E3 ubiquitin ligase UBR5 modulates circadian rhythm by facilitating the ubiquitination and degradation of the key clock transcription factor BMAL1.

The circadian clock is the inner rhythm of life activities and is controlled by a self-sustained and endogenous molecular clock, which maintains a ~ 24 h internal oscillation. As the core element of the circadian clock, BMAL1 is susceptible to degradation through the ubiquitin-proteasome system (UPS). Nevertheless, scant information is available regarding the UPS enzymes that intricately modulate both the stability and transcriptional activity of BMAL1, affecting the cellular circadian rhythm. In this work, we identify and validate UBR5 as a new E3 ubiquitin ligase that interacts with BMAL1 by using affinity purification, mass spectrometry, and biochemical experiments. UBR5 overexpression induced BMAL1 ubiquitination, leading to diminished stability and reduced protein level of BMAL1, thereby attenuating its transcriptional activity. Consistent with this, UBR5 knockdown increases the BMAL1 protein. Domain mapping discloses that the C-terminus of BMAL1 interacts with the N-terminal domains of UBR5. Similarly, cell-line-based experiments discover that HYD, the UBR5 homolog in Drosophila, could interact with and downregulate CYCLE, the BMAL1 homolog in Drosophila. PER2-luciferase bioluminescence real-time reporting assay in a mammalian cell line and behavioral experiments in Drosophila reveal that UBR5 or hyd knockdown significantly reduces the period of the circadian clock. Therefore, our work discovers a new ubiquitin ligase UBR5 that regulates BMAL1 stability and circadian rhythm and elucidates the underlying molecular mechanism. This work provides an additional layer of complexity to the regulatory network of the circadian clock at the post-translational modification level, offering potential insights into the modulation of the dysregulated circadian rhythm.

Protective Effect of the Eluting Fraction of Da-Yuan-Yin Decoction on Acute Lung Injury.

Context: The Da-yuan-yin (DYY) decoction is a classical prescription of traditional Chinese medicine that has antipyretic and anti-inflammatory effects. Network Pharmacology (NP) is an emerging discipline based on system-biology theory and biosystem network analysis that researchers can use to predict drug-action targets and mechanisms. Objective: The study intended to use NP evaluate the protective effects of the fifth eluting fraction of the supernatant of the DYY decoction (DYY-5) for mice induced with acute lung injury (ALI) using lipopolysaccharide (LPS) and to explore DYY-5's mechanisms. Design: The research team performed an animal study. Setting: The study took place at the College of Pharmaceutical Science at Soochow University in Suzhou, China. Animals: The animals were 42 male Balb/c mice, about 20 to 25 g in weight. Intervention: The research team: instilled 2 mg/kg of LPS intratracheally (i.t.) to induce ALI. The team divided the mice into seven groups of six mice: (1) a control group; (2) a negative control group-the DYY-5 group with mice treated only with a high dosage, 60 mg/kg, of DYY-5 to investigate the effects of DYY-5 on normal mice; (3) the positive control group, the LPS group, with induced ALI but no treatments; (4) the LPS+60 mg/kg-DYY-5 group with induced ALI treated with a high dosage of DYY-5; (5) the LPS+30 mg/kg-DYY-5 group with induced ALI treated with a medium dosage of DYY-5; (6) the LPS+15 mg/kg-DYY-5 group with induced ALI treated with a low dosage of DYY-5; and (7) a reference drug control group, the LPS+DXM group, with induced ALI treated with 5 mg/kg of dexamethasone (DXM). Outcome Measures: The research team: (1) determined the chemical components of DYY; (2) identified the anticomplementary activities of DYY-5; (3) took lung specimens, serum, and bronchoalveolar lavage fluid (BALF) from the mice for histopathological examination, Western blot, and biochemical analysis; (4) measured total protein concentrations and lung W/D ratios; (5) measured the expressions of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) using quantitative real-time polymerase chain reaction (PCR); (6) measured the levels of pro-inflammatory and anti-inflammatory factors, the activity of myeloperoxidase (MPO) and superoxide dismutase (SOD), and the levels of complements, including complements 3 (C3), C3c, C5a, C5aR1, and C5b-9, using kits; (7) analyzed the levels of nuclear factor-kappa B (NF-κB) and IkB kinase (IKK) using Western blot; and (8) used network pharmacology (NP) to predict DYY-5's mechanisms and potential targets. Results: The study's results were consistent with the NP analysis, which reflected the multitarget and multipathway characteristics of DYY-5 in alleviating ALI. The LPS+30 mg/kg-DYY-5 group had significantly lower lung wet-to-dry (W/D) ratios and total protein concentrations in BALF than the LPS group did, with P < .01 and P < .0001, respectively as did the LPS+60 mg/kg-DYY-5 group (both P < .0001). The 60 mg/kg of DYY-5 compared to the LPS group: (1) regulated the levels tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and interleukin-1 beta (IL-1ß), with all P < .0001, anti-inflammatory factors-IL-4 (P < .05), IL-10 (P < .001), and IL-13 (P < .001); (2) increased the activity of SOD (P < .0001) and decreased the activity of MPO (P < .0001) and the expressions of iNOS and COX-2 mRNA (both P < .01); (3) blocked the activation of NF-κB and IKK; and (4) alleviated the pathological changes in the lung tissue, by reducing the depositions of C3c and decreasing the levels of C3, C5a and C5aR1 (all P < .0001), C5b-9 (P < .001) and C3c (P < .01) in serum. Conclusions: The protective effects of DYY-5 on ALI were related to antioxidation, anti-complementary activities, and regulation of inflammatory factors through the IKK/NF-κB signal pathway. DYY-5 may be useful as a potential therapeutic agent for treating ALI in clinics.

