Design and Screening of Fluorescent Probes Based upon Hemicyanine Dyes for Monitoring Mitochondrial Viscosity in Living Cells.

Viscosity, at the subcellular level, plays a crucial role as a physicochemical factor affecting microenvironment homeostasis. Abnormal changes in mitochondrial viscosity often lead to various diseases in the organism. Based on the twisted intramolecular charge transfer mechanism, four hemicyanine dye fluorescent probes (HT-SA, HT-SA-S, HT-Bzh, and HT-NA) were designed and synthesized for viscosity response. The single bond between the nitrogen-containing heterocycle and the carbon-carbon double in the structure of the probe bond served as the viscosity response site. Finally, the probe HT-Bzh was screened as the optimal mitochondrial viscosity probe according to its responsiveness, targeting, and interference resistance. The fluorescence intensity of the probe HT-Bzh increased 22-fold when the viscosity was increased from 13.75 to 811.2 cP. In summary, all four viscosity probes we have developed can be used in different applications depending on the external environment, providing a valuable reference for the design of potential tools to address viscosity monitoring in biological systems.

G-quadruplex in mitochondria as a possible biomarker for mitophagy detection.

Mitochondrial autophagy (mitophagy) is a key physiological process that maintains the homeostasis of mitochondrial quality and quantity. Monitoring mitophagy is of great significance for detecting cellular abnormalities and developing therapeutic drugs. However, there are still very few biomarkers specifically developed for monitoring mitophagy. Here, we propose for the first time that mitochondrial G-quadruplex may serve as a biomarker for mitophagy detection, and develope a fluorescent light-up probe AMTC to monitor mitophagy in live cells. During mitophagy, AMTC fluorescence is significantly enhanced, but once mitophagy is inhibited, its fluorescence immediately decreases. The fluorescence behavior of AMTC implicates an increase in the formation of mitochondrial G-quadruplex during mitophagy. This inference has also been supported by the other two G-quadruplex probes. Taken together, this work provides a new possible biomarker and detection tool for the study of mitophagy.

Development of a one-pot and sequence-specific LAMP assay based on the self-quenching probe integrated by the complementary oligonucleotide for an enhanced fluorescence quenching.

Single-modified fluorogenic primer (Sfp) enables accurate identification of LAMP amplicons without being affected by non-specific products. However, the fluorescence self-quenching by nucleobases for Sfp is generally of low efficiency, and the high background signal makes it a great challenge to achieve visual inspection with naked eyes. In the present study, the oligonucleotide (Ao) complementary to Sfp was designed, which would hybridize to Sfp and dramatically heighten the quenching effect, leading to a low background signal in negative reaction. Instead, for positive reaction, Sfp is incorporated into the double-stranded amplicons, resulting in dequenching and consequently, enhanced fluorescence. The detection scheme can be further improved by a dual-color fluorescence strategy, allowing visual detection of 1 pg rainbow trout DNA in a closed-tube format within 30 min. Therefore, our LAMP-Ao-Sfp assay represents a useful tool for rapid and sensitive detection, and can serve as a reliable method for on-site detection in low-resource settings.

Novel fluorescence strategy based on G-quadruplex structure-switching aptamer for enrofloxacin detection in food and environmental samples.

Enrofloxacin (ENR) is widely used in the prevention and treatment of animal infectious diseases, so it is necessary to strengthen the residue detection of this drug in animal-derived food and water environments. In this work, for the first time, we engineered assembly a split ENR aptamer into the G-quadruplex (G4) region to form a new aptamer (G4-ENRA) that provides a more sensitive signal-reporting function while retaining target-specific recognition ability of the aptamer. This rational design effectively overcomes the issue of difficulty in identification probe development. Under the optimized conditions, a response range of 0.05-20 µM and limit of detection of 26.7 nM were obtained by directly detecting fluorescence signals, displaying a comparative advantage over the previously reported methods. Moreover, this method demonstrated satisfactory performance for the ENR detection in various real food and environmental samples, with the detection recoveries ranging from 95.87 % to 104.36 %, illustrating promising applicability prospects.

Advances and prospects of natural dietary polyphenols as G-quadruplex stabilizers in biomedical applications.

