[Clinical analysis of denture rehabilitation after mandibular fibula free-flap reconstruction].

OBJECTIVE: To evaluate the postoperative denture restoration and denture function in patients with mandibular defect reconstructed with vascularized free fibula flap. METHODS: In the study, 154 patients who underwent mandibular segment resection and used vascularized free fibula flap to repair mandibular defects due to inflammation, trauma and tumor from January 2015 to December 2020 were collected. These patients had common inclusion criteria which were stable occlusal relationship before operation, segmental defects of mandibular bone caused by lesions of mandible and adjacent parts (such as floor of mouth, tongue, cheek), free fibula flap used for repair and surviving after operation. Relevant data were reviewed and situation of denture restoration was followed up. A questionnaire related to denture functional evaluation had been proposed for those who had completed the denture rehabilitation. The evaluation index of denture restoration function was assigned by expert authority to obtain the denture function score. SPSS 18.0 software was used for statistical analysis of the basic information of the patients included in the study and the denture restoration of the patients. RESULTS: The rate of postoperative denture restoration in the patients with mandibular defects repaired by free fibula flap was 17.5%, and the rate of postoperative denture restoration in the patients with benign mandibular tumors was 25.0% (18/72), which was significantly greater than that in the patients with malignant tumors 11.0% (9/82, P < 0.05). There was no significant difference in denture function score between the patients with condylar defect and those without condylar defect in denture repair rate and denture function score (P>0.05). The functional score of implant denture was significantly greater than that of removable denture (P < 0.05). According to Brown classification, the denture function score of the patients with the defect invo-lving the anterior mandibular region was significantly greater than that of the patients without the anterior mandibular region involved (P < 0.05). The poor oral conditions, such as less amount of remaining teeth, insufficient retention strength, large mobility of soft tissue in the surgical area, poor oral vestibular groove condition became the main reason of not receiving denture restoration (37.86%). CONCLUSION: The denture rehabilitation of mandibular defect reconstructed with vascularized free fibula flap is closely rela-ted to pathological properties and oral conditions. The clinical outcome of implant denture has been confirmed effectively and it is a better choice for future denture restoration after mandibular reconstruction.

Exosomes derived from bladder epithelial cells infected with uropathogenic Escherichia coli increase the severity of urinary tract infections (UTIs) by impairing macrophage function.

Uropathogenic Escherichia coli (UPEC) is the primary causative agent of urinary tract infections (UTIs) in humans. Moreover, as one of the most common bacterial pathogens, UPEC imposes a substantial burden on healthcare systems worldwide. Epithelial cells and macrophages are two major components of the innate immune system, which play critical roles in defending the bladder against UPEC invasion. Yet, the routes of communication between these cells during UTI pathogenesis are still not fully understood. In the present study, we investigated the role of membrane-bound nanovesicles (exosomes) in the communication between bladder epithelial cells and macrophages during UPEC infection, using an array of techniques such as flow cytometry, miRNA profiling, RNA sequencing, and western blotting. Moreover, our in vitro findings were validated in a mouse model of UPEC-induced cystitis. We found that UPEC infection induced the bladder epithelial MB49 cell line to secrete large numbers of exosomes (MB49-U-Exo), which were efficiently absorbed by macrophages both in vivo and in vitro. Assimilation of MB49-U-Exo induced macrophages to produce proinflammatory cytokines, including tumor necrosis factor (TNF)α. Exposure of macrophages to MB49-U-Exo reduced their phagocytic activity (by downregulating the expression of phagocytosis-related genes) and increased their rate of apoptosis. Mechanistically, we showed that MB49-U-Exo were enriched in miR-18a-5p, which induced TNFα expression in macrophages by targeting PTEN and activating the MAPK/JNK signaling pathway. Moreover, administration of the exosome secretion inhibitor GW4869 or a TNFα-neutralizing antibody alleviated UPEC-mediated tissue damage in mice with UPEC-induced cystitis by reducing the bacterial burden of the bladder and dampening the associated inflammatory response. Collectively, these findings suggest that MB49-U-Exo regulate macrophage function in a way that exacerbates UPEC-mediated tissue impairment. Thus, targeting exosomal -release or TNFα signaling during UPEC infection may represent promising non-antibiotic strategies for treating UTIs.

