Mass Spectrometry-Based Precise Identification of Citrullinated Histones via Limited Digestion and Biotin Derivative Tag Enrichment.

Histone citrullination is an essential epigenetic post-translational modification (PTM) that affects many important physiological and pathological processes, but effective tools to study histone citrullination are greatly limited due to several challenges, including the small mass shift caused by this PTM and its low abundance in biological systems. Although previous studies have reported frequent occurrences of histone citrullination, these methods failed to provide a high-throughput and site-specific strategy to detect histone citrullination. Recently, we developed a biotin thiol tag that enabled precise identification of protein citrullination coupled with mass spectrometry. However, very few histone citrullination sites were identified, likely due to the highly basic nature of these proteins. In this study, we develop a novel method utilizing limited digestion and biotin derivative tag enrichment to facilitate direct in vivo identification of citrullination sites on histones. We achieve improved coverage of histone identification via partial enzymatic digestion and lysine block by dimethylation. With biotin tag-assisted chemical derivatization and enrichment, we also achieve precise annotation of histone citrullination sites with high confidence. We further compare different fragmentation methods and find that the electron-transfer-dissociation-based approach enables the most in-depth analysis and characterization. In total, we unambiguously identify 18 unique citrullination sites on histones in human astrocytoma U87 cells, including 15 citrullinated sites being detected for the first time. Some of these citrullination sites are observed to exhibit noticeable alterations in response to DNA damage, which demonstrates the superiority of our strategy in understanding the roles of histone citrullination in critical biological processes.

11-Plex DiLeu Isobaric Labeling Enables Quantitative Assessment of Brain Region Protein Association Networks Impacted by the Gut Microbiome.

Gut microbiota can regulate host brain functions and influence various physiological and pathological processes through the brain-gut axis. To systematically elucidate the intervention of different gut environments on different brain regions, we implemented an integrated approach that combines 11-plex DiLeu isobaric tags with a "BRIDGE" normalization strategy to comparatively analyze the proteome of six brain regions in germ-free (GF)- and conventionally raised (ConvR)-mice. A total of 5945 proteins were identified and 5656 were quantifiable, while 1906 of them were significantly changed between GF- and ConvR-mice; 281 proteins were filtered with FC greater than 1.2 in at least one brain region, of which heatmap analysis showed clear protein profile disparities, both between brain regions and gut microbiome conditions. Gut microbiome impact is most overt in the hypothalamus and the least in the thalamus region. Collectively, this approach allows an in-depth investigation of the induced protein changes by multiple gut microbiome environments in a brain region-specific manner. This comprehensive proteomic work improves the understanding of the brain region protein association networks impacted by the gut microbiome and highlights the critical roles of the brain-gut axis.

Trap Depth Engineering from Persistent Luminescence Phosphors Mg2-xZnxSnO4 for Dynamic Optical Information Encryption Application.

Traditional information encryption materials that rely on fluorescent/phosphorescent molecules are facing an increasing risk of counterfeiting or tampering due to their static reading mode and advances in counterfeiting technology. In this study, a series of Mg2-xZnxSnO4 (x = 0.55, 0.6, 0.65, 0.7 0.75, 0.8) that realizes the writing, reading, and erasing of dynamic information is developed. When heated to 90 °C, the materials exhibit a variety of dynamic emission changes with the concentration of Zn2+ ions. As the doping concentration increased, the ratio of the shallow trap to deep trap changed from 7.77 to 20.86. When x = 0.55, the proportion of deep traps is relatively large, resulting in a higher temperature and longer time required to read the information. When x = 0.80, the proportion of shallow traps is larger and the encrypted information is easier to read. Based on the above features, encryption binary codes device was designed, displaying dynamic writing, reading, and erasing of information under daylight and heating conditions. Accordingly, this work provides reliable guidance on advanced dynamic information encryption.

BBOX1-AS1 mediates trophoblast cells dysfunction via regulating hnRNPK/GADD45A axis†.