G-quadruplexes (G4s) have arrested continuous interest in cancer research, and targeting G4s with small molecules has become an ideal approach for drug development. Plant-based dietary polyphenols have attracted much attention for their remarkable anti-cancer effects. Studies have suggested that polyphenols exhibit interesting scaffolds to bind G4s, which can effectively downregulate the proto-oncogenes by stabilizing those G4 structures. Therefore, this review not only summarizes studies on natural dietary polyphenols (including analogs) as G4 stabilizers, but also reveals their anti-cancer activities. Furthermore, the structural and antioxidant insights of polyphenols with G4s are discussed, and future development is proposed. These insights may pave the way for the development of the next generation of anti-cancer drugs targeting nucleic acids.

Preschool or/and kindergarten? The long-term benefits of different types of early childhood education on pupils' skills.

BACKGROUND: Developing countries have witnessed great progress in early childhood education (ECE) enrollment rate over the past three decades. Preschool and kindergarten are the two most common types of ECE in developing countries. Questions remain as to which of the two types of ECE is more effective in promoting child development in developing countries, including both cognitive and non-cognitive skills. The objective of this paper is to examine the long-term benefits of attending preschool or/and kindergarten on pupils' cognitive and non-cognitive skills in rural China. METHODOLOGY: We pooled data from two large-scale surveys conducted by the authors themselves at 136 rural primary schools in 20 counties from three provinces in northwestern China in 2009. The final study sample consisted of 9,839 pupils who both reported their ECE experience and completed cognitive and non-cognitive tests. We measured pupils' cognitive skills by standardized math test scores and grade retention, and their non-cognitive skills by both self-reported self-efficacy, mental health, and teacher-reported behaviors. Inverse Probability Weighting (IPW) was used to balance the pre-treatment variables between the treatment (Any ECE, Preschool Only, Kindergarten Only, or Preschool+Kindergarten) and comparison (No ECE) groups. RESULTS: Results from IPW show that compared with their peers without any ECE experience, pupils with any ECE experience perform better in cognitive skills (0.118 standard deviations (s.d.) increase in the TIMSS, 7.1 percentage point (pp) decrease in the probability of grade retention) but not in non-cognitive skills. By ECE types, attending kindergarten only is associated with a 0.150 s.d. increase in the TIMSS, a 7.0 pp decrease in the probability of grade retention, and a 0.059 s.d. decrease in the index of behavioral problems of pupils. Moreover, attending both preschool and kindergarten predicts a lower probability of grade retention, but attending preschool only has few benefits. Heterogenous analyses suggest that the long-term benefits of ECE are more prominent among the Han pupils from households with higher socio-economic status. CONCLUSIONS: Our findings imply that increasing access to ECE can be an effective instrument to improve pupils' skills in less-developed rural areas of China, especially their cognitive skills. Among different types of ECE, attending kindergarten contributes more to pupils' skill development in rural China than other types. We call for strengthened efforts to ensure equal access to quality ECE for preschool-aged children in rural China.

Template-Free and Stretchable Conductive Fiber with a Built-In Helical Structure for Strain-Insensitive Signal Transmission.

With the rapid development of intelligent electronic devices, conductive fibers have become very critical to signal transmission devices. However, metal-based rigid conductive wires, such as high-modulus copper and silver wires, are prone to signal failure owing to tensile breakage under large strain conditions. Therefore, strain-insensitive stretchable conductive fibers for signal transmission are critical for next-generation wearable devices. Herein, a stretchable conductive fiber with a built-in helical structure is constructed by a "speed discrepancy" fiber-coating strategy with mass scalable production (60 cm/min). Such a "speed discrepancy" strategy is the key mechanism to template-free fabricate a built-in helical structure of the stretchable conductive fiber. The resultant fiber exhibits high conductivity (873 S/cm), stable insensitive signal transmission with a high quality factor (47.4), and a low relative resistance change (∼6%) under large strain. The built-in helical structure inspired by loofah whiskers endows the fiber with excellent strain insensitivity, and it can withstand large strains. On the proof of concept, our fiber can be seamlessly knitted, woven, and braided into smart textiles as an ideal signal transmission device under large strains, which will undoubtedly promote the development of intelligent electronic textiles and next-generation wearable devices.

Design and Screening of Fluorescent Probes Based upon Hemicyanine Dyes to Sensitively Respond to HSO3- in Living Cells.

