Pressure Cycling Technology Assisted Mass Spectrometric Quantification of Gingival Tissue Reveals Proteome Dynamics during the Initiation and Progression of Inflammatory Periodontal Disease

Understanding the progression of periodontal tissue destruction is at the forefront of periodontal research. The authors aimed to capture the dynamics of gingival tissue proteome during the initiation and progression of experimental (ligature‐induced) periodontitis in mice. Pressure cycling technology (PCT), a recently developed platform that uses ultra‐high pressure to disrupt tissues, is utilized to achieve efficient and reproducible protein extraction from ultra‐small amounts of gingival tissues in combination with liquid chromatography‐tandem mass spectrometry (MS). The MS data are processed using Progenesis QI and the regulated proteins are subjected to METACORE, STRING, and WebGestalt for functional enrichment analysis. A total of 1614 proteins with ≥2 peptides are quantified with an estimated protein false discovery rate of 0.06%. Unsupervised clustering analysis shows that the gingival tissue protein abundance is mainly dependent on the periodontitis progression stage. Gene ontology enrichment analysis reveals an overrepresentation in innate immune regulation (e.g., neutrophil‐mediated immunity and antimicrobial peptides), signal transduction (e.g., integrin signaling), and homeostasis processes (e.g., platelet activation and aggregation). In conclusion, a PCT‐assisted label‐free quantitative proteomics workflow that allowed cataloging the deepest gingival tissue proteome on a rapid timescale and provided novel mechanistic insights into host perturbation during periodontitis progression is applied.     

Elicitor hydrophobin Hyd1 interacts with Ubiquilin1‐like to induce maize systemic resistance

Trichoderma harzianum is a plant-beneficial fungus that secretes small cysteine-rich proteins that induce plant defense responses; however, the molecular mechanism involved in this induction is largely unknown.Here, we report that the class II hydrophobin Th Hyd1 acts as an elicitor of induced systemic resistance(ISR) in plants. Immunogold labeling and immunofluorescence revealed Th Hyd1 localized on maize(Zea mays) root cell plasma membranes. To identify host plant protein interactors of Hyd1, we screened a maize B73 root c DNA library. Th Hyd1 interacted directly with ubiquilin1-like(UBL). Furthermore, the N-terminal fragment of UBL was primarily responsible for binding with Hyd1 and the eight-cysteine amino acid of Hyd1 participated in the protein-protein interactions. Hyd1 from T. harzianum(Thhyd1) and ubl from maize were co-expressed in Arabidopsis thaliana, they synergistically promoted plant resistance against Botrytis cinerea. RNA-sequencing analysis of global gene expression in maize leaves 24 h after spraying with Curvularia lunata spore suspension showed that Thhyd1-induced systemic resistance was primarily associated with brassinosteroid signaling, likely mediated through BAK1. Jasmonate/ethylene(JA/ET)signaling was also involved to some extent in this response. Our results suggest that the Hyd1-UBL axis might play a key role in inducing systemic resistance as a result of Trichoderma-plant interactions.     

Prognostic value and immune cell infiltration of hypoxic phenotype‐related gene signatures in glioblastoma microenvironment

Glioblastoma (GBM) is a malignant intracranial tumour with the highest proportion and lethality. It is characterized by invasiveness and heterogeneity. However, the currently available therapies are not curative. As an essential environmental cue that maintains glioma stem cells, hypoxia is considered the cause of tumour resistance to chemotherapy and radiation. Growing evidence shows that immunotherapy focusing on the tumour microenvironment is an effective treatment for GBM; however, the current clinicopathological features cannot predict the response to immunotherapy and provide accurate guidance for immunotherapy. Based on the ESTIMATE algorithm, GBM cases of The Cancer Genome Atlas (TCGA) data set were classified into high‐ and low‐immune/stromal score groups, and a four‐gene tumour environment‐related model was constructed. This model exhibited good efficiency at forecasting short‐ and long‐term prognosis and could also act as an independent prognostic biomarker. Additionally, this model and four of its genes (CLECL5A, SERPING1, CHI3L1 and C1R) were found to be associated with immune cell infiltration, and further study demonstrated that these four genes might drive the hypoxic phenotype of perinecrotic GBM, which affects hypoxia‐induced glioma stemness. Therefore, these might be important candidates for immunotherapy of GBM and deserve further exploration.     

Metabolic phenotyping to monitor chronic enteritis canceration

Introduction

Untargeted metabolomics intends to objectively analyze a wide variety of compounds. Their diverse physicochemical properties make it difficult to choose an appropriate reconstitution solvent after sample evaporation without influencing the chromatography or hamper column sorbent integrity.

Objectives

The study aimed to identify the most appropriate reconstitution solvent for blood plasma samples in terms of feature recovery, four endogenous compounds, and one selected internal standard.