Coupling Z-Scheme g-C3N4/rGO/MoS2 Ternary Heterojunction as an Efficient Visible Light Photocatalyst for Hydrogen Evolution and RhB Degradation.

Coupling heterostructures to synergistically improve the light adsorption and promote the charge carrier separation has been regarded as an operative approach to advance the photocatalytic performances. However, it is still challenging to construct heterostructures with appropriate optical properties and interfacial energy structures at the same time. In this work, a Z-scheme g-C3N4/rGO/MoS2 ternary composite photocatalyst is successfully synthesized via an effective hydrothermal method. The as-synthesized g-C3N4/rGO/MoS2 composite photocatalyst exhibited significant improvement for visible light absorption and boosted the separation efficiency of photoinduced electron-hole pairs. The g-C3N4/rGO/MoS2 system exhibited optimum visible-light-induced photocatalytic activity in hydrogen (H2) from water splitting and degrading pollutant rhodamin B (RhB), which is 22 times and 5 times higher than that of pure g-C3N4, respectively. The excellent photocatalytic activities are attributed to the synergetic effects of coupling rGO, g-C3N4, and MoS2 ternary structures to the composite photocatalyst. These combinations of intimate two-dimensional nanoconjugations can effectively inhibit charge recombination and accelerate charge transfer kinetics, forming a Z-scheme-assisted photocatalytic mechanism, thereby exhibiting superior photocatalytic activity.

Unraveling the Impact of Curvature on Electrocatalytic Performance of Carbon Materials: A State-of-the-Art Review.

Curvature of carbon materials has gained significant attention as catalysts due to their distinctive properties and potential applications. This review comprehensively summarizes how the bending of carbon materials can improve electrocatalytic performance, with special attention to the applications of various bent carbon materials (such as carbon nanotubes, graphene, and fullerene) in electrocatalysts and a large number of related density functional theory (DFT) theoretical calculations. Extensive mechanism research has provided a wealth of evidence indicating that the curvature of carbon materials has a profound impact on catalytic activity. This improvement in catalytic performance by curved carbon materials is attributed to factors like a larger active surface area, modulation of electronic structure, and better dispersal of catalytic active sites. A comprehensive understanding and utilization of these effects enable the design of highly efficient carbon-based catalysts for applications in energy conversion, environmental remediation, and chemical synthesis.

The preparation and characterization of self-healing hydrogels based on polypeptides with a dual response to light and hydrogen peroxide.

Injectable self-healing hydrogels are being widely used in drug delivery, tissue engineering, and other fields. Because of their excellent biocompatibility and biodegradability, polypeptides are an ideal candidate for preparing injectable self-healing hydrogels. In this study, a polypeptide-based hydrogel with dual response to hydrogen peroxide and light was obtained by copolymerizing 4-arm PEG-amine, N-(p-nitrophenoxycarbonyl)-l-methionine, and N-(p-nitrophenoxycarbonyl)-γ-o-nitrobenzyl-l-glutamate. The hydrogel exhibits injectable self-healing behavior due to the hydrophobic interactions among peptide blocks, which also act as the reservoir of hydrophobic drug molecules. In the presence of hydrogen peroxide or under light irradiation, the thioether bond in methionine was oxidized to sulfoxide, whereas the o-nitro benzyl ester bond was broken to form glutamic acid. As a result, the corresponding hydrophobic blocks of polypeptide become hydrophilic, accelerating the release of drug molecules loaded in the polypeptide hydrophobic blocks. Using this technique, the controlled release of hydrophobic drug molecules was achieved. Our efforts could provide a new strategy for the preparation of self-healing hydrogels based on polypeptides with a dual response to hydrogen peroxide and light. In this view, the practical application of polypeptides in drug delivery, tissue engineering, and other fields, could be expanded and advanced.