Recurrent pregnancy loss (RPL) is a common pathological problem during pregnancy, and its clinical etiology is complex and unclear. Dysfunction of trophoblasts may cause a series of pregnancy complications, including preeclampsia, fetal growth restriction, and RPL. Recently, lncRNAs have been found to be closely related to the occurrence and regulation of pregnancy-related diseases, but few studies have focused on their role in RPL. In this study, we identified a novel lncRNA BBOX1-AS1 that was significantly upregulated in villous tissues and serum of RPL patients. Functionally, BBOX1-AS1 inhibited proliferation, migration, invasion, tube formation and promoted apoptosis of trophoblast cells. Mechanistically, overexpression of BBOX1-AS1 activated the p38 and JNK MAPK signaling pathways by upregulating GADD45A expression. Further studies indicated that BBOX1-AS1 could increase the stability of GADD45A mRNA by binding hnRNPK and ultimately cause abnormal trophoblast function. Collectively, our study highlights that the BBOX1-AS1/hnRNPK/GADD45A axis plays an important role in trophoblast-induced RPL and that BBOX1-AS1 may serve as a potential target for the diagnosis of RPL.

The translocase of the inner mitochondrial membrane 22-2 is required for mitochondrial membrane function during Arabidopsis seed development.

The carrier translocase (also known as translocase of the inner membrane 22; TIM22 complex) is an important component of the mitochondrial protein import apparatus. However, the biological functions of AtTIM22-2 in Arabidopsis remain poorly defined. Here, we report studies on two tim22-2 mutants that exhibit defects in embryo and endosperm development, leading to seed abortion. AtTIM22-2, which was localized in mitochondria, was widely expressed in embryos and in various seedling organs. Loss of AtTIM22-2 function resulted in irregular mitochondrial cristae, decreased respiratory activity, and a lower membrane potential, together with changes in gene expression and enzyme activity related to reactive oxygen species (ROS) metabolism, leading to increased accumulation of ROS in the embryo. The levels of transcripts encoding mitochondrial protein import components were also altered in the tim22-2 mutants. Furthermore, mass spectrometry, bimolecular fluorescence complementation and co-immunoprecipitation assays revealed that AtTIM22-2 interacted with AtTIM23-2, AtB14.7 (a member of Arabidopsis OEP16 family encoded by At2G42210), and AT5G27395 (mitochondrial inner membrane translocase complex, subunit TIM44-related protein). Taken together, these results demonstrate that AtTIM22-2 is essential for maintaining mitochondrial membrane functions during seed development. These findings lay the foundations for a new model of the composition and functions of the TIM22 complex in higher plants.

Evaluation of oilseed proteins as precursors of antimicrobial peptides using bioinformatics method.

In this study, the potential of oilseed proteins from soybean, peanut, sesame, sunflower seed and flaxseed as antimicrobial peptide (AMP) precursors was assessed using the bioinformatics method. Thirty-four novel potential AMPs were obtained by in silico hydrolysis of 12 oilseed protein sequences, and 11 of them were positive in all four algorithm tests in CAMPR3. Among the six proteases analyzed, trypsin cleaved soybean, peanut, sesame and sunflower seed proteins most effectively to generate AMPs, with three, four, two and two AMPs obtained, respectively. Subtilisin was most effective for flaxseed AMPs release, obtaining three AMPs. More than 85% of AMPs were predicted to be cationic peptides, and some AMPs were hydrophobic. These potential AMPs were classified as non-toxic peptides, and 15 peptides were non-allergenic. All the AMPs were unstable to digestive enzymes according to in silico simulated digestion. The results of this study provide a theoretical basis for further development of AMPs using oilseed proteins.

Sunlight-induced degradation of COVID-19 antivirals arbidol in natural aquatic environments: Mechanisms, pathways and toxicity.

Insights into COVID-19 antivirals' environmental fate and ecological risk are urgently required due to their increasing concentrations in aquatic environments, which have rarely been studied. Herein, we first investigated the photochemical transformation and the resulting alterations in toxicity of arbidol, an antiviral drug with relatively higher toxicity. The photolysis of arbidol was rapid with a rate constant of 0.106 min-1 due to its superior ultraviolet light absorption, in which the direct photolysis was predominated with a contribution of 91.5%. Despite its substantial photolysis, only 14.45% of arbidol was mineralized after 100 min, implying that arbidol and its products might have a long-term impact on aquatic environment. It was inferred that arbidol was photolyzed mainly via the loss of thiophenol, bromine, and alkylamine, based on twelve photolytic products identified. Notably, the experimental results demonstrated that the photolysis process increased the acute toxicity of arbidol, and the toxicity prediction indicated that the ecotoxicity of two photolytic products was very high with LC50 values below 0.1 mg/L. Due to the co-effect of multiple constituents, the photolytic rate observed in wastewater treatment plant effluent and in river water was comparable to that in ultra-pure water, while it was slightly enhanced in lake water. The presence of dissolved organic matter suppressed arbidol photolysis, while NO3- exhibited a promotion effect. These results would be of great significance to assess the fate and risk of COVID-19 antivirals in natural aquatic environments.

