ALOX5-mediated ferroptosis acts as a distinct cell death pathway upon oxidative stress in Huntington's disease.

Although it is well established that Huntington's disease (HD) is mainly caused by polyglutamine-expanded mutant huntingtin (mHTT), the molecular mechanism of mHTT-mediated actions is not fully understood. Here, we showed that expression of the N-terminal fragment containing the expanded polyglutamine (HTTQ94) of mHTT is able to promote both the ACSL4-dependent and the ACSL4-independent ferroptosis. Surprisingly, inactivation of the ACSL4-dependent ferroptosis fails to show any effect on the life span of Huntington's disease mice. Moreover, by using RNAi-mediated screening, we identified ALOX5 as a major factor required for the ACSL4-independent ferroptosis induced by HTTQ94. Although ALOX5 is not required for the ferroptotic responses triggered by common ferroptosis inducers such as erastin, loss of ALOX5 expression abolishes HTTQ94-mediated ferroptosis upon reactive oxygen species (ROS)-induced stress. Interestingly, ALOX5 is also required for HTTQ94-mediated ferroptosis in neuronal cells upon high levels of glutamate. Mechanistically, HTTQ94 activates ALOX5-mediated ferroptosis by stabilizing FLAP, an essential cofactor of ALOX5-mediated lipoxygenase activity. Notably, inactivation of the Alox5 gene abrogates the ferroptosis activity in the striatal neurons from the HD mice; more importantly, loss of ALOX5 significantly ameliorates the pathological phenotypes and extends the life spans of these HD mice. Taken together, these results demonstrate that ALOX5 is critical for mHTT-mediated ferroptosis and suggest that ALOX5 is a potential new target for Huntington's disease.

Inhibition of Gpx4-mediated ferroptosis alleviates cisplatin-induced hearing loss in C57BL/6 mice.

Cisplatin-induced hearing loss is a common side effect of cancer chemotherapy in clinics; however, the mechanism of cisplatin-induced ototoxicity is still not completely clarified. Cisplatin-induced ototoxicity is mainly associated with the production of reactive oxygen species, activation of apoptosis, and accumulation of intracellular lipid peroxidation, which also is involved in ferroptosis induction. In this study, the expression of TfR1, a ferroptosis biomarker, was upregulated in the outer hair cells of cisplatin-treated mice. Moreover, several key ferroptosis regulator genes were altered in cisplatin-damaged cochlear explants based on RNA sequencing, implying the induction of ferroptosis. Ferroptosis-related Gpx4 and Fsp1 knockout mice were established to investigate the specific mechanisms associated with ferroptosis in cochleae. Severe outer hair cell loss and progressive damage of synapses in inner hair cells were observed in Atoh1-Gpx4-/- mice. However, Fsp1-/- mice showed no significant hearing phenotype, demonstrating that Gpx4, but not Fsp1, may play an important role in the functional maintenance of HCs. Moreover, findings showed that FDA-approved luteolin could specifically inhibit ferroptosis and alleviate cisplatin-induced ototoxicity through decreased expression of transferrin and intracellular concentration of ferrous ions. This study indicated that ferroptosis inhibition through the reduction of intracellular ferrous ions might be a potential strategy to prevent cisplatin-induced hearing loss.

NRF2 Is a Major Target of ARF in p53-Independent Tumor Suppression.

Although ARF can suppress tumor growth by activating p53 function, the mechanisms by which it suppresses tumor growth independently of p53 are not well understood. Here, we identified ARF as a key regulator of nuclear factor E2-related factor 2 (NRF2) through complex purification. ARF inhibits the ability of NRF2 to transcriptionally activate its target genes, including SLC7A11, a component of the cystine/glutamate antiporter that regulates reactive oxygen species (ROS)-induced ferroptosis. As a consequence, ARF expression sensitizes cells to ferroptosis in a p53-independent manner while ARF depletion induces NRF2 activation and promotes cancer cell survival in response to oxidative stress. Moreover, the ability of ARF to induce p53-independent tumor growth suppression in mouse xenograft models is significantly abrogated upon NRF2 overexpression. These results demonstrate that NRF2 is a major target of p53-independent tumor suppression by ARF and also suggest that the ARF-NRF2 interaction acts as a new checkpoint for oxidative stress responses.