A deep learning and radiomics fusion model based on contrast-enhanced computer tomography improves preoperative identification of cervical lymph node metastasis of oral squamous cell carcinoma.

OBJECTIVES: In this study, we constructed and validated models based on deep learning and radiomics to facilitate preoperative diagnosis of cervical lymph node metastasis (LNM) using contrast-enhanced computed tomography (CECT). MATERIALS AND METHODS: CECT scans of 100 patients with OSCC (217 metastatic and 1973 non-metastatic cervical lymph nodes: development set, 76 patients; internally independent test set, 24 patients) who received treatment at the Peking University School and Hospital of Stomatology between 2012 and 2016 were retrospectively collected. Clinical diagnoses and pathological findings were used to establish the gold standard for metastatic cervical LNs. A reader study with two clinicians was also performed to evaluate the lymph node status in the test set. The performance of the proposed models and the clinicians was evaluated and compared by measuring using the area under the receiver operating characteristic curve (AUC), accuracy (ACC), sensitivity (SEN), and specificity (SPE). RESULTS: A fusion model combining deep learning with radiomics showed the best performance (ACC, 89.2%; SEN, 92.0%; SPE, 88.9%; and AUC, 0.950 [95% confidence interval: 0.908-0.993, P < 0.001]) in the test set. In comparison with the clinicians, the fusion model showed higher sensitivity (92.0 vs. 72.0% and 60.0%) but lower specificity (88.9 vs. 97.5% and 98.8%). CONCLUSION: A fusion model combining radiomics and deep learning approaches outperformed other single-technique models and showed great potential to accurately predict cervical LNM in patients with OSCC. CLINICAL RELEVANCE: The fusion model can complement the preoperative identification of LNM of OSCC performed by the clinicians.

Interfacial electron modulation of 2D nanopetal ZnIn2S4 with edge-decorated Ni clusters for accelerated photocatalytic H2 evolution.

Heterostructure interfacial engineering between photocatalyst and co-catalyst to obtain an optimized electronic structure is a promising approach to improving their performance in the photocatalytic hydrogen evolution reaction (HER). In this work, two-dimensional nanopetal-like ZnIn2S4 (ZIS) with an adequately exposed active (110) edge facet-decorated Ni cluster heterostructure was prepared via chemical bath deposition, followed by photodeposition. In the catalyst preparation, the ZIS microstructure was modulated to sufficiently expose the active sites of the (110) edge for the HER, on which spontaneous interfacial engineering with an additional Ni cluster co-catalyst would be triggered via photodeposition in situ. The hydrogen production rate of the composite photocatalyst was excellent, at up to 26.80 mmol g-1 h-1 under simulated sunlight, which was 15.4 times greater than that of pristine ZIS. The optimized photocatalyst achieved a state-of-the-art apparent quantum yield of 61.68% at a single wavelength of 420 nm. Combined with systematic experimental characterization and density functional theory calculation, it was demonstrated that the separation and migration of charge carriers were significantly enhanced via the Ni cluster-induced interfacial electron redistribution, which contributed to the near-zero Gibbs free energy barrier and favored intermediate (*H) adsorption and desorption behavior, resulting in the superior photocatalytic performance. In summary, this work enables tuning of the interfacial electronic properties via spontaneous photodeposition of metallic cluster co-catalyst on the edge active sites, through which the separation of photogenerated charge carriers and surface redox reactions can be synergistically facilitated.

Increased G3BP2-Tau interaction in tauopathies is a natural defense against Tau aggregation.

Many RNA-binding proteins (RBPs), particularly those associated with RNA granules, promote pathological protein aggregation in neurodegenerative diseases. Here, we demonstrate that G3BP2, a core component of stress granules, directly interacts with Tau and inhibits Tau aggregation. In the human brain, the interaction of G3BP2 and Tau is dramatically increased in multiple tauopathies, and it is independent of neurofibrillary tangle (NFT) formation in Alzheimer's disease (AD). Surprisingly, Tau pathology is significantly elevated upon loss of G3BP2 in human neurons and brain organoids. Moreover, we found that G3BP2 masks the microtubule-binding region (MTBR) of Tau, thereby inhibiting Tau aggregation. Our study defines a novel role for RBPs as a line of defense against Tau aggregation in tauopathies.