12-Plex DiLeu Isobaric Labeling Enabled High-Throughput Investigation of Citrullination Alterations in the DNA Damage Response.

Protein citrullination is a key post-translational modification (PTM) that leads to the loss of positive charge on arginine and consequent protein structural and functional changes. Though it has been indicated to play critical roles in various physiological and pathological processes, effective analytical tools are largely limited due to a few challenges such as the small mass shift induced by this PTM and its low-abundance nature. Recently, we developed a biotin thiol tag, which enabled large-scale profiling of protein citrullination from complex biological samples via mass spectrometry. However, a high-throughput quantitative approach is still in great need to further improve the understanding of this PTM. In this study, we report an efficient pipeline using our custom-developed N,N-dimethyl leucine isobaric tags to achieve a multiplexed quantitative analysis of citrullination from up to 12 samples for the first time. We then apply this strategy to investigating citrullination alterations in response to DNA damage stress using human cell lines. We unveil important biological functions regulated by protein citrullination and observe hypercitrullination on RNA-binding proteins and DNA repair proteins, respectively. Our results reveal the involvement of citrullination in DNA damage pathways and may provide new insights into DNA-damage-related disease pathogenesis.

Enabling Global Analysis of Protein Citrullination via Biotin Thiol Tag-Assisted Mass Spectrometry.

Citrullination is a key post-translational modification (PTM) that affects protein structures and functions. Although it has been linked to various biological processes and disease pathogenesis, the underlying mechanism remains poorly understood due to a lack of effective tools to enrich, detect, and localize this PTM. Herein, we report the design and development of a biotin thiol tag that enables derivatization, enrichment, and confident identification of citrullination via mass spectrometry. We perform global mapping of the citrullination proteome of mouse tissues. In total, we identify 691 citrullination sites from 432 proteins which represents the largest data set to date. We discover novel distribution and functions of this PTM. This study depicts a landscape of protein citrullination and lays the foundation for further deciphering their physiological and pathological roles.

The lysosomal membrane protein Sidt2 is a vital regulator of mitochondrial quality control in skeletal muscle.

The role of Sidt2 in the process of glucose and lipid metabolism has been recently reported. However, whether Sidt2 is involved in the metabolic regulation in skeletal muscle remains unknown. In this study, for the first time, using skeletal muscle-selective Sidt2 knockout mice, we found that Sidt2 was vital for the quality control of mitochondria in mouse skeletal muscle. These mice showed significantly reduced muscle tolerance and structurally abnormal mitochondria. Deletion of the Sidt2 gene resulted in decreased expression of mitochondrial fusion protein 2 (Mfn2) and Dynamin-related protein 1 (Drp1), as well as peroxisome proliferator-activated receptor γ coactivator-1 (PGC1-α). In addition, the clearance of damaged mitochondria in skeletal muscle was inhibited upon Sidt2 deletion, which was caused by blockade of autophagy flow. Mechanistically, the fusion of autophagosomes and lysosomes was compromised in Sidt2 knockout skeletal muscle cells. In summary, the deletion of the Sidt2 gene not only interfered with the quality control of mitochondria, but also inhibited the clearance of mitochondria and caused the accumulation of a large number of damaged mitochondria, ultimately leading to the abnormal structure and function of skeletal muscle.

Efficacy and safety of osimertinib for patients with EGFR-mutated NSCLC: a systematic review and meta-analysis of randomized controlled studies.