Structural basis and molecular mechanism of biased GPBAR signaling in regulating NSCLC cell growth via YAP activity.

The G protein-coupled bile acid receptor (GPBAR) is the membrane receptor for bile acids and a driving force of the liver-bile acid-microbiota-organ axis to regulate metabolism and other pathophysiological processes. Although GPBAR is an important therapeutic target for a spectrum of metabolic and neurodegenerative diseases, its activation has also been found to be linked to carcinogenesis, leading to potential side effects. Here, via functional screening, we found that two specific GPBAR agonists, R399 and INT-777, demonstrated strikingly different regulatory effects on the growth and apoptosis of non-small cell lung cancer (NSCLC) cells both in vitro and in vivo. Further mechanistic investigation showed that R399-induced GPBAR activation displayed an obvious bias for ß-arrestin 1 signaling, thus promoting YAP signaling activation to stimulate cell proliferation. Conversely, INT-777 preferentially activated GPBAR-Gs signaling, thus inactivating YAP to inhibit cell proliferation and induce apoptosis. Phosphorylation of GPBAR by GRK2 at S310/S321/S323/S324 sites contributed to R399-induced GPBAR-ß-arrestin 1 association. The cryoelectron microscopy (cryo-EM) structure of the R399-bound GPBAR-Gs complex enabled us to identify key interaction residues and pivotal conformational changes in GPBAR responsible for the arrestin signaling bias and cancer cell proliferation. In summary, we demonstrate that different agonists can regulate distinct functions of cell growth and apoptosis through biased GPBAR signaling and control of YAP activity in a NSCLC cell model. The delineated mechanism and structural basis may facilitate the rational design of GPBAR-targeting drugs with both metabolic and anticancer benefits.

Phosphine-Capped Effects Enable Full-Color Clusteroluminescence in Nonconjugated Polyesters.

Full-color luminophores have advanced applications in materials and engineering, but constructing color-tunable clusteroluminescence (CL) from nonconjugated polymers based on through-space interactions remains a huge challenge. Herein, we develop phosphine-capped nonconjugated polyesters exhibiting blue-to-red CL (400-700 nm) based on phosphine-initiated copolymerization of epoxides and cyclic anhydrides, especially P1-0.5TPP, which exhibits red CL (610 nm) with a high quantum yield of 32%. Experiments and theoretical calculations disclose that the phosphine-capped effect in polyesters brings about conformational changes and induces phosphine-ester clusters by through-space (n,π*) interactions. Moreover, CL colors and efficiencies can be easily tailored by types of phosphines, compositions and structures of polyesters, and concentration. Significantly, the role of polymer motions (group, segmental, and chain motions) on CL originating from microregions inside polyesters is revealed. Further, phosphine-capped nonconjugated polyesters are demonstrated to be nonconjugated dyes and fluorescent fibers and are also used for multicolor light-emitting diodes including white light. This work not only provides an engineering strategy based on the end-group effect to prepare full-color clusteroluminogens but also broadens the prospects for material applications.

Formation and removal of 1,N6-dimethyladenosine in mammalian transfer RNA.

RNA molecules harbor diverse modifications that play important regulatory roles in a variety of biological processes. Over 150 modifications have been identified in RNA molecules. N6-methyladenosine (m6A) and 1-methyladenosine (m1A) are prevalent modifications occurring in various RNA species of mammals. Apart from the single methylation of adenosine (m6A and m1A), dual methylation modification occurring in the nucleobase of adenosine, such as N6,N6-dimethyladenosine (m6,6A), also has been reported to be present in RNA of mammals. Whether there are other forms of dual methylation modification occurring in the nucleobase of adenosine other than m6,6A remains elusive. Here, we reported the existence of a novel adenosine dual methylation modification, i.e. 1,N6-dimethyladenosine (m1,6A), in tRNAs of living organisms. We confirmed that m1,6A is located at position 58 of tRNAs and is prevalent in mammalian cells and tissues. The measured level of m1,6A ranged from 0.0049% to 0.047% in tRNAs. Furthermore, we demonstrated that TRMT6/61A could catalyze the formation of m1,6A in tRNAs and m1,6A could be demethylated by ALKBH3. Collectively, the discovery of m1,6A expands the diversity of RNA modifications and may elicit a new tRNA modification-mediated gene regulation pathway.