Design of a Novel Trabecular Acetabular Cup and Selective Laser Melting Fabrication.

The acetabular cups used in total hip arthroplasty are mostly made of dense metal materials with an elastic moduli much higher than that of human bone. This leads to stress shielding after implantation, which may cause aseptic loosening of the implant. Selective laser melting (SLM) technology allows us to produce tiny and complex porous structures and to reduce the elastic moduli of dense metals, thereby avoiding stress shielding. In the present study, rhombic dodecahedron porous structures with cell sizes of 1 mm, 1.5 mm, and 2 mm were designed. The strut diameter was changed to ensure that the porosity and pore size would meet the bone ingrowth requirements. Then, porous Ti6Al4V alloy specimens were printed using SLM, and compressive tests were carried out. The results showed that the compressive strength and elastic modulus values of the specimens with a cell size of 1.5 mm were in the range of 78.16-242.94 MPa and 1.74-4.17 GPa, respectively, which are in line with the mechanical properties of human cortical bone. Finite element analysis of a total hip joint model was carried out to simulate gait, and the surface of the trabecular acetabular cup was divided into 10 regions according to the stress distribution, with the stress interval in the range of 37.44-219.24 MPa. According to the compression test results, the gradient structure of Ti6Al4V alloy with different porosity was designed for trabecular coating. The gradient porous structure meets the mechanical requirements and is closer to the natural structure of human bone than the uniformly distributed porous structure.

Identification and characterization of a MAPT-targeting locked nucleic acid antisense oligonucleotide therapeutic for tauopathies.

Tau is a microtubule-associated protein (MAPT, tau) implicated in the pathogenesis of tauopathies, a spectrum of neurodegenerative disorders characterized by accumulation of hyperphosphorylated, aggregated tau. Because tau pathology can be distinct across diseases, a pragmatic therapeutic approach may be to intervene at the level of the tau transcript, as it makes no assumptions to mechanisms of tau toxicity. Here we performed a large library screen of locked-nucleic-acid (LNA)-modified antisense oligonucleotides (ASOs), where careful tiling of the MAPT locus resulted in the identification of hot spots for activity in the 3' UTR. Further modifications to the LNA design resulted in the generation of ASO-001933, which selectively and potently reduces tau in primary cultures from hTau mice, monkey, and human neurons. ASO-001933 was well tolerated and produced a robust, long-lasting reduction in tau protein in both mouse and cynomolgus monkey brain. In monkey, tau protein reduction was maintained in brain for 20 weeks post injection and corresponded with tau protein reduction in the cerebrospinal fluid (CSF). Our results demonstrate that LNA-ASOs exhibit excellent drug-like properties and sustained efficacy likely translating to infrequent, intrathecal dosing in patients. These data further support the development of LNA-ASOs against tau for the treatment of tauopathies.

Functional imaging of brain organoids using high-density microelectrode arrays.