BACKGROUND: Osimertinib is a recently approved third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) that selectively inhibits both EGFR-TKI-sensitizing and EGFR-T790M resistance mutations. The aim of the present meta-analysis was to investigate the efficacy and safety of osimertinib for patients with EGFR-mutated non-small-cell lung cancer (NSCLC). MATERIALS AND METHODS: Databases were searched for randomized controlled studies that reported the efficacy and safety of osimertinib versus other treatments (chemotherapy, other EGFR-TKIs, etc.) in treating EGFR-mutated NSCLC. The measured effects included objective response rate (ORR), disease control rate (DCR), progression-free survival (PFS), central nervous system progression-free survival (CNS-PFS), and overall survival (OS). Additional outcome was the incidence of adverse event. Relative risk (RR) for incidence and hazard ratio (HR) for survival outcomes were pooled. RESULTS: Seven studies containing 3335 participants were finally included. Osimertinib tended to improve ORR and DCR (RRs >1) as compared with other treatments. Osimertinib was also a significant protective factor for PFS, CNS-PFS, and OS (HRs <1 and p < .05). Osimertinib showed similar advantages in improving tumor response and patient survival when used as first-line, second-line, and third-line/adjuvant therapy, respectively, as compared with other treatments (RRs >1 for ORR and DCR; HRs <1 for PFS, CNS-PFS, and OS). Osimertinib also had better therapeutic effects as compared with chemotherapy, other EGFR TKIs, docetaxel + bevacizumab, and placebo, respectively. The five most common adverse events with pooled incidence > 20% were diarrhea, rash, nail effects, dry skin, and stomatitis, yet the pooled incidence of serious adverse events was less than 2%. CONCLUSIONS: This meta-analysis suggests that osimertinib has a positive effect in disease control and survival for patients with EGFR-mutated NSCLC with acceptable toxicities.

Association between long non-coding RNA H19 polymorphisms and breast cancer risk: a meta-analysis.

Common genes mutation was demonstrated associating with the risk of breast cancer (BC) recently, while the role of long non-coding RNA (lncRNA) polymorphism is still controversial. A meta-analysis was designed to discuss the association between lncRNA H19 polymorphisms and susceptibility to BC. The related databases were systematically reviewed up to April 13, 2021. Estimates were summarized as ORs and 95 percent CIs for each included study. The heterogeneity was assessed by the I2 test and subgroup analysis. Ten studies with 10354 BC patients and 11,177 control cases were included in our study. LncRNA H19 single nucleotide polymorphism (SNP) rs2839698 C/T significantly increases the susceptibility of BC (OR = 1.717 , 95 percent CI = 1.052-2.803, P = 0.031). LncRNA H19 polymorphism rs3741219 and rs217727 also increase the risk of ER-positive BC (OR = 1.128 , 95 percent CI = 1.010-1.259, P = 0.0032 for rs3741219, and OR = 1.297, 95 percent CI = 1.027-1.639, P = 0.029 for rs217727). Our results demonstrated that lncRNA H19 SNP rs2839698 C/T was significantly associated with the susceptibility of BC. LncRNA H19 SNP rs217727 and rs3741219 were associated with the risks of ER-positive BC. However, further studies are needed to reach a robust conclusion.

Partnering with immigrant families to promote language justice and equity in education.

Despite US federal legislation mandates institutions to provide meaningful access and participation to students and families in educational settings, culturally and linguistically diverse (CLD) families and caregivers of children in special education experience cultural and linguistic barriers. A Community Advisory Team (CAT) of parents, advocates, community interpreters and translators, researchers, and teachers explored CLD families' experiences and advocacy efforts. Critical bifocality and circuits of dispossession, privilege, and resistance informed the documentation of inequities and resistance to understand the linkages of structural arrangements of power. Focus groups with families (n = 21) speakers of Spanish, Portuguese, and Cantonese were conducted. Findings indicate perceived discrimination, poor and inadequate interpretation and translation services impact children's access to special education services, hinder family's communication with schools and reduce the perceptions of schools as trustworthy institutions. Families advocate relentlessly for their children and recommend schools listen to families and hire culturally and linguistically competent interpreters and translators. Community psychologists can make significant contributions to promote language justice in education settings through participatory approaches to inquiry that value CLD families' knowledge and expertise.

Effects of Bedaquiline on Antimicrobial Activity and Cytokine Secretion of Macrophages Infected with Multidrug-Resistant Mycobacterium tuberculosis Strains.