Near-Infrared Emission Beyond 900†nm from Stable Radicals in Nonconjugated Poly(diphenylmethane).

Organic radicals with narrow energy gaps are highly sought-after for the production of near-infrared (NIR) fluorophores. However, the current repertoire of developed organic radicals is notably limited, facing challenges related to stability and low fluorescence efficiency. This study addresses these limitations by achieving stable radicals in nonconjugated poly(diphenylmethane) (PDPM). Notably, PDPM exhibits a well-balanced structural flexibility and rigidity, resulting in a robust intra-/inter-chain through-space conjugation (TSC). The stable radicals within PDPM, coupled with strong TSC, yield a remarkable full-spectrum emission spanning from blue to NIR beyond 900 nm. This extensive tunability is achieved through careful adjustments of concentration and excitation wavelength. The findings highlight the efficacy of polymerization in stabilizing radicals and introduce a novel approach for developing nonconjugated NIR emitters based on triphenylmethane subunits.

A large-scale investigation and identification of methicillin-resistant Staphylococcus aureus based on peaks binning of matrix-assisted laser desorption ionization-time of flight MS spectra.

Recent studies have demonstrated that the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) could be used to detect superbugs, such as methicillin-resistant Staphylococcus aureus (MRSA). Due to an increasingly clinical need to classify between MRSA and methicillin-sensitive Staphylococcus aureus (MSSA) efficiently and effectively, we were motivated to develop a systematic pipeline based on a large-scale dataset of MS spectra. However, the shifting problem of peaks in MS spectra induced a low effectiveness in the classification between MRSA and MSSA isolates. Unlike previous works emphasizing on specific peaks, this study employs a binning method to cluster MS shifting ions into several representative peaks. A variety of bin sizes were evaluated to coalesce drifted or shifted MS peaks to a well-defined structured data. Then, various machine learning methods were performed to carry out the classification between MRSA and MSSA samples. Totally 4858 MS spectra of unique S. aureus isolates, including 2500 MRSA and 2358 MSSA instances, were collected by Chang Gung Memorial Hospitals, at Linkou and Kaohsiung branches, Taiwan. Based on the evaluation of Pearson correlation coefficients and the strategy of forward feature selection, a total of 200 peaks (with the bin size of 10 Da) were identified as the marker attributes for the construction of predictive models. These selected peaks, such as bins 2410-2419, 2450-2459 and 6590-6599 Da, have indicated remarkable differences between MRSA and MSSA, which were effective in the prediction of MRSA. The independent testing has revealed that the random forest model can provide a promising prediction with the area under the receiver operating characteristic curve (AUC) at 0.8450. When comparing to previous works conducted with hundreds of MS spectra, the proposed scheme demonstrates that incorporating machine learning method with a large-scale dataset of clinical MS spectra may be a feasible means for clinical physicians on the administration of correct antibiotics in shorter turn-around-time, which could reduce mortality, avoid drug resistance and shorten length of stay in hospital in the future.

Large-scale and tunable transparent displays based on silver nanoparticles metasurface.

We report a transparent display based on a metasurface of silver nanoparticles (Ag NPs), consisting of a transparent substrate and a layer of Ag NPs deposited by a dielectric film. The Ag NPs metasurface is prepared by a simple and direct annealing process. It presents a deep transmission valley at the wavelength ofλ= 468 nm and enables desired transparent display by projecting the monochromatic image onto the metasurface. We also demonstrate that the formed Ag NPs can be approximated as truncated nanospheres, which have obvious directional scattering properties, and can radiate most of the scattered energy into the backward hemisphere with a relatively large angular beamwidth (the full width at half maximum of the scattered intensity) of ∼90°. Therefore, the fabricated displays possess wide viewing angles and high brightness characteristics. Additionally, the transmission modes can be red-shifted to the wavelength ofλ= 527 nm by controlling the thickness of the deposited dielectric film. This approach using traditional thin film deposition and moderate annealing processing techniques enables simple, low-cost, and scalable fabrication in large areas for transparent displays.

