Lactate dehydrogenase A promotes nasopharyngeal carcinoma progression through the TAK1/NF-κB Axis.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a malignant tumor that originates in the nasopharyngeal mucosa and is common in China and Southeast Asian countries. Cancer cells reprogram glycolytic metabolism to promote their growth, survival and metastasis. Glycolysis plays an important role in NPC development, but the underlying mechanisms remain incompletely elucidated. Lactate dehydrogenase A (LDHA) is a crucial glycolytic enzyme, catalyzing the last step of glycolysis. This study aims to investigate the exact role of LDHA, which catalyzes the conversion of pyruvate into lactate, in NPC development. METHODS AND RESULTS: The western blot and immunohistochemical (IHC) results indicated that LDHA was significantly upregulated in NPC cells and clinical samples. LDHA knockdown by shRNA significantly inhibited NPC cell proliferation and invasion. Further knockdown of LDHA dramatically weakened the tumorigenicity of NPC cells in vivo. Mechanistic studies showed that LDHA activated TGF-ß-activated kinase 1 (TAK1) and subsequent nuclear factor κB (NF-κB) signaling to promote NPC cell proliferation and invasion. Exogenous lactate supplementation restored NPC cell proliferation and invasion inhibited by LDHA knockdown, and this restorative effect was reversed by NF-κB inhibitor (BAY 11-7082) or TAK1 inhibitor (5Z-7-oxozeaenol) treatment. Moreover, clinical sample analyses showed that LDHA expression was positively correlated with TAK1 Thr187 phosphorylation and poor prognosis. CONCLUSIONS: Our results suggest that LDHA and its major metabolite lactate drive NPC progression by regulating TAK1 and its downstream NF-κB signaling, which could become a therapeutic target in NPC.

A nanopore counter for highly sensitive evaluation of DNA methylation and its application in in vitro diagnostics.

DNA methylation has been considered an essential epigenetic biomarker for diagnosing various diseases, such as cancer. A simple and sensitive way for DNA methylation level detection is necessary. Inspired by the label-free and ultra-high sensitivity of solid-state nanopores to double-stranded DNA (dsDNA), we proposed a nanopore counter for evaluating DNA methylation by integrating a dual-restriction endonuclease digestion strategy coupled with polymerase chain reaction (PCR) amplification. Simultaneous application of BstUI/HhaI endonucleases can ensure the full digestion of the unmethylated target DNA but shows no effect on the methylated ones. Therefore, only the methylated DNA remains intact and can trigger the subsequent PCR reaction, producing a large quantity of fixed-length PCR amplicons, which can be directly detected through glassy nanopores. By simply counting the event rate of the translocation signals, the concentration of methylated DNA can be determined to range from 1 aM to 0.1 nM, with the detection limit as low as 0.61 aM. Moreover, a 0.01% DNA methylation level was successfully distinguished. The strategy of using the nanopore counter for highly sensitive DNA methylation evaluation would be a low-cost but reliable alternative in the analysis of DNA methylation.

Bare glassy nanopore for length-resolution reading of PCR amplicons from various pathogenic bacteria and viruses.

In this study, it is confirmed that without addition of organic solvent and embedding polymer hydrogel into glass nanopore, bare glass nanopore can faithfully measure various lengths of DNA duplexes from 200 to 3000 base pairs with 200 base pairs resolution, showing well-separated peak amplitudes of blockage currents. Furthermore, motivated by this readout capability of duplex DNA, amplicons from Polymerase Chain Reaction (PCR) amplification are straightforwardly discriminated by bare glassy nanopore without fluorescent labeling. Except simultaneous discrimination of up to 7 different segments of the same lambda genome, various pathogenic bacteria and viruses including SARS-CoV-2 and its mutants in clinical samples can be discriminated at high resolution. Moreover, quantitative measurement of PCR amplicons is obtained with detection range spanning from 0.75 aM to 7.5 pM and detection limit of 7.5 aM, which reveals that bare glass nanopore can faithfully disclose PCR results without any extra labeling.

A signal on-off strategy based on the digestion of DNA cubes assisted by the CRISPR-Cas12a system for ultrasensitive HBV detection in solid-state nanopores.