Background: Bedaquiline (Bdq) exerts bactericidal effects against drug-susceptible and drug-resistant Mycobacterium tuberculosis strains, including multidrug-resistant M. tuberculosis strains (MDR-MTBs). However, few reported investigations exist regarding Bdq effects on MDR-MTBs-infected macrophages activities and cytokine secretion. Here, Bdq bactericidal activities against MDR-MTBs and related cellular immune mechanisms were explored. Methods: Macrophages infected with MDR-MTBs or H37Rv received Bdq treatments (4 h/8 h/24 h/48 h) at 1 × the minimum inhibitory concentration (1 × MIC), 10 × MIC and 20 × MIC. Intracellular colony-forming units (CFUs) and culture supernatant IL-12/23 p40, TNF-α, IL-6, and IL-10 were determined using the Luminex® 200TM system. Normally distributed continuous data (mean ± standard deviation) were analyzed using t-test or F-test (SPSS 25.0, P < 0.05 deemed statistically significant). Results: (1) 100% of Bdq-treated macrophages (all doses applied over 4-48 h) survived with 0% inhibition of proliferation observed. (2) Intracellular CFUs of Bdq-treated MDR-MTBs-infected macrophages decreased over 4-48 h of treatment, were lower than preadministration and control CFUs, decreased with increasing Bdq dose, and resembled H37Rv-infected group CFUs (48 h). (3) For MDR-MTBs-infected macrophages (various Bdq doses), IL-12/23 p40 levels resembled preadministration group levels and exceeded controls (4 h); TNF-α levels exceeded preadministration group levels (24 h/48 h) and controls (24 h); IL-12/23 p40 and TNF-α levels resembled H37Rv-infected group levels (4 h/8 h/24 h/48 h); IL-6 levels exceeded preadministration and H37Rv-infected group levels (24 h/48 h) and controls (24 h); IL-10 levels resembled preadministration and H37Rv-infected group levels (4 h/8 h/24 h/48 h) and were lower than controls (24 h/48 h); IL-12/23 p40 and IL-10 levels remained unchanged as intracellular CFUs changed, with IL-12/23 p40 levels exceeding controls (4 h) and IL-10 levels remaining lower than controls (24 h/48 h); TNF-α and IL-6 levels increased as intracellular CFUs decreased (24 h/48 h) and exceed controls (24 h). Conclusion: Bdq was strongly bactericidal against intracellular MDR-MTBs and H37Rv in a time-dependent, concentration-dependent manner. Bdq potentially exerted immunomodulatory effects by inducing high-level Th1 cytokine expression (IL-12/23 p40, TNF-α) and low-level Th2 cytokine expression (IL-10).

[Mechanism of Shenling Baizhu Powder in alleviation of ulcerative colitis in mice based on high-throughput transcriptome sequencing].

High-throughput transcriptome sequencing was used to study the mechanism of Shenling Baizhu Powder(SLBZP) in the alleviation of the dextran sulfate sodium(DSS)-induced ulcerative colitis(UC) in mice. The mouse model of DDS-induced UC was treated with SLBZP by gavage. The changes in general state, disease activity index(DAI), and colon length were observed. The hematoxylin-eosin(HE) staining was used to observe the pathological changes in the colon tissues of mice. Enzyme-linked immunosorbent assay(ELISA) was used to determine the expression levels of tumor necrosis factor-α(TNF-α), interleukin(IL)-1ß, IL-6, IL-4, and IL-10 in the serum and tissues of mice. The differentially expressed genes in the control group, the model group, and the SLBZP group were analyzed by high-throughput transcriptome sequencing, and the Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses were conducted on the differentially expressed genes. The results showed that after intragastric administration of SLBZP, the symptoms of diarrhea and bloody stool were improved, and the disease active index(DAI) score was reduced. SLBZP effectively reduced the inflammatory cell infiltration and goblet cell loss in the colonic mucosal tissue, reduced the levels of TNF-α, IL-1ß, IL-6 in the serum and colon tissue, and increased the levels of IL-4 and IL-10 in the serum and colon tissue. There were 25 differential genes in SLBZP vs the model group, which were significantly enriched in immune response, immune system process, immunoglobulin production, and other biological processes. KEGG pathway analysis showed that the differential genes were enriched in signaling pathways such as neomycin, kanamycin, and gentamicin biosynthesis, cytokine-cytokine receptor interaction, primary immunodeficiency, and IgA synthesis of the intestinal immune network. This study shows that SLBZP may alleviate UC through immune regulation.

