cancer cells. Based on a mathematical modeling and experimental results, this mini-review proposes a hypothesis that the formation of G-quadruplex in telomere may constitute a significant contribution to the incomplete end-replication of telomere DNA by preventing the priming of DNA synthesis near the 3' end during telomere replication. According to this, stabilization of telomere G-quadruplex by chemical ligand may promise to accelerate telomere shortening in proliferating cells.