Abstract: Studies have provided evidence that human cerebral organoids (hCOs) recapitulate fundamental milestones of early brain development, but many important questions regarding their functionality and electrophysiological properties persist. High-density microelectrode arrays (HD-MEAs) represent an attractive analysis platform to perform functional studies of neuronal networks at the cellular and network scale. Here, we use HD-MEAs to derive large-scale electrophysiological recordings from sliced hCOs. We record the activity of hCO slices over several weeks and probe observed neuronal dynamics pharmacologically. Moreover, we present results on how the obtained recordings can be spike-sorted and subsequently studied across scales. For example, we show how to track single neurons across several days on the HD-MEA and how to infer axonal action potential velocities. We also infer putative functional connectivity from hCO recordings. The introduced methodology will contribute to a better understanding of developing neuronal networks in brain organoids and provide new means for their functional characterization. Impact statement: Human cerebral organoids (hCOs) represent an attractive in vitro model system to study key physiological mechanisms underlying early neuronal network formation in tissue with healthy or disease-related genetic backgrounds. Despite remarkable advances in the generation of brain organoids, knowledge on the functionality of their neuronal circuits is still scarce. Here, we used complementary metal-oxide-semiconductor (CMOS)-based high-density microelectrode arrays (HD-MEAs) to perform large-scale recordings from sliced hCOs over several weeks and quantified their activity across scales. Using single-cell and network metrics, we were able to probe aspects of hCO neurophysiology that are more difficult to obtain with other techniques, such as patch clamping (lower yield) and calcium imaging (lower temporal resolution). These metrics included, for example, extracellular action potential (AP) waveform features and axonal AP velocity at the cellular level, as well as functional connectivity at the network level. Analysis was enabled by the large sensing area and the high spatiotemporal resolution provided by HD-MEAs, which allowed recordings from hundreds of neurons and spike sorting of their activity. Our results demonstrate that HD-MEAs provide a multi-purpose platform for the functional characterization of hCOs, which will be key in improving our understanding of this model system and assessing its relevance for translational research. Supplementary Information: The online version contains supplementary material available at 10.1557/s43577-022-00282-w.

Compound Structure-Composition Control on the Mechanical Properties of Selective Laser-Melted Titanium Alloys.

In the performance optimization of the additive manufacturing of Ti6Al4V components, conventional control methods have difficulty taking into account the requirements of quality and mechanical properties of components, resulting in insufficient mechanical properties and a small control range. Therefore, combining the advantages of porous structure and alloy composition control, this paper proposed a structure-composition composite control method for selective laser-fused titanium alloy components by coupling the effects of porous structure parameters and boron content on the properties of Ti6Al4V components. Based on the Gibson-Ashby formula, the compression test of porous Ti6Al4V alloy and the tensile test of boron-containing Ti6Al4V alloy were carried out by SLM forming technology. The parameters C and n related to the pore parameters of porous structure were solved by the experimental data, and the analytical relationship between the pore parameters and the mechanical properties of Ti6Al4V alloy was established. The analytical relationship between boron content (t wt%) and mechanical properties of the alloy was established by tensile test. Finally, the Gibson-Ashby formula was used to combine the above analytical relationship, and a composite regulation model of compressive strength was obtained. The results show that the control range of the composite model ranges from 19.46-416.47 MPa, which was 45.53% higher than that obtained by controlling only pore parameters, and performance improved by 42.49%. The mechanical properties of the model are verified and the deviation between calculated values and experimental values was less than 1.3%. Taking aviation rocker arm as an example, the optimized design can improve the strength and reduce the mass of rocker arm by 51.94%. This method provides a theoretical basis for expanding the application of Ti6Al4V additive manufacturing components in aerospace and other fields.

A cross-species spatiotemporal proteomic analysis identifies UBE3A-dependent signaling pathways and targets.

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by the loss of neuronal E3 ligase UBE3A. Restoring UBE3A levels is a potential disease-modifying therapy for AS and has recently entered clinical trials. There is paucity of data regarding the molecular changes downstream of UBE3A hampering elucidation of disease therapeutics and biomarkers. Notably, UBE3A plays an important role in the nucleus but its targets have yet to be elucidated. Using proteomics, we assessed changes during postnatal cortical development in an AS mouse model. Pathway analysis revealed dysregulation of proteasomal and tRNA synthetase pathways at all postnatal brain developmental stages, while synaptic proteins were altered in adults. We confirmed pathway alterations in an adult AS rat model across multiple brain regions and highlighted region-specific differences. UBE3A reinstatement in AS model mice resulted in near complete and partial rescue of the proteome alterations in adolescence and adults, respectively, supporting the notion that restoration of UBE3A expression provides a promising therapeutic option. We show that the nuclear enriched transketolase (TKT), one of the most abundantly altered proteins, is a novel direct UBE3A substrate and is elevated in the neuronal nucleus of rat brains and human iPSC-derived neurons. Taken together, our study provides a comprehensive map of UBE3A-driven proteome remodeling in AS across development and species, and corroborates an early UBE3A reinstatement as a viable therapeutic option. To support future disease and biomarker research, we present an accessible large-scale multi-species proteomic resource for the AS community ( https://www.angelman-proteome-project.org/ ).