Systematic Evaluation of Mycobacterium tuberculosis Proteins for Antigenic Properties Identifies Rv1485 and Rv1705c as Potential Protective Subunit Vaccine Candidates.

The lack of efficacious vaccines against Mycobacterium tuberculosis (MTB) infection is a limiting factor in the prevention and control of tuberculosis (TB), the leading cause of death from an infectious agent. Improvement or replacement of the BCG vaccine with one that reliably protects all age groups is urgent. Concerns exist that antigens currently being evaluated are too homogeneous. To identify new protective antigens, we screened 1,781 proteins from a high-throughput proteome-wide protein purification study for antigenic activity. Forty-nine antigens (34 previously unreported) induced antigen-specific gamma interferon (IFN-γ) release from peripheral blood mononuclear cells (PBMCs) derived from 4,452 TB and suspected TB patients and 167 healthy donors. Three (Rv1485, Rv1705c, and Rv1802) of the 20 antigens evaluated in a BALB/c mouse challenge model showed protective efficacy, reducing lung CFU counts by 66.2%, 75.8%, and 60%, respectively. Evaluation of IgG2a/IgG1 ratios and cytokine release indicated that Rv1485 and Rv1705c induce a protective Th1 immune response. Epitope analysis of PE/PPE protein Rv1705c, the strongest candidate, identified a dominant epitope in its extreme N-terminal domain accounting for 90% of its immune response. Systematic preclinical assessment of antigens Rv1485 and Rv1705c is warranted.

Inhibiting microRNA-424 in bone marrow mesenchymal stem cells-derived exosomes suppresses tumor growth in colorectal cancer by upregulating TGFBR3.

OBJECTIVE: MicroRNAs (miRNAs) have been demonstrated to be differently expressed in colorectal cancer (CRC) and were identified as biomarkers and therapeutic targets for CRC. We aimed to identify the effect of microRNA-424 (miR-424) on process of CRC. METHODS: Exosomes were obtained from bone marrow mesenchymal stem cells (BMSCs). MiR-424, transforming growth factor-ß receptor 3 (TGFBR3) vimentin, S100A4, p-Smad1 expression in tissues and cells was measured. After treated with miR-424 inhibitor or TGFBR3 overexpression plasmid, the migration, invasion, cell cycle distribution and apoptosis of Lovo cells and exosomes-transfected Lovo cells were determined. The subcutaneous tumor models were established and the tumor growth was observed. The target relation between miR-424 and TGFBR3 was confirmed. RESULTS: MiR-424 was upregulated while TGFBR3 was downregulated in CRC tissues. TGFBR3 was targeted by miR-424. Inhibited miR-424 or elevated TGFBR3 upregulated p-Smad1, indicating that TGFBR3 mediated the Smad1 pathway, thus regulating CRC progression. MiR-424 inhibition or TGFBR3 restoration also suppressed migration and invasion of CRC cells, arrested the CRC cells at G0/G1 phase, and promoted CRC cell apoptosis. Moreover, exosomal miR-424 from BMSCs promoted CRC development. CONCLUSION: Inhibited exosomal miR-424 from BMSCs inhibited malignant behaviors of CRC cells by targeting TGFBR3, thus suppressing the progression of CRC.

Integrated Label-Free and 10-Plex DiLeu Isobaric Tag Quantitative Methods for Profiling Changes in the Mouse Hypothalamic Neuropeptidome and Proteome: Assessment of the Impact of the Gut Microbiome.

Gut microbiota can regulate host physiological and pathological status through gut-brain communications or pathways. However, the impact of the gut microbiome on neuropeptides and proteins involved in regulating brain functions and behaviors is still not clearly understood. To address the problem, integrated label-free and 10-plex DiLeu isobaric tag-based quantitative methods were implemented to compare the profiling of neuropeptides and proteins in the hypothalamus of germ-free (GF)- vs conventionally raised (ConvR)-mice. A total of 2943 endogenous peptides from 63 neuropeptide precursors and 3971 proteins in the mouse hypothalamus were identified. Among these 368 significantly changed peptides (fold changes over 1.5 and a p-value of <0.05), 73.6% of the peptides showed higher levels in GF-mice than in ConvR-mice, and 26.4% of the peptides had higher levels in ConvR-mice than in GF-mice. These peptides were mainly from secretogranin-2, phosphatidylethanolamine-binding protein-1, ProSAAS, and proenkephalin-A. A quantitative proteomic analysis employing DiLeu isobaric tags revealed that 282 proteins were significantly up- or down-regulated (fold changes over 1.2 and a p-value of <0.05) among the 3277 quantified proteins. These neuropeptides and proteins were mainly involved in regulating behaviors, transmitter release, signaling pathways, and synapses. Interestingly, pathways including long-term potentiation, long-term depression, and circadian entrainment were involved. In the present study, a combined label-free and 10-plex DiLeu-based quantitative method enabled a comprehensive profiling of gut microbiome-induced dynamic changes of neuropeptides and proteins in the hypothalamus, suggesting that the gut microbiome might mediate a range of behavioral changes, brain development, and learning and memory through these neuropeptides and proteins.