Da-Yuan-Yin decoction polyphenol fraction attenuates acute lung injury induced by lipopolysaccharide.

CONTEXT: Da-Yuan-Yin is a Chinese traditional prescription. OBJECTIVE: This study explores the therapeutic effects of the Da-Yuan-Yin decoction polyphenol fraction (DYY-4) on acute lung injury (ALI) in mice induced by lipopolysaccharide (LPS). MATERIALS AND METHODS: The mice (n = 10) were orally administrated with DYY-4 (15, 30, and 60 mg/kg) or DXM (5 mg/kg), half an hour after LPS (2 mg/kg) instilled intratracheally. The protein content and the levels of inflammatory factors, the levels of complements, the mRNA expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), the level of myeloperoxidase (MPO) and superoxide dismutase (SOD), the expression of the IkB kinase (IKK) and nuclear factor-kappa B (NF-κB), the lung wet-to-dry weight (W/D) ratio and lung tissue were evaluated, 24 h after LPS challenge. Network pharmacology predicted potential targets. RESULTS: DYY-4 (30, 60 mg/kg, p < 0.01, p < 0.01) decreased the lung W/D ratio, total protein concentration, the levels of C3, C3c and C5a, the levels of TNF-α, IL-6, and IL-1ß, while increased the levels of IL-4 and IL-10. DYY-4 (60 mg/kg) decreased the levels of C5aR1, C5b-9 and COX-2 mRNA (p < 0.05), the levels of MPO and iNOS mRNA, the activation of the IKK/NF-κB pathway (p < 0.01), and increased the levels of IL-13 and SOD (p < 0.01). DYY-4 (60 mg/kg) relieved the lung tissue pathological changes and reduced the C3c deposition. DISCUSSION AND CONCLUSIONS: Network pharmacology combined with animal experiments revealed the targets of DYY-4 alleviating ALI.

Targeted Proteomic Approaches for Proteome-Wide Characterizations of the AMP-Binding Capacities of Kinases.

Kinases play important roles in cell signaling, and adenosine monophosphate (AMP) is known to modulate cellular energy homeostasis through AMP-activated protein kinase (AMPK). Here, we explored novel AMP-binding kinases by employing a desthiobiotin-conjugated AMP acyl-phosphate probe to enrich efficiently AMP-binding proteins. Together with a parallel-reaction monitoring-based targeted proteomic approach, we uncovered 195 candidate AMP-binding kinases. We also enriched desthiobiotin-labeled peptides from adenine nucleotide-binding sites of kinases and analyzed them using LC-MS/MS in the multiple-reaction monitoring mode, which resulted in the identification of 44 peptides derived from 43 kinases displaying comparable or better binding affinities toward AMP relative to adenosine triphosphate (ATP). Moreover, our proteomic data revealed a potential involvement of AMP in the MAPK pathway through binding directly to the relevant kinases, especially MEK2 and MEK3. Together, we revealed the AMP-binding capacities of a large number of kinases, and our work built a strong foundation for understanding how AMP functions as a second messenger to modulate cell signaling.