Identification and screening of fungicides against Piper nigrum basal Fusarium wilt disease in Hainan, China.

Fusarium wilt has occurred in the main Piper nigrum cultivation regions, which seriously affects the yield and quality of P. nigrum. To identify the pathogen of this disease, the diseased roots were collected from a demonstration base in Hainan Province. The pathogen was obtained by tissue isolation method and confirmed by pathogenicity test. Based on the morphological observation, sequence analyses of TEF1-α nuclear gene, Fusarium solani was identified as the pathogen causing P. nigrum Fusarium wilt and induced symptoms on inoculated plants, including chlorosis, necrotic spots, wilt, drying, and root rot. The experiments for the antifungal activity showed that all the 11 fungicides selected in this study showed certain inhibitory effects on the colony growth of F. solani, where 2% kasugamycin AS, 45% prochloraz EW, 25 g·L-1 fludioxonil SC and 430 g·L-1 tebuconazole SC exhibited relative higher inhibitory effects with EC50 as 0.065, 0.205, 0.395, and 0.483 mg·L-1 , respectively, and were selected to perform SEM analysis and test in seeds in vitro. The SEM analysis showed that kasugamycin, prochloraz, fludioxonil, and tebuconazole might have exerted their antifungal effect by damaging F. solani mycelia or microconidia. These preparations were applied as a seed coating of P. nigrum Reyin-1. The kasugamycin treatment was most effective in reducing the harmful impact of F. solani on the seed germination. These results presented herein provide useful guidance for the effective control of P. nigrum Fusarium wilt.

NIR Clusteroluminescence of Non-conjugated Phenolic Resins Enabled by Through-Space Interactions.

Clusteroluminescence (CL) and through-space interactions (TSIs) of non-conjugated molecules have drawn more attention due to their unique photophysical behaviors that are different from largely conjugated luminogens. However, achieving red and even near-infrared (NIR) emission from such systems is still challenging due to the intrinsic drawbacks of non-conjugated molecules and the lack of theories for structure-property relationships. In this work, six phenolic resins are designed and synthesized based on two molecule-engineering strategies: increasing the number of TSIs units and introducing electron-donating/-withdrawing groups. All phenolic resins are verified as luminogens with CL property (CLgens), and the first example of CLgens with NIR emission (maximum emission wavelength ≥680 nm) and high absolute quantum yield (47 %) is reported. Experiments and theoretical analysis reveal that two TSIs types, through-space locally excited state and through-space charge transfer state, play essential roles in achieving CL from these non-conjugated polymers, which could be manipulated via changing structural conformation and electron density or altering electron transition behaviors. This work not only provides an approach to manipulate TSIs and CL of non-conjugated polymers but also endows commercially available phenolic resins with high practical value as luminescence materials.

Aliphatic Polyesters with White-Light Clusteroluminescence.

Single-molecule white-light emission (SMWLE) has many advantages in practical applications; however, the fabrication of SMWLE from nonconjugated luminescent polymers, namely, clusteroluminogens (CLgens), is still a big challenge. Herein, the first example of linear nonconjugated polyesters with SMWLE is reported. Twenty-four kinds of nonconjugated aliphatic polyesters with tunable clusteroluminescence (CL) colors and efficiency were synthesized by the copolymerization of six epoxides and four anhydrides. Experimental and calculation results prove that, at the primary structure level, the balance of structural flexibility and rigidity via adjusting the side-chain length significantly enhances the efficiency of CL without wavelength change. However, altering the chemical structures of the monomer from succinic anhydride to trans-maleic anhydride (MA), cis-MA, and citraconic anhydride (CA), secondary structures of these polyesters change from helix to straight and folding sheet accompanied by gradually red-shifted CL from 460 to 570 nm due to the increase in through-space n-π* interactions, as demonstrated by the computational and experimental results. Then, pure SMWLE with CIE coordination (0.30, 0.32) based on overlapped short-wavelength and long-wavelength CL is achieved in CA-based polyesters. This work not only provides further insights into the emission mechanism of CL but also provides a new strategy to manipulate the properties of CL by regulating the hierarchical structures of CLgens.