On-Tissue Derivatization with Girard's Reagent P Enhances N-Glycan Signals for Formalin-Fixed Paraffin-Embedded Tissue Sections in MALDI Mass Spectrometry Imaging.

Glycosylation is a major protein post-translational modification whose dysregulation has been associated with many diseases. Herein, an on-tissue chemical derivatization strategy based on positively charged hydrazine reagent (Girard's reagent P) coupled with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was developed for analysis of N-glycans from FFPE treated tissue sections. The performance of the proposed approach was evaluated by analysis of monosaccharides, oligosaccharides, N-glycans released from glycoproteins, as well as MS imaging of N-glycans from human cancer tissue sections. The results demonstrated that the signal-to-noise ratios for target saccharides were notably improved after chemical derivatization, in which signals were enhanced by 230-fold for glucose and over 28-fold for maltooctaose. Improved glycome coverage was obtained for N-glycans derived from glycoproteins and tissue samples after chemical derivatization. Furthermore, on-tissue derivatization was applied for MALDI-MSI of N-glycans from human laryngeal cancer and ovarian cancer tissues. Differentially expressed N-glycans among the tumor region, adjacent normal tissue region, and tumor proximal collagen stroma region were imaged, revealing that high-mannose type N-glycans were predominantly expressed in the tumor region. Overall, our results indicate that the on-tissue labeling strategy coupled with MALDI-MSI shows great potential to spatially characterize N-glycan expression within heterogeneous tissue samples with enhanced sensitivity. This study provides a promising approach to better understand the pathogenesis of cancer related aberrant glycosylation, which is beneficial to the design of improved clinical diagnosis and therapeutic strategies.

Mycobacterium tuberculosis type III-A CRISPR/Cas system crRNA and its maturation have atypical features.

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems are prokaryotic adaptive immune systems against invading nucleic acids. CRISPR locus variability has been exploited in evolutionary and epidemiological studies of Mycobacterium tuberculosis, the causative agent of tuberculosis, for over 20 yr, yet the biological function of this type III-A system is largely unexplored. Here, using cell biology and biochemical, mutagenic, and RNA-seq approaches, we show it is active in invader defense and has features atypical of type III-A systems: mature CRISPR RNA (crRNA) in its crRNA-CRISPR/Cas protein complex are of uniform length (∼71 nt) and appear not to be subject to 3'-end processing after Cas6 cleavage of repeat RNA 8 nt from its 3' end. crRNAs generated resemble mature crRNA in type I systems, having both 5' (8 nt) and 3' (28 nt) repeat tags. Cas6 cleavage of repeat RNA is ion dependent, and accurate cleavage depends on the presence of a 3' hairpin in the repeat RNA and the sequence of its stem base nucleotides. This study unveils further diversity among CRISPR/Cas systems and provides insight into the crRNA recognition mechanism in M. tuberculosis, providing a foundation for investigating the potential of a type III-A-based genome editing system.-Wei, W., Zhang, S., Fleming, J., Chen, Y., Li, Z., Fan, S., Liu, Y., Wang, W., Wang, T., Liu, Y., Ren, B., Wang, M., Jiao, J., Chen, Y., Zhou, Y., Zhou, Y., Gu, S., Zhang, X., Wan, L., Chen, T., Zhou, L., Chen, Y., Zhang, X.-E., Li, C., Zhang, H., Bi, L. Mycobacterium tuberculosis type III-A CRISPR/Cas system crRNA and its maturation have atypical features.