1.7 µm - 1.73 µm tunable ultrafast Raman fiber laser pumped by 1.6 µm dissipative soliton pulses.

Here, we report an all-fiber tunable ultrafast Raman laser synchronously pumped by a home-made 1.6 µm dissipative soliton (DS) picosecond (ps) laser, which produces Stokes light beyond 1.7 µm. The Raman gain medium is a segment of highly germanium-doped (Ge-doped) fiber offering a high Raman gain coefficient at the target wavelength. Once the Raman conversion cavity is synchronized with the pump light, a stable 1.7 µm Raman laser (the first Stokes light) can be obtained at a low pump threshold. The maximum output power of the 1.7 µm Raman laser can reach ∼ 22.62 mW. The wavelength tuning operation is independent of tunable pump source and intra-cavity filter. By adjusting the intra-cavity delay line simply, the different spectral component within the broad Raman gain bandwidth can be selectively synchronized with the pump light so that the Raman laser wavelength can be tuned continuously from 1702.6 nm ∼ 1728.84 nm. This tunable 1.7 µm waveband ultrafast laser will have potential applications in multiphoton microscopy for e.g. deep bio-imaging.

Twin-tube terahertz fiber for a polarization filter.

A simple polymer twin-tube terahertz (THz) fiber that can be used as a polarization filter is proposed and investigated using the finite element method in this paper. The twin-tube THz fiber consists of two closely spaced identical tubes located symmetrically inside the protecting jacket. The simulation results show that the y-polarization fundamental mode (YPFM) can be well confined between the two tube walls near the fiber center, while the x-polarization fundamental mode (XPFM) has a huge confinement loss due to the coupling with the tube mode. For the fundamental mode (FM), a polarization extinction ratio (PER) of 30 dB can be realized after a 1.3 cm length of the fiber, and the insertion loss of the YPFM is less than 0.5 dB at 1 THz. In addition, higher order modes (HOMs) can be effectively suppressed by further increasing the fiber length. Simulation results indicate that all HOMs have powers being 30 dB lower than that of the supported YPFM after a 7.44 cm length of the fiber, and the insertion loss of the YPFM is less than 2.7 dB at 1 THz. Furthermore, the effects of fiber structure parameters on the loss properties are investigated, proving that the proposed fiber has a good fabrication tolerance. Owing to the simple structure, the proposed fiber polarization filter is easy to be fabricated and low-cost, which makes it a potential application in commercial THz systems.

Centrosomal protein Dzip1l binds Cby, promotes ciliary bud formation, and acts redundantly with Bromi to regulate ciliogenesis in the mouse.

The primary cilium is a microtubule-based organelle required for Hedgehog (Hh) signaling and consists of a basal body, a ciliary axoneme and a compartment between the first two structures, called the transition zone (TZ). The TZ serves as a gatekeeper to control protein composition in cilia, but less is known about its role in ciliary bud formation. Here, we show that centrosomal protein Dzip1l is required for Hh signaling between Smoothened and Sufu. Dzip1l colocalizes with basal body appendage proteins and Rpgrip1l, a TZ protein. Loss of Dzip1l results in reduced ciliogenesis and dysmorphic cilia in vivo Dzip1l interacts with, and acts upstream of, Cby, an appendage protein, in ciliogenesis. Dzip1l also has overlapping functions with Bromi (Tbc1d32) in ciliogenesis, cilia morphogenesis and neural tube patterning. Loss of Dzip1l arrests ciliogenesis at the stage of ciliary bud formation from the TZ. Consistent with this, Dzip1l mutant cells fail to remove the capping protein Cp110 (Ccp110) from the distal end of mother centrioles and to recruit Rpgrip1l to the TZ. Therefore, Dzip1l promotes ciliary bud formation and is required for the integrity of the TZ.

New phorbol ester derivatives as potent anti-HIV agents.

Tigliane esters show many biological activities, including anti-HIV-1 activity. Our aim in this study was to establish structure-anti-HIV activity relationships for four series of tigliane-type diterpenoids. We synthesized and evaluated 29 new phorbol ester derivatives for anti-HIV activity and for cytotoxicity against human tumor cell lines. Among them, three derivatives, two phorbol-13-monoesters (5d and 5e) and a phorbol-12,13-diester (6a), showed significant anti-HIV activity. We found that better anti-HIV activity was often associated with a shorter acyl ester at C-13. Particularly, compounds with a phenyl ring in the ester side chain exhibited excellent anti-HIV activity and had good safety indexes. Due to its significant anti-HIV potency with a high selectivity index, phorbol-12,13-dicinnamoate (6a) was chosen as the potential candidate for further preclinical trials.