Altering Chain Flexibility of Aliphatic Polyesters for Yellow-Green Clusteroluminescence in 38 % Quantum Yield.

Preparation of non-conjugated polymers with long-wavelength emission and high quantum yield (QY) is still a huge challenge. Herein, we report the first example of linear non-conjugated polyester exhibiting yellow-green clusteroluminescence (CL) and a high QY of 38 %. We discovered that the polyester P3 with balanced flexibility and rigidity showed the longest CL wavelength and highest QY. Systematically photophysical characterization unravel the key role of ester cluster in the CL and the cluster formation via the aggregate of ester units was visualized. Moreover, P3 was demonstrated to be a highly selective, quick-responsive (ca. 1.2 min) and sensitive detector (detection limit is 0.78 µM) for irons owing to the fast disassociation of clusters by irons. This work not only gains further mechanistic insight into CL but also provides a new strategy to design high-efficiency and long-wavelength CL, meanwhile, enlightens the glorious application prospect of luminescent polyester.

Sensitive and Simultaneous Determination of Uridine Thiolation and Hydroxylation Modifications in Eukaryotic RNA by Derivatization Coupled with Mass Spectrometry Analysis.

The discovery of dynamic and reversible modifications in RNA expands their functional repertoires. Now, RNA modifications have been viewed as new regulators involved in a variety of biological processes. Among these modifications, thiolation is one kind of special modification in RNA. Several thiouridines have been identified to be present in RNA, and they are essential in the natural growth and metabolism of cells. However, detection of these thiouridines generally is challenging, and few studies could offer the quantitative levels of uridine modifications in RNA, which limits the in-depth elucidation of their functions. Herein, we developed a chemical derivatization in combination with mass spectrometry analysis for the sensitive and simultaneous determination of uridine thiolation and hydroxylation modifications in eukaryotic RNA. The chemical derivatization strategy enables the addition of easily ionizable groups to the uridine thiolation and hydroxylation modifications, leading up to a 339-fold increase in detection sensitivities of these modifications by mass spectrometry analysis. The limits of detection of these uridine modifications can be down to 17 amol. With the established method, we discovered and confirmed that a new modification of 5-hydroxyuridine (ho5U) was widely present in small RNAs of mammalian cells, expanding the diversity of RNA modifications. The developed method shows superior capability in determining low-abundance RNA modifications and may promote identifying new modifications in RNA, which should be valuable in uncovering the unknown functions of RNA modifications.

Antitumor Activities for Two Pt(II) Complexes of Tropolone and 8-Hydroxyquinoline Derivative.

The reactions of cis-Pt(DMSO)2Cl2 and tropolone (HL) with 8-hydroxyquinoline (HQ) or 2-methyl-8-hydroxyquinoline (HMQ) gave [Pt(Q)(L)] (1) and [Pt(MQ)(L)] (2), which present mononuclear structures with their Pt(II) ions four-coordinated in square planar geometries. Their in vitro biological properties were evaluated by MTT assay, which showed a remarkable cytotoxic activity on the cancer cell lines. 1 shows higher cytotoxic activities on tumor cells such as T24, HeLa, A549, and NCI-H460 than complex 2 and cisplatin, with IC50 values <16 µM. Among them, an IC50 value of 3.6 ± 0.63 µM was found for complex 1 against T24 cells. It presented a tuning cytotoxic activity by substitution groups on 8-hydroxyquinoline skeleton. In our case, the substitution groups of -H are much superior to -CH3 against tumor cells. It revealed that both complexes can induce cell apoptosis by decreasing the potential of a mitochondrial membrane, enhancing reactive oxygen species and increasing Ca2+ levels of T24 cells. The T24 cell cycle can be arrested at G2 and G1 phases by complexes 1 and 2, respectively, with an upregulation for P21 and P27 expression levels and a down-regulation for cyclin A, CDK1, Cdc25A, and cyclin B expression levels. Furthermore, complex 1 exhibits satisfactory in vivo antitumor activity as revealed by the tumor inhibitory rate and the tumor weight change as well as by the cute toxicity assay and renal pathological examinations, which is close to cisplatin and much better than complex 2. All of these suggest that 1 might be a potential candidate for developing into a safe and effective anticancer agent.