Freestanding Surface Disordered NiCu Solid Solution as Ultrastable High Current Density Hydrogen Evolution Reaction Electrode.

The poor performance of conventional powdery catalysts under large current density and the slow kinetics of the Volmer step limit the large-scale application of alkaline hydrogen generation. Here, we report the preparation of freestanding surface disordered NiCu solid solution as an ultrastable hydrogen evolution reaction electrode. The introduction of ammonium ion could tailor the reduction/nucleation rate of metal ions during the hydrothermal process, thus contributing to its unique intertwined 3D microstructure. The catalyst exhibits superior HER activity with an overpotential of 322 mV at 1000 mA cm-2, and limited degradation after 110 h continuous operation at 1000 mA cm-2. Density functional theory calculations confirm that the substitution of Cu could accelerate the hydroxyl desorption process (OHads + e- → OH-) and thereby enhance the overall kinetics of the Volmer step. Our work demonstrates the strong efficacy of optimizing catalysts' structures and facilitating intermediate desorption for boosting industrial-scale alkaline HER performance.

Secreted retrovirus-like GAG-domain-containing protein PEG10 is regulated by UBE3A and is involved in Angelman syndrome pathophysiology.

Angelman syndrome (AS) is a neurodevelopmental disorder caused by the loss of maternal UBE3A, a ubiquitin protein ligase E3A. Here, we study neurons derived from patients with AS and neurotypical individuals, and reciprocally modulate UBE3A using antisense oligonucleotides. Unbiased proteomics reveal proteins that are regulated by UBE3A in a disease-specific manner, including PEG10, a retrotransposon-derived GAG protein. PEG10 protein increase, but not RNA, is dependent on UBE3A and proteasome function. PEG10 binds to both RNA and ataxia-associated proteins (ATXN2 and ATXN10), localizes to stress granules, and is secreted in extracellular vesicles, modulating vesicle content. Rescue of AS patient-derived neurons by UBE3A reinstatement or PEG10 reduction reveals similarity in transcriptome changes. Overexpression of PEG10 during mouse brain development alters neuronal migration, suggesting that it can affect brain development. These findings imply that PEG10 is a secreted human UBE3A target involved in AS pathophysiology.

Graphene-supported single-atom catalysts and applications in electrocatalysis.

Supported metal nanostructures are the most extensively studied heterogeneous catalysts, benefiting from easy separation, regeneration and affordable cost. The size of the supported metal species is one of the decisive factors in determining the activity of heterogeneous catalysts. Particularly, the unsaturated coordination environment of metal atoms preferably act as the active centers, minimizing these metal species can significantly boost the specific activity of every single metal atom. Single-atom catalysts/catalysis (SACs), containing isolated metals atomically dispersed on or coordinated with the surface of a support material, represent the ultimate utilization of supported metals and maximize metal usage efficiency. Graphene, a two-dimensional star material, exhibiting extraordinary physical and chemical properties, has been approved as an excellent platform for constructing SACs. When atomically dispersed metal atoms are strongly anchored on the graphene surface, featuring ultra-high surface area and excellent electronic properties, SACs offer a great potential to significantly innovate the conventional heterogeneous catalysis, especially in the field of electrocatalysis. In this review, a detailed discussion of graphene-supported SACs, including preparation approaches, characterization techniques and applications on typical electrocatalytic reactions is provided. The advantages and unique features of graphene-supported SACs as efficient electrocatalysts and the upcoming challenges for improving their performance and further practical applications are also highlighted.

Isolated Fe atoms dispersed on cellulose-derived nanocarbons as an efficient electrocatalyst for the oxygen reduction reaction.