Methods

We investigated several reconstitution solvent mixtures containing acetonitrile and methanol to resolve human plasma extract and evaluated them concerning the peak areas of tryptophan-d₅, glucose, creatinine, palmitic acid, and the phophatidylcholine PC(P-16:0/P-16:0), as well as the total feature count

Results

Results indicated that acetonitrile containing 30% methanol was best suited to match all tested criteria at least for human blood plasma samples.

Conclusion

Despite identifying the mixture of acetonitrile and methanol being suitable as solvent for human blood plasma extracts, we recommend to systematically test for an appropriate reconstitution solvent for each analyzed biomatrix.

     

Dracorhodin perchlorate inhibits osteoclastogenesis through repressing RANKL-stimulated NFATc1 activity

Osteolytic skeletal disorders are caused by an imbalance in the osteoclast and osteoblast function. Suppressing the differentiation and resorptive function of osteoclast is a key strategy for treating osteolytic diseases. Dracorhodin perchlorate (D.P), an active component from dragon blood resin, has been used for facilitating wound healing and anti-cancer treatments. In this study, we determined the effect of D.P on osteoclast differentiation and function. We have found that D.P inhibited RANKL-induced osteoclast formation and resorbed pits of hydroxyapatite-coated plate in a dose-dependent manner. D.P also disrupted the formation of intact actin-rich podosome structures in mature osteoclasts and inhibited osteoclast-specific gene and protein expressions. Further, D.P was able to suppress RANKL-activated JNK, NF-κB and Ca²⁺ signalling pathways and reduces the expression level of NFATc1 as well as the nucleus translocation of NFATc1. Overall, these results indicated a potential therapeutic effect of D.P on osteoclast-related conditions.     

Deficiency of Macf1 in osterix expressing cells decreases bone formation by Bmp2/Smad/Runx2 pathway

Microtubule actin cross‐linking factor 1 (Macf1) is a spectraplakin family member known to regulate cytoskeletal dynamics, cell migration, neuronal growth and cell signal transduction. We previously demonstrated that knockdown of Macf1 inhibited the differentiation of MC3T3‐E1 cell line. However, whether Macf1 could regulate bone formation in vivo is unclear. To study the function and mechanism of Macf1 in bone formation and osteogenic differentiation, we established osteoblast‐specific Osterix (Osx) promoter‐driven Macf1 conditional knockout mice (Macf1^f/fOsx‐Cre). The Macf1^f/fOsx‐Cre mice displayed delayed ossification and decreased bone mass. Morphological and mechanical studies showed deteriorated trabecular microarchitecture and impaired biomechanical strength of femur in Macf1^f/fOsx‐Cre mice. In addition, the differentiation of primary osteoblasts isolated from calvaria was inhibited in Macf1^f/fOsx‐Cre mice. Deficiency of Macf1 in primary osteoblasts inhibited the expression of osteogenic marker genes (Col1, Runx2 and Alp) and the number of mineralized nodules. Furthermore, deficiency of Macf1 attenuated Bmp2/Smad/Runx2 signalling in primary osteoblasts of Macf1^f/fOsx‐Cre mice. Together, these results indicated that Macf1 plays a significant role in bone formation and osteoblast differentiation by regulating Bmp2/Smad/Runx2 pathway, suggesting that Macf1 might be a therapeutic target for bone disease.     

MiR‐155 promotes interleukin‐1β‐induced chondrocyte apoptosis and catabolic activity by targeting PIK3R1‐mediated PI3K/Akt pathway

Osteoarthritis (OA) is a common joint disease characterized by progressive cartilage degradation, in which elevated chondrocyte apoptosis and catabolic activity play an important role. MicroRNA‐155 (miR‐155) has recently been shown to regulate apoptosis and catabolic activity in some pathological circumstances, yet, whether and how miR‐155 is associated with OA pathology remain unexplored. We report here that miR‐155 level is significantly up‐regulated in human OA cartilage biopsies and also in primary chondrocytes stimulated by interleukin‐1β (IL‐1β), a pivotal pro‐catabolic factor promoting cartilage degradation. Moreover, miR‐155 inhibition attenuates and its overexpression promotes IL‐1β‐induced apoptosis and catabolic activity in chondrocytes in vitro. We also demonstrate that the PIK3R1 (p85α regulatory subunit of phosphoinositide 3‐kinase (PI3K)) is a target of miR‐155 in chondrocytes, and more importantly, PIK3R1 restoration abrogates miR‐155 effects on chondrocyte apoptosis and catabolic activity. Mechanistically, PIK3R1 positively regulates the transduction of PI3K/Akt pathway, and a specific Akt inhibitor reverses miR‐155 effects on promoting chondrocyte apoptosis and catabolic activity, phenocopying the results obtained via PIK3R1 knockdown, hence establishing that miR‐155 promotes chondrocyte apoptosis and catabolic activity through targeting PIK3R1‐mediated PI3K/Akt pathway activation. Altogether, our study discovers novel roles and mechanisms of miR‐155 in regulating chondrocyte apoptosis and catabolic activity, providing an implication for therapeutically intervening cartilage degradation and OA progression.