Narrow-linewidth high-efficiency single-frequency ytterbium-doped fiber laser with highly linear polarization at 1064 nm.

We present a linearly polarized single-longitudinal-mode (SLM) ytterbium-doped fiber laser with a polarization extinction ratio (PER) of over 35 dB and a narrow linewidth of less than 4.5 kHz. The very high PER is obtained by utilizing the polarization evolution effect at the optical fiber and the high-performance polarizing beam splitter. The SLM is achieved by using a segment of polarization-maintaining ytterbium-doped fiber as a narrowband filter. In addition, a high optical signal-to-noise ratio of 50 dB and a good slope efficiency of 60.5% are achieved. Using an ytterbium-doped fiber amplifier, a linearly polarized SLM laser system with a high power of over 2.5 W is demonstrated at 1064 nm.

SILAC-based proteomic profiling of the suppression of TGF-ß1-induced lung fibroblast-to-myofibroblast differentiation by trehalose.

Fibroblast-to-myofibroblast differentiation is one of the most important characteristics of pulmonary fibrosis, and screening natural compounds targeting fibroblast differentiation is always a promising approach to discover drug candidates for treatment of pulmonary fibrosis. Trehalose reportedly has many potential medical applications, especially in treating neurodegeneration diseases. However, it remains unclear whether trehalose suppresses lung fibroblast differentiation. In this work, we found that trehalose decreased the expression levels of α-smooth muscle actin (α-SMA) following the induction of transforming growth factor ß1 (TGF-ß1) in pretreatment, co-treatment, and post-treatment groups. Trehalose also reduced the production of type I collagen, lung fibroblast-containing gel contractility and cell filament formation in TGF-ß1-stimulated MRC-5 cells. Although trehalose is a known autophagy inducer, our results showed that its suppressive effect on fibroblast differentiation was not via trehalose-induced autophagy. And it did not affect canonical TGFß/Smad2/3 pathway. By applying proteomic profiling technology, we demonstrated that the downregulation of ß-catenin was involved in the trehalose-repressive action on fibroblast differentiation. The ß-catenin agonist, SKL2001, reversed the suppressive effect of trehalose on fibroblast differentiation. Overall, these experiments demonstrated that trehalose suppressed fibroblast differentiation via the downregulation of ß-catenin, but not through canonical autophagy and TGFß/Smad2/3 pathway, which is not only a novel understanding of trehalose, but also quite helpful for in vivo research of trehalose on pulmonary fibrosis in future.

Cereblon suppresses the lipopolysaccharide-induced inflammatory response by promoting the ubiquitination and degradation of c-Jun.

Chronic inflammation is associated with multiple human disorders, such as rheumatoid arthritis, metabolic diseases, and neurodegenerative diseases. Therefore, alleviation of inflammation induced by environmental stimuli is important for disease prevention or treatment. Cereblon (CRBN) functions as a substrate receptor of the cullin-4 RING E3 ligase to mediate protein ubiquitination and degradation. Although it has been reported that CRBN reduces the inflammatory response through its nonenzymatic function, its role as a substrate receptor of the E3 ligase is not explored in mediating this process. Here we used a quantitative proteomics approach to find that the major component of the activator protein 1 (AP-1) complex, c-Jun, is significantly down-regulated upon CRBN expression. Biochemical approaches further discover that CRBN interacts and partially colocalizes with c-Jun and promotes the formation of Lys48-linked polyubiquitin chains on c-Jun, enhancing c-Jun degradation. We further reveal that CRBN attenuates the transcriptional activity of the AP-1 complex and reduces the mRNA expression and protein level of several pro-inflammatory cytokines. Moreover, flow cytometry analyses show that CRBN attenuates lipopolysaccharide-induced apoptosis in differentiated THP-1 cells. Through genetic manipulation and pharmacological inhibition, we uncover a new molecular mechanism by which CRBN regulates the inflammatory response and apoptosis induced by lipopolysaccharide. Our work and previous studies demonstrate that CRBN suppresses the inflammatory response by promoting or inhibiting the ubiquitination of two key molecules at different levels of the inflammatory cascade through its enzymatic function as a substrate receptor and its nonenzymatic function as a protein binding partner.

400 mW narrow linewidth single-frequency fiber ring cavity laser in 2 um waveband.