Cost-effective preparation of efficient electrocatalysts is vitally important for energy storage and conversion. Here, a facile chemical activation strategy using biomass cellulose as the carbon feedstock to fabricate isolated Fe atoms dispersed on a nitrogen doped graphene/nanocarbon hybrid is reported. This new single atom catalyst aFe-NGC worked as an excellent electrocatalyst towards the ORR compared to commercial Pt/C with 30 mV higher positive half-wave potential, larger current density, better stability and stronger methanol-tolerance. The key active sites for enhancing the ORR activity originated from the constructed high loading Fe-N/C configuration coupled with doped nitrogens, as explored by optimizing the activation temperatures and characterized by state-of-the-art techniques including aberration-corrected STEM and synchrotron XANES. This strategy could be developed into a general approach to prepare highly efficient atomic metal electrocatalysts using abundant biomass as a cost-effective carbon source.

Atomic zinc dispersed on graphene synthesized for active CO2 fixation to cyclic carbonates.

CO2 fixation to cyclic carbonates is important but depends on the catalyst. Here, atomic zinc (1.62 at%) dispersed on graphene was synthesized as a high-performance heterocatalyst for the cycloaddition reaction of epoxides and CO2. High yield (99%) and high selectivity (98%) of propylene carbonate with a TOF of 2889 h-1 were achieved. [ZnN3.76±0.2] was the active site, which was proved by advanced characterization, including synchrotron XANES, EXAFS and XPS and comparative performance tests.

Dual-Ion-Mode MALDI MS Detection of Small Molecules with the O-P,N-Doped Carbon/Graphene Matrix.

It is challenging to realize a dual-ion mode of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for detecting small molecules. Herein, graphene coated by porous amorphous carbon with P-O surface group and codoped by phosphorus and nitrogen (O-P,N-C/G) was synthesized from an aerogel formed by phytic acid, polyaniline, and electrochemically exfoliated graphene. The carbon material synthesized has the feature of large surface area (583 m2/g), good electrical conductivity, strong UV absorption, heteroatom doping, and surface functional groups suitable for laser- induced desorption/ionization. It was employed as a novel matrix suitable for both positive-ion and negative-ion modes in MALDI-TOF MS for the analysis of various small molecules including amino acids, small peptides, saccharides, drugs, and environmental pollutants, significantly outperforming control materials and a traditional CHCA (α-cyano-4-hydroxycinnamic acid) or 2,5-dihydroxybenzoic (DHB) matrix. Remarkably, the detection limit of the anticancer drugs (5-fluorouracil and ellagic acid) reaches 50 pmol. In addition, nice MALDI-TOF MS images can be mapped to detect mixed amino acids corresponding to homogeneous distribution of ion intensity. The monosaccharides and disaccharides can be distinguished by using the new matrix. Last but not least, it can be used to quantitatively detect glucose in human serum and soft drinks (glucose/fructose, 203.1 mM) without adding standards.

Preparation of Sequencing RNA Libraries through Chemical Cross-linking Coupled to Affinity Purification (cCLAP) in Saccharomyces cerevisiae.

Ribonucleoprotein particles (mRNPs) are complexes consisting of mRNAs and RNA-binding proteins (RBPs) which control mRNA transcription localization, turnover, and translation. Some mRNAs within the mRNPs have been shown to undergo degradation or storage. Those transcripts can lack general mRNA elements, like the poly(A) tail or 5' cap structure, which prevent their identification through the application of widely-used approaches like oligo(dT) purification. Here, we describe a modified cross-linking affinity purification protocol (cCLAP) based on existing cross-linking and immunoprecipitation (CLIP) methods to isolate mRNAs which could be deadenylated, decapped and/or partially degraded in mRNPs, opening the possibility to detect different types of non-coding RNAs (ncRNAs). Once isolated, the RNAs are subjected to adapter ligation and subsequently proceeded to Next-generation sequencing (NGS). Due to the fast and efficient cross-linking and quenching steps, this protocol is also suitable for transiently induced mRNP granules. Examples include processing bodies (PBs) or stress granules (SGs) triggered by extrinsic stressors. Its reproducibility and broad applications make this protocol a useful and powerful tool to study the RNA compositions of specific RNPs.