SO2, a gas signaling molecule, can be produced endogenously in mitochondria. Its hydrolysate, HSO3-, plays a key role in food preservation, cardiovascular relaxation, and other fields, suggesting that it is important to achieve its detection. Here, based on the Michael addition mechanism, four hemicyanine dye fluorescent probes (ETN, ETB, STB, and EIB) were designed and synthesized for responding to HSO3-. We evaluated the reaction ability of different probes with HSO3- and tried to explain the reasons for the significantly different response effects between probes and HSO3- according to the structure-activity relationship. The influence of different substituents of probes on the properties of mitochondria-targeting was also discussed. Finally, we screened out ETN as the optimal HSO3- probe due to its high sensitivity, rapid reactivity, and good mitochondria-targeting, and it could sensitively respond to HSO3- in living cells. The LODs of ETN for HSO3- were calculated by both absorption and fluorescence methods, respectively, which were 2.727 and 0.823 µM. Our work provided valuable references for designing strategies and potential tools for response to SO2 derivatives in biosystems.

"One stone, two birds": a mitochondria-targeted fluorescent probe for the detection of viscosity and HSO3- in living cells.

The material transport and physiological events of mitochondria need to be supported by a suitable microenvironment. For example, high viscosity will seriously hinder material exchange, and SO2, as the precursor of HSO3-, is an endogenous signal molecule that plays a key role in information transmission. It is very important to detect viscosity and HSO3- in mitochondria. Here, we developed a dual-responsive fluorescent probe (named Hcy-NT) to image the changes in mitochondrial viscosity and HSO3- in a "killing two birds with one stone" manner. Hcy-NT showed an OFF-ON fluorescence signal for the increase in cell viscosity induced by nystatin, while an ON-OFF fluorescence signal for intracellular and endogenous HSO3-. Its limits of detection for HSO3- were calculated by both absorption and fluorescence methods, which were 1.200 and 1.291 µM, respectively. This work provides a valuable tool for the study of viscosity and HSO3- related physiological processes and the diagnosis of potential diseases.

Glutaminase 1 deficiency confined in forebrain neurons causes autism spectrum disorder-like behaviors.

An abnormal glutamate signaling pathway has been proposed in the mechanisms of autism spectrum disorder (ASD). However, less is known about the involvement of alterations of glutaminase 1 (GLS1) in the pathophysiology of ASD. We show that the transcript level of GLS1 is significantly decreased in the postmortem frontal cortex and peripheral blood of ASD subjects. Mice lacking Gls1 in CamKIIα-positive neurons display a series of ASD-like behaviors, synaptic excitatory and inhibitory (E/I) imbalance, higher spine density, and glutamate receptor expression in the prefrontal cortex, as well as a compromised expression pattern of genes involved in synapse pruning and less engulfed synaptic puncta in microglia. A low dose of lipopolysaccharide treatment restores microglial synapse pruning, corrects synaptic neurotransmission, and rescues behavioral deficits in these mice. In summary, these findings provide mechanistic insights into Gls1 loss in ASD symptoms and identify Gls1 as a target for the treatment of ASD.

Effectiveness of a Game-Based Mobile Application in Educating Nursing Students on Venous Blood Specimen Collection: A Randomized Controlled Trial.

Aim: Incorporating mobile applications into traditional clinical teaching methods to assess the impact of game-based mobile applications on the practical knowledge and skill levels of venous blood specimen collection among nursing students. Background: Although game-based mobile applications are recognized as teaching aids that replicate clinical practice in a safe environment, their impact and effectiveness are relatively unknown in the education of nursing students. Design: In September 2021, a single-blind randomized controlled trial was conducted in a university-affiliated hospital in China. Methods: One hundred five nursing students were randomly divided into the control group (n = 53) and the experimental group (n = 52). All participants received the same theoretical and operational training. For the next 7 days, the experimental group used a game-based mobile application, and the control group practiced venous blood specimen collection using traditional teaching methods. We observed the before-and-after comparison of the skill performance and learning curve of both groups of participants. Results: The final skill performance scores of the nursing students in the experimental group were higher than that of the nursing students in the control group (P < 0.001). Analysis of the learning curve showed that to master the skills, the experimental and control groups needed an average of 8 and 10 repetitions, respectively. Conclusion: This mobile application has a positive learning effect on nursing students' venous blood specimen collection skills in the short term. It should be applied to the training of clinical nursing skills.

A label-free G-quadruplex aptamer fluorescent aptasensor for visual and real-time kanamycin detection in lake and human samples.