We present a single-frequency thulium-doped fiber laser (TDFL) with a narrow linewidth of 20 kHz. Stable single-longitudinal-mode (SLM) lasing operation at 1957 nm is achieved using a segment of un-pumped polarization-maintaining thulium-doped fiber (PM-TDF) as an ultra-narrow bandwidth filter. A high optical signal-to-noise ratio (OSNR) of over 60 dB is obtained and a high power of over 400 mW is achieved with a high slope-efficiency (~45.8%) thulium-doped fiber amplifier (TDFA).

Generation of high-power 780 nm femtosecond pulses by an all-polarization-maintaining Er-doped fiber amplification system.

A 187 fs pulse laser with 2.3 W of output power at 1560 nm is built for second-harmonic generation (SHG), which is the highest average power output reported from an all-polarization-maintaining single-mode Er-doped chirped pulse amplification system. By subsequently using an MgO-doped periodically poled lithium niobate (MgO:PPLN) crystal as frequency doubler, a 183 fs pulse laser with 1.1 W output power at 778 nm is generated. Benefitting from a polarization-stable fundamental laser input and the temperature control of the PPLN crystal, the power fluctuation of the frequency doubled laser is less than 0.5% during 24 h. To the best of our knowledge, the 1.1 W output at 780 nm band is also the highest average power generated from a frequency doubled femtosecond Er-doped fiber laser.

Proteomic and Biochemical Analyses Reveal a Novel Mechanism for Promoting Protein Ubiquitination and Degradation by UFBP1, a Key Component of Ufmylation.

Protein post-translational modification by ubiquitin-fold modifier 1, UFM1, regulates many biological processes such as response to endoplasmic reticulum stress and regulation of tumor progression. A recent study has indicated that the UFM1-binding and PCI domain-containing protein 1 (UFBP1) is required for the conjugation of UFM1 to a substrate. However, other biological functions of UFBP1 have not been explored. Here, we use immunoprecipitation and label-free quantitative proteomics to identify UFBP1-interacting proteins in a mammalian cell line. About 80 potential interacting proteins are obtained from MS analyses of three biological replicates. Bioinformatics analyses of these proteins suggest that UFBP1 may participate in the regulation of protein folding, stability, and trafficking. Biochemical experiments discover that UFBP1 expression downregulates the protein level and reduces the stability of several of its interacting proteins, while UFBP1 knockdown increases their protein levels. Protein synthesis inhibition and proteasomal inhibition experiments reveal that UFBP1 promotes their ubiquitination and degradation. Experiments using a model UFBP1-interacting protein ANT3 demonstrate that UFBP1 enhances the interaction between ANT3 and its E3 ligase and thus promotes its ubiquitination and degradation. Our work elucidates a novel molecular mechanism by which UFBP1 regulates protein ubiquitination and degradation.

Baicalein represses TGF-ß1-induced fibroblast differentiation through the inhibition of miR-21.

Fibroblast-to-myofibroblast differentiation is a highly important pathological characteristic of pulmonary fibrosis. In this study, we aimed to investigate the effects and mechanisms of baicalein on the differentiation of human lung fibroblasts. Baicalein reduced the levels of α-smooth muscle actin (α-SMA) mRNA and protein expression in TGF-ß1-treated human lung fibroblasts. It also decreased the contents of collagen type I and fibronectin in time- and dose-dependent manners, and retarded TGF-ß1-stimulated α-SMA filament formation. Baicalein diminished the expression of miR-21, and miR-21 mimics partially antagonized the effects of baicalein. Additionally, Baicalein inhibited the miR-21 transcriptor STAT3 activity but not AP-1 activity. Moreover, the expression of Spry 1 protein, a miR-21 known target, was improved by baicalein treatment, but the level of Smurf2 protein, another miR-21 target, was not interfered. Collectively, these results demonstrated that baicalein can attenuate TGF-ß1-induced human lung fibroblast differentiation by inhibiting the miR-21 expression.

Elevated serum levels of TNF-α, IL-8, and ECP can be involved in the development and progression of bronchial asthma.