Diazo Reagent Labeling with Mass Spectrometry Analysis for Sensitive Determination of Ribonucleotides in Living Organisms.

Ribonucleotide analogues and their related phosphorylated metabolites play critical roles in tumor metabolism. However, determination of the endogenous ribonucleotides from the complex biological matrix is still a challenge due to their high structural similarity and high polarity that will lead to the low retention and low detection sensitivities by liquid chromatogram mass spectrometry analysis. In this study, we developed the diazo reagent labeling strategy with mass spectrometry analysis for sensitive determination of ribonucleotides in the living organism. A pair of light and heavy stable isotope labeling reagents, 2-(diazomethyl)-N-methyl-N-phenyl-benzamide (2-DMBA) and d5-2-(diazomethyl)-N-methyl-N-phenyl-benzamide (d5-2-DMBA), were synthesized to label ribonucleotides. 2-DMBA showed high specificity and high efficiency for the labeling of ribonucleotides. Our results demonstrated that the detection sensitivities of 12 ribonucleotides increased by 17-174-fold upon 2-DMBA labeling. The obtained limits of detection (LODs) of ribonucleotides ranged from 0.07 fmol to 0.41 fmol. Using this method, we achieved the sensitive and accurate detection of ribonucleotides from only a few cells (8 cells). To the best of our knowledge, this is the highest detection sensitivity for ribonucleotides ever reported. In addition, we found that the contents of almost all of these ribonucleotides were significantly increased in human breast carcinoma tissues compared to tumor-adjacent normal tissues, suggesting that endogenous ribonucleotides may play certain functional roles in the regulation of cancer development and formation. This method also can be potentially applied in the analysis of phosphorylated compounds.

Improving Flow Bead Assay: Combination of Near-Infrared Optical Tweezers Stabilizing and Upconversion Luminescence Encoding.

To enhance signal acquisition stability and diminish background interference for conventional flow bead-based fluorescence detection methods, we demonstrate here an exceptional microfluidic chip assisted platform by integrating near-infrared optical tweezers with upconversion luminescence encoding. For the former, a single 980 nm laser is employed to perform optical trapping and concurrently excite upconversion luminescence, avoiding the fluctuation of the signals and the complexity of the apparatus. By virtue of the favorable optical properties of upconversion nanoparticles (UCNPs), the latter is carried out by employing two-color UCNPs (Er-UCNPs and Tm-UCNPs) with negligible spectral overlaps. With the assistance of the double key techniques, we fabricated complex microbeads referred to a UCNPs-miRNAs-microbead sandwich construct by a one-step nucleic acid hybridization process and then obtained uniform terrace peaks for the automatic and simultaneous quantitative determination of miRNA-205 and miRNA-21 sequences with a detection limit of pM level on the basis of a special home-built flow bead platform. Furthermore, the technique was successfully applied for analyzing complex biological samples such as cell lysates and human tissue lysates, holding certain potential for disease diagnosis. In addition, it is expected that the flow platform can be utilized to investigate many other biomolecules of single cells and to allow analysis of particle heterogeneity in biological fluid by means of optical tweezers.

Spectrally Combined Encoding for Profiling Heterogeneous Circulating Tumor Cells Using a Multifunctional Nanosphere-Mediated Microfluidic Platform.