Antibiotic abuse is considered a serious problem affecting human health, necessitating that great attention be paid to explore robust, simple and sensitive methods for rapid evaluation. In this paper, we developed a fluorescent aptasensor for visual and real-time kanamycin detection by taking advantage of the label-free strategy based on H-aggregate disassembly of a chiral cyanine dye induced by a G-quadruplex aptamer. The good sensitivity and selectivity enabled this aptasensor to have a detection limit as low as 43 nM and have high specificity for kanamycin recognition. Furthermore, this assay was successfully applied for the detection of kanamycin in lake water and urine with excellent recoveries.

RNA G-quadruplex in live cells lighted-up by a thiazole orange analogue for SCA36 identification.

SCA36 is a neurodegenerative disease mainly caused by the abnormal expansion of the GGGCCT repeat sequence in intron 1 of NOP56. The RNA sequences of this gene are expected to form large amounts of G-quadruplexes in the cytoplasm, which may be a potential intervention and detection target for SCA36. Here, we have developed a small-molecular compound named TCB-1, which shows good selectivity to the G-quadruplex structure, and its fluorescence can be enhanced by hundreds of folds. Interestingly, TCB-1 can avoid lysosome capture, evenly disperse in the cytoplasm, and selectively light up the cytoplasmic RNA G-quadruplexes. This property allows TCB-1 to sensitively detect the increased formation of cytoplasmic RNA G-quadruplexes in SCA36 model cells. This work not only provides new ideas for the design of small-molecule compounds targeting RNA G-quadruplexes in living cells, but also intuitively demonstrates the increased formation of RNA G-quadruplexes caused by NOP56 gene mutation, providing a possible tool for the detection of SCA36.

Microglial glutaminase 1 deficiency mitigates neuroinflammation associated depression.

Glutaminase 1 (GLS1) has recently been reported to be expressed in microglia and plays a crucial role in neuroinflamation. Significantly increased level of GLS1 mRNA expression together with neuroinflammation pathway were observed in postmortem prefrontal cortex from depressed patients. To find out the function of microglial GLS1 in depression and neuroinflammation, we generated transgenic mice (GLS1 cKO), postnatally losing GLS1 in microglia, to detect changes in the lipopolysaccharide (LPS)-induced depression model. LPS-induced anxiety/depression-like behavior was attenuated in GLS1 cKO mice, paralleled by a significant reduction in pro-inflammatory cytokines and an abnormal microglia morphological phenotype in the prefrontal cortex. Reduced neuroinflammation by GLS1 deficient microglia was a result of less reactive astrocytes, as GLS1 deficiency enhanced miR-666-3p and miR-7115-3p levels in extracellular vesicles released from microglia, thus suppressing astrocyte activation via inhibiting Serpina3n expression. Together, our data reveal a novel mechanism of GLS1 in neuroinflammation and targeting GLS1 in microglia may be a novel strategy to alleviate neuroinflammation-related depression and other disease.

An organic molecular compound for in situ identification of mitochondrial G-quadruplexes in live cells.

Emerging studies have shown that mitochondrial G-quadruplex plays a critical role in regulating mitochondrial gene replication and transcription, which makes it a promising target for the diagnosis and treatment of cancer or other major diseases. Molecular compounds that can highly target the mitochondrial G-quadruplexes in live cells are essential for further revealing the function and mechanism of these G-quadruplexes. Here, we have developed an organic molecular compound that can highly target the mitochondria of living cells by virtue of the membrane potential mechanism. Then it shows high selectivity to the G-quadruplex structure in the mitochondria, and its fluorescence overlaps well with that of the BG4 antibody. Moreover, the compound has extremely low cytotoxicity and does not interfere with the natural state of G-quadruplex structure. With these good properties, this compound will have great potential in mitochondrial G-quadruplex tracking research or targeted drug screening.

G4LDB 2.2: a database for discovering and studying G-quadruplex and i-Motif ligands.

Noncanonical nucleic acid structures, such as G-quadruplex (G4) and i-Motif (iM), have attracted increasing research interests because of their unique structural and binding properties, as well as their important biological activities. To date, thousands of small molecules that bind to varying G4/iM structures have been designed, synthesized and tested for diverse chemical and biological uses. Because of the huge potential and increasing research interests on G4-targeting ligands, we launched the first G4 ligand database G4LDB in 2013. Here, we report a new version, termed G4LDB 2.2 (http://www.g4ldb.com), with upgrades in both content and function. Currently, G4LDB2.2 contains >3200 G4/iM ligands, ∼28 500 activity entries and 79 G4-ligand docking models. In addition to G4 ligand library, we have also added a brand new iM ligand library to G4LDB 2.2, providing a comprehensive view of quadruplex nucleic acids. To further enhance user experience, we have also redesigned the user interface and optimized the database structure and retrieval mechanism. With these improvements, we anticipate that G4LDB 2.2 will serve as a comprehensive resource and useful research toolkit for researchers across wide scientific communities and accelerate discovering and validating better binders and drug candidates.