Solid-state nanopores have been proven as a powerful platform for label-free single-molecule analysis. However, due to its relatively low resolution and selectivity, developing biosensors with good translocation signals faces two significant challenges: (1) small-sized chemical or biological targets show difficulty in producing recognizable translocation signals because of their weak interaction with the nanopore and (2) protein interferents that widely exist in biological samples or buffers would considerably deteriorate the noise level of the nanopore, submerging the translocation signal. Herein, we demonstrate an effective way to overcome both the challenges. DNA cubes were used as signal transducers that can achieve an ultra-high (>50 : 1) signal-to-noise ratio (SNR) translocation signal, which is maintained even in protein interferent-rich buffers. A sensing strategy was constructed via hepatitis B virus (HBV) target-triggered cleavage of the component elements of the DNA cube with the assistance of the CRISPR-Cas12a technology, which caused a great drop in the translocation rate. The elements to cleave were optimized, and the sensor performance was tested in different protein stabilizer-rich buffers and human serum. Coupling with the polymerase chain reaction (PCR) pre-amplification technology, HBV-positive or -negative classification was achieved with the detection limit reaching 5 aM. It is worth noting that in our method, all reaction buffers were directly used without further optimization, which is of great help for the practical application of solid-state nanopores.

Comprehensive analysis of lncRNAs N6-methyladenosine modification in colorectal cancer.

BACKGROUND: Long non-coding RNAs (lncRNAs) and their N6-methyladenosine (M6A) modifications are involved in cancer occurrence and development. METHODS: lncRNA M6A modification in colorectal cancer (CRC) was comprehensively analyzed for the first time. RESULTS: M6A levels of lnRNAs in CRC tissues were higher than those in tumor-adjacent normal tissues. A total of 8,332 M6A peaks were detected in 6,690 lncRNAs in CRC tissues. Approximately 91% of the modified lncRNAs had unique M6A modification peaks. A total of 383 lncRNAs were differentially methylated in CRC, of which 48.24% had a length of 1-1,000 bp. Most of these were located on chromosomes 1, 2, 7, 11, 16 and 19; 42.3% were within a sense-overlapping exon. RNA sequencing identified 163 differentially expressed lncRNAs in CRC. GO and KEGG analyses revealed that genes near differentially-methylated or -expressed lncRNAs were associated with CRC occurrence and development. Methylation was positively correlated with lncRNA expression levels in CRC and tumor-adjacent normal tissues. More unmethylated than M6A methylated lncRNA molecules were detected. A competing endogenous RNA (ceRNA) and lncRNA-mRNA expression-regulation network revealed a regulatory relationship between lncRNAs, microRNAs (miRNAs), and mRNAs. CONCLUSIONS: The findings may help improve our understanding of lncRNA function in colorectal cancer.

Chemical Probes Reveal Sirt2's New Function as a Robust "Eraser" of Lysine Lipoylation.

Lysine lipoylation, a highly conserved lysine post-translational modification, plays a critical role in regulating cell metabolism. The catalytic activity of a number of vital metabolic proteins, such as pyruvate dehydrogenase (PDH), depends on lysine lipoylation. Despite its important roles, the detailed biological regulatory mechanism of lysine lipoylation remains largely unexplored. Herein we designed a powerful affinity-based probe, KPlip, to interrogate the interactions of lipoylated peptide/proteins under native cellular environment. Large-scale chemical proteomics analysis revealed a number of binding proteins of KPlip, including sirtuin 2 (Sirt2), an NAD+-dependent protein deacylase. To explore the potential activity of Sirt2 toward lysine lipoylation, we designed a single-step fluorogenic probe, KTlip, which reports delipoylation activity in a continuous manner. The results showed that Sirt2 led to significant delipoylation of KTlip, displaying up to a 60-fold fluorescence increase in the assay. Further kinetic experiments with different peptide substrates revealed that Sirt2 can catalyze the delipoylation of peptide (DLAT-PDH, K259) with a remarkable catalytic efficiency (kcat/Km) of 3.26 × 103 s-1 M-1. The activity is about 400-fold higher than that of Sirt4, the only mammalian enzyme with known delipoylation activity. Furthermore, overexpression and silencing experiments demonstrated that Sirt2 regulates the lipoylation level and the activity of endogenous PDH, thus unequivocally confirming that PDH is a genuine physiological substrate of Sirt2. Using our chemical probes, we have successfully established the relationship between Sirt2 and lysine lipoylation in living cells for the first time. We envision that such chemical probes will serve as useful tools for delineating the roles of lysine lipoylation in biology and diseases.

Determinants and the Moderating Effect of Perceived Policy Effectiveness on Residents' Separation Intention for Rural Household Solid Waste.