OBJECTIVE: This study aimed to explore the value of elevated serum levels of tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), and eosinophil cationic protein (ECP) in the diagnosis of bronchial asthma (BA). METHODS: A total of 170 patients with BA (case group, 85 patients in acute attack and 85 patients in clinical remission) and 150 healthy individuals (control group) were enrolled in this study. Enzyme-linked immunosorbent assay and receiver operating characteristic (ROC) curves were calculated for the contents and diagnostic values of serum TNF-α, IL-8, and ECP in BA. RESULTS: Compared with the control group, patients in acute attack and clinical remission had higher TNF-α, IL-8, and ECP levels (p < 0.05). The serum level of TNF-α was positively correlated with IL-8 and ECP (p < 0.05). ROC curves showed that the diagnostic threshold value of IL-8 was 13.53 ng/ml, its area under the curve (AUC) was 0.87, its specificity was 99.3%, and its sensitivity was 57.6%. The diagnostic threshold value of TNF-α was 1.29 ng/ml with AUC being 0.94, specificity was 89.3%, and sensitivity was 83.5%. ECP showed 7.22 ng/ml diagnostic threshold value (AUC = 0.88, specificity = 74.0%, sensitivity = 86.5%). The FEV1/pre(%) and FEV1/FVC were negatively correlated and the Z5/pre(%) and resonance frequency (Fres) were positively correlated with the serum levels of TNF-α, IL-8, and ECP in patients in acute attack and in clinical remission (all p < 0.05). CONCLUSION: Our findings reveal that elevated serum levels of TNF-α, IL-8, and ECP can be involved in the development and progression of BA.

Wavelength-tunable all-fiber mode-locked laser based on supermode interference in a seven-core fiber.

A wavelength-tunable all-fiber mode-locked erbium-doped fiber laser has been proposed and realized by using a supermode interference filter (SMIF). The SMIF is fabricated by splicing a segment of seven-core fiber (SCF) to two standard single-mode fibers. Since two supermodes of the propagating light are excited in the SCF, the transmission spectrum of the SMIF shows a clean broadband comb-shape characteristic. By bending the SMIF in the proposed mode-locked laser, the output spectrum can be continuously tuned in a wavelength range up to 22 nm while keeping mode-locking operation. The self-starting laser produces 230 fs pulses with a spectral width of 14 nm.

Nucleolar stress and impaired stress granule formation contribute to C9orf72 RAN translation-induced cytotoxicity.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are the two common neurodegenerative diseases that have been associated with the GGGGCC·GGCCCC repeat RNA expansion in a noncoding region of C9orf72. It has been previously reported that unconventional repeat-associated non-ATG (RAN) translation of GGGGCC·GGCCCC repeats produces five types of dipeptide-repeat proteins (referred to as RAN proteins): poly-glycine-alanine (GA), poly-glycine-proline (GP), poly-glycine-arginine (GR), poly-proline-arginine (PR) and poly-proline-alanine (PA). Although protein aggregates of RAN proteins have been found in patients, it is unclear whether RAN protein aggregation induces neurotoxicity. In the present study, we aimed to understand the biological properties of all five types of RAN proteins. Surprisingly, our results showed that none of these RAN proteins was aggregate-prone in our cellular model and that the turnover of these RAN proteins was not affected by the ubiquitin-proteasome system or autophagy. Moreover, poly-GR and poly-PR, but not poly-GA, poly-GP or poly-PA, localized to the nucleolus and induced the translocation of the key nucleolar component nucleophosmin, leading to nucleolar stress and cell death. This poly-GR- and poly-PR-mediated defect in nucleolar function was associated with the suppression of ribosomal RNA synthesis and the impairment of stress granule formation. Taken together, the results of the present study suggest a simple model of the molecular mechanisms underlying RAN translation-mediated cytotoxicity in C9orf72-linked ALS/FTD in which nucleolar stress, but not protein aggregation, is the primary contributor to C9orf72-linked neurodegeneration.

Automated platform of µLC-MS/MS using SAX trap column for highly efficient phosphopeptide analysis.

In the current study, a specially designed automated nanoflow liquid chromatography coupled with tandem mass spectrometer (µLC-MS/MS) system for phosphopeptide analysis was reported. The system was established by applying a strong anion exchange (SAX) trap column. At the beginning of the analysis, phosphopeptides were loaded onto SAX trap column at high flow rate. Next, the retained phosphopeptides were eluted onto a C18 analytical capillary column by an acidic buffer with high ionic strength. At last, its performance was evaluated by analyzing the phosphopeptides enriched from the tryptic digest of mouse liver lysate. Compared with conventional automated µLC-MS/MS system using C18 trap column, much more phosphopeptides could be identified by the SAX trap column system. This system was fully automated and had promising application in high throughput phosphoproteomic analysis.