Comprehensive phenotypic profiling of heterogeneous circulating tumor cells (CTCs) at single-cell resolution has great importance for cancer management. Herein, a novel spectrally combined encoding (SCE) strategy was proposed for multiplex biomarker profiling of single CTCs using a multifunctional nanosphere-mediated microfluidic platform. Different cellular biomarkers uniquely labeled by multifunctional nanosphere barcodes, possessing identical magnetic tags and distinct optical signatures, enabled isolation of heterogeneous CTCs with over 91.6 % efficiency and in situ SCE of phenotypes. By further trapping individual CTCs in ordered microstructures on chip, composite single-cell spectral signatures were conveniently and efficiently obtained, allowing reliable spectral-readout for multiplex biomarker profiling. This SCE strategy exhibited great potential in multiplex profiling of heterogeneous CTC phenotypes, offering new avenues for cancer study and precise medicine.

4-Plex Chemical Labeling Strategy Based on Cinchona Alkaloid-Derived Primary Amines for the Analysis of Chiral Carboxylic Acids by Liquid Chromatography-Mass Spectrometry.

Chiral carboxylic acids play important roles in energy metabolism and signal transduction in the human body. These enantiomers usually possess different bioactivities and are also associated with the development of some diseases. Therefore, simultaneous determination of multiple chiral carboxylic acids is vital for study of the pathogenesis of related diseases. However, it is still challenging to simultaneously detect the enantiomers of multiple chiral carboxylic acids in biological samples. Here, we developed a novel 4-plex chemical labeling strategy based on 4 analogues of cinchona alkaloid-derived primary amines (CAPAs) for simultaneous determination of 16 enantiomers of 8 chiral carboxylic acids by liquid chromatography-mass spectrometry (LC-MS). To achieve high-throughput analysis, one CAPA analogue was used to label chiral carboxylic acid standards and served as internal standards (ISs), while the other 3 CAPA analogues were used to label endogenous chiral carboxylic acids in 3 different biological samples. After CAPAs labeling, the 16 chiral carboxylic acid enantiomers could be detected by LC-MS, and their detection sensitivity was greatly enhanced by up to 3 orders of magnitude compared to intact analytes. Further, the developed method for the determination of 16 chiral carboxylic acid enantiomers was validated in human serums and mammalian cells. Finally, the proposed method was applied to the determination of chiral carboxylic acids in the serum samples from type 2 diabetes mellitus (T2DM) and colorectal cancer (CRC) patients. We found that 5 chiral carboxylic acid enantiomers in T2DM serum samples and 4 chiral carboxylic acid enantiomers in CRC serum samples exhibited significant change compared to the healthy control (HC).

Determination of RNA Hydroxylmethylation in Mammals by Mass Spectrometry Analysis.

RNA molecules harbor diverse chemical modifications that play important regulatory roles in a variety of biological processes. Up to date, more than 150 modifications have been identified in various RNA species. Most of these modifications occurring in nucleic acids are the methylation of nucleic acids. It has been demonstrated that many of these methylation are reversible and undergo dynamic demethylation. Previous studies established that the demethylation of the two most important and prevalent modifications of 5-methylcytidine (m5C) and N6-methyladenosine (m6A) in nucleic acids is through the hydroxylation of m5C and m6A, forming 5-hydroxymethylcytidine (hm5C) and N6-hydroxymethyladenosine (hm6A), respectively. This indicates the hydroxylation of the methylated nucleosides may be a general pathway for the demethylation of nucleic acid methylation. However, few other hydroxylmethylation modifications have yet to be reported in existence in mammals. In the current study, we developed a neutral enzymatic digestion method for the mild digestion of nucleic acids, followed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. With the established method, we reported the existence of a new hydroxylmethylated nucleosides, N2-hydroxymethylguanosine (hm2G), in mammalian RNA. In addition, we found that the contents of hm2G, as well as N2-methylguanosine (m2G), showed significant differences between thyroid carcinoma tissues and tumor-adjacent normal tissues, indicating that m2G and hm2G in RNA may play certain roles in the carcinogenesis of thyroid carcinoma. Collectively, our study suggests that RNA hydroxylmethylation may be a new prevalent group of modifications existing in RNA, which expands the diversity of nucleic acid modifications and should exert regulatory functions in living organisms.