Currently, villages "besieged with garbage" have become a serious problem in rural areas of China. Separation of rural residential solid waste (RRSW) is one of the main strategies for waste reduction. Although previous studies have analyzed the social and psychological motivations of residents' separation intention for municipal solid waste (MSW), little attention has been paid to the situation in rural areas. This paper investigates key factors influencing rural residents' separation intention, as well as analyzing the moderating effects of perceived policy effectiveness on the relationship between the determinants and the intention, using survey data of 538 rural residents in the province of Sichuan in China. The results show that all the proposed key factors influence the separation intention significantly. Furthermore, the policies were divided into two types and the moderating effects were tested for each type. The results show that the perceived effectiveness of both the inducement policy and the capacity building policy moderated the relationship between attitude and separation intention positively, while the perceived effectiveness of the inducement policy moderated the relationship between subjective norms and intention negatively. The findings provide insightful information for policymakers to design effective RRSW separation policies.

Fluorescent Probes for Single-Step Detection and Proteomic Profiling of Histone Deacetylases.

Histone deacetylases (HDACs) play important roles in regulating various physiological and pathological processes. Developing fluorescent probes capable of detecting HDAC activity can help further elucidate the roles of HDACs in biology. In this study, we first developed a set of activity-based fluorescent probes by incorporating the Kac residue and the O-NBD group. Upon enzymatic removal of the acetyl group in the Kac residue, the released free amine reacted intramolecularly with the O-NBD moiety, resulting in turn-on fluorescence. These designed probes are capable of detecting HDAC activity in a continuous fashion, thereby eliminating the extra step of fluorescence development. Remarkably, the amount of turn-on fluorescence can be as high as 50-fold, which is superior to the existing one-step HDAC fluorescent probes. Inhibition experiments further proved that the probes can serve as useful tools for screening HDAC inhibitors. Building on these results, we moved on and designed a dual-purpose fluorescent probe by introducing a diazirine photo-cross-linker into the probe. The resulting probe was not only capable of reporting enzymatic activity but also able to directly identify and capture the protein targets from the complex cellular environment. By combining a fluorometric method and in-gel fluorescence scanning technique, we found that epigenetic readers and erasers can be readily identified and differentiated using a single probe. This is not achievable with traditional photoaffinity probes. In light of the prominent properties and the diverse functions of this newly developed probe, we envision that it can provide a robust tool for functional analysis of HDACs and facilitate future drug discovery in epigenetics.

[Clinical observation of sleeping disorder in children with encephalopathy treated with acupuncture at head points and seed-pressure at ear points].

OBJECTIVE: To compare the difference of clinical efficacy on sleeping disorder in the children with encephalopathy between the combined therapy of acupuncture at head points and seed-pressure at ear points and the simple acupuncture at head points. METHODS: Thirty cases of sleeping disorder induced by encephalopathy werei randomized into an observation group and a control group, 15 cases in each one. In the observation group, the combined therapy of acupuncture at head points and seed-pressure at ear points was adopted. The head points in cluded Sishencong (EX-HN 1), Shenting (GV 24) and Benshen (GB 13). The ear points were the positive reactive sites in the cymba and cavum conchae. In the control group, acupuncture was applied simply to the acupoints on the head. The treatment was given once on every Tuesday and Friday a week separately, 30 min each time. Totally, 16 treatments were required. Children's sleeping habit questionnaire (CSHQ) was used to observe the sleep improvements and the efficacy in the patients of the two groups. RESULTS: In the observation group, the results of sleep resistance, sleep anxiety, night sleep wake, parasomnias, sleep dyspnea, daytime somnolence and the total score after treatment were all improved apparently as compared with those before treatment (all P<0. 05). In the control group, the results of night sleep wake, parasomnias, daytime somnolence and the total score after treatment were improved apparently than those before treatment (all P<0. 05). In the observation group, the results of sleep resistance, sleep dyspnea and the total score after treatment were better than those in the control group (all P<0. 05) and the scores of sleep anxiety and daytime somnolence in the control group were better than those in the observation group after treatment (both P<0. 05). CONCLUSION: The combined therapy of acupuncture at head points and seed-pressure at the positive reactive sites in the cymba and cavum conchae achieves the superior efficacy on sleep resistance and sleep dyspnea as compared with the simple acupuncture. The efficacy of simple acupuncture is more satisfactory on sleep anxiety and daytime somnolence.