Clinical Characteristics of 195 Cases of COVID-19 with Gastrointestinal Symptoms COVID-19 with Gastrointestinal Symptoms.

BACKGROUND: Patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), have fever, dry cough, dyspnea, and fatigue. The disease has now become a global pandemic. The purpose of this study was to explore the relationship between COVID-19 and gastrointestinal (GI) symptoms. METHODS: We collected and analyzed data on patients with laboratory-confirmed COVID-19 by high-throughput sequencing or reverse transcription-polymerase chain reaction. We reviewed electronic medical records of 405 hospitalized COVID-19 patients in the Third Hospital of Wuhan. RESULTS: Among the 405 confirmed patients, 210 had no GI symptoms, 195 had GI symptoms, and the first symptom of 155 patients was GI. The prevalence of vascular and digestive diseases in the group with GI symptoms was significantly higher than in the group without GI symptoms. In patients with GI symptoms, the proportion with fever, cough, dysphoria, chest tightness, poor appetite, chest pain, and pharyngeal pain was significantly higher than in those without GI symptoms. There was no significant difference in imaging between the 2 groups. In patients with GI symptoms, the proportion with increased procalcitonin (PCT) level and decreased lymphocyte count was significantly higher than in those without GI symptoms. CONCLUSION: COVID-19 patients with GI symptoms had significantly more vascular and digestive system diseases and were more likely to have clinical manifestations of fever, cough, poor appetite, chest tightness, chest pain, insomnia, and pharyngeal pain. There were more patients with diarrhea, nausea, and vomiting. Patients with GI symptoms were more likely to have increased PCT and decreased lymphocyte count.

An increase in DNA G-quadruplex formation in acute myelocytic leukemia is detected by a supramolecular probe.

Acute myeloid leukemia (AML) is a common acute leukemia in both adults and children, with poor early detection and diagnosis. Therefore, identifying new indicators for AML detection is significant for effective treatment. Here, we developed a supramolecular probe that exhibits high specificity and sensitivity to G-quadruplex structures in physiological buffer solution, chromosomes, and cells. Using this probe, we tested the DNA extracted from different types of cells and found that the DNA extracted from human acute myeloid leukemia cells HL-60 and KG-1 enhanced the probe fluorescence more significantly than the DNA extracted from other cells. This phenomenon may be related to a large number of G-quadruplexes in acute myeloid leukemia cells, implicating that G-quadruplex levels may be a potential indicator for the detection of acute myeloid leukemia.

A hybrid aggregate FRET probe from the mixed assembly of cyanine dyes for highly specific monitoring of mitochondria autophagy.

Mitochondria autophagy, also known as mitophagy, is a process in which mitochondria are wrapped by autophagosomes and fused with lysosomes for degradation. This process is essential for mitochondrial quality control. Here, we developed a hybrid aggregate FRET probe through mixed assembly of two cyanine dyes FMOTY and AMTC. In live cells, FMOTY and AMTC exist independently in lysosomes and mitochondria and will not produce interfering FRET background signals. The FRET signal is only generated when mitochondria is transported to lysosomes during mitophagy. This allows the hybridized aggregate to be used as a highly specific probe for monitoring mitophagy.

c-Myc G-quadruplex is sensitively and specifically recognized by a fluorescent probe.

The G-quadruplex structure formed by the c-myc gene sequence has attracted much attention due to its important physiological function in biology and wide application in nanotechnology. So far, probes capable of recognition of c-myc G-quadruplex with both high specificity and sensitivity are still scarce. This work presented a cyanine dye fluorescent probe named Cy-1, which has almost no fluorescence in aqueous solution, but showing more than 1000-fold fluorescence enhancement for recognizing c-myc G-quadruplex. Cy-1 also has good specificity and can selectively recognize c-myc G-quadruplex from other a variety of G-quadruplex and non-G-quadruplex structures. These properties make Cy-1 a promising probe for c-myc G-quadruplex recognition in nanotechnology or biology.