Proteomic Analysis Research Articles

Glycoproteomic and proteomic analysis of Burkholderia cenocepacia reveals glycosylation events within FliF and MotB are dispensable for motility.

Burkholderia cenocepacia is an opportunistic pathogen of concern within the Cystic Fibrosis community. Despite a greater appreciation of the unique physiology of B. cenocepacia gained over the last 20 years a complete understanding of the proteome and especially the O-glycoproteome, is lacking. In this study, we utilize systems biology approaches to expand the known B. cenocepacia glycoproteome as well as track the dynamics of glycoproteins across growth phases, culturing media and in response to the loss of glycosylation. We show that the glycoproteome of B. cenocepacia is largely stable across conditions and that the loss of glycosylation only impacts five glycoproteins including the motility associated proteins FliF and MotB. Examination of MotB and FliF shows, while these proteins are essential for motility, glycosylation is dispensable. Combined this work supports that B. cenocepacia glycosylation can be dispensable for protein function and may influence protein properties beyond stability.

A novel Mediterranean diet-inspired supplement ameliorates cognitive, microbial, and metabolic deficits in a mouse model of low-grade inflammation

ABSTRACT The Mediterranean diet (MD) and its bioactive constituents have been advocated for their neuroprotective properties along with their capacity to affect gut microbiota speciation and metabolism. Mediated through the gut brain axis, this modulation of the microbiota may partly contribute to the neuroprotective properties of the MD. To explore this potential interaction, we evaluated the neuroprotective properties of a novel bioactive blend (Neurosyn240) resembling the Mediterranean diet in a rodent model of chronic low-grade inflammation. Behavioral tests of cognition, brain proteomic analysis, 16S rRNA sequencing, and 1H NMR metabolomic analyses were employed to develop an understanding of the gut-brain axis interactions involved. Recognition memory, as assessed by the novel object recognition task (NOR), decreased in response to LPS insult and was restored with Neurosyn240 supplementation. Although the open field task performance did not reach significance, it correlated with NOR performance indicating an element of anxiety related to this cognitive change. Behavioral changes associated with Neurosyn240 were accompanied by a shift in the microbiota composition which included the restoration of the Firmicutes: Bacteroidota ratio and an increase in Muribaculum, Rikenellaceae Alloprevotella, and most notably Akkermansia which significantly correlated with NOR performance. Akkermansia also correlated with the metabolites 5-aminovalerate, threonine, valine, uridine monophosphate, and adenosine monophosphate, which in turn significantly correlated with NOR performance. The proteomic profile within the brain was dramatically influenced by both interventions, with KEGG analysis highlighting oxidative phosphorylation and neurodegenerative disease-related pathways to be modulated. Intriguingly, a subset of these proteomic changes simultaneously correlated with Akkermansia abundance and predominantly related to oxidative phosphorylation, perhaps alluding to a protective gut-brain axis interaction. Collectively, our results suggest that the bioactive blend Neurosyn240 conferred cognitive and microbiota resilience in response to the deleterious effects of low-grade inflammation.

Comprehensive Characterization of the Viscoelastic Properties of Bovine Submaxillary Mucin (BSM) Hydrogels and the Effect of Additives.

This study presents a comprehensive characterization of the viscoelastic and structural properties of bovine submaxillary mucin (BSM), which is widely used as a commercial source to conduct mucus-related research. We conducted concentration studies of BSM and examined the effects of various additives, NaCl, CaCl2, MgCl2, lysozyme, and DNA, on its rheological behavior. A notable connection between BSM concentration and viscoelastic properties was observed, particularly under varying ionic conditions. The rheological spectra could be well described by a fractional Kelvin-Voigt model with a minimum of model parameters. A detailed proteomics analysis provided insight into the protein, especially mucin composition within BSM, showing MUC19 as the main component. Cryo-scanning electron microscopy enabled the visualization of the porous BSM network structure. These investigations give us a more profound comprehension of the BSM properties, especially those pertaining to viscoelasticity, and how they are influenced by concentration and environmental conditions, aspects relevant to the field of mucus research.

Increasing Proteome Coverage Through a Reduction in Analyte Complexity in Single-Cell Equivalent Samples.

The advancement of sophisticated instrumentation in mass spectrometry has catalyzed an in-depth exploration of complex proteomes. This exploration necessitates a nuanced balance in experimental design, particularly between quantitative precision and the enumeration of analytes detected. In bottom-up proteomics, a key challenge is that oversampling of abundant proteins can adversely affect the identification of a diverse array of unique proteins. This issue is especially pronounced in samples with limited analytes, such as small tissue biopsies or single-cell samples. Methods such as depletion and fractionation are suboptimal to reduce oversampling in single cell samples, and other improvements on LC and mass spectrometry technologies and methods have been developed to address the trade-off between precision and enumeration. We demonstrate that by using a monosubstrate protease for proteomic analysis of single-cell equivalent digest samples, an improvement in quantitative accuracy can be achieved, while maintaining high proteome coverage established by trypsin. This improvement is particularly vital for the field of single-cell proteomics, where single-cell samples with limited number of protein copies, especially in the context of low-abundance proteins, can benefit from considering analyte complexity. Considerations about analyte complexity, alongside chromatographic complexity, integration with data acquisition methods, and other factors such as those involving enzyme kinetics, will be crucial in the design of future single-cell workflows.

NEDD4L mediates ITGB4 ubiquitination and degradation to suppress esophageal carcinoma progression

The ubiquitination-mediated protein degradation exerts a vital role in the progression of multiple tumors. NEDD4L, which belongs to the E3 ubiquitin ligase NEDD4 family, is related to tumor genesis, metastasis and drug resistance. However, the anti-tumor role of NEDD4L in esophageal carcinoma, and the potential specific recognition substrate remain unclear. Based on public esophageal carcinoma database and clinical sample data, it was discovered in this study that the expression of NEDD4L in esophageal carcinoma was apparently lower than that in atypical hyperplastic esophageal tissue and esophageal squamous epithelium. Besides, patients with high expression of NEDD4L in esophageal carcinoma tissue had longer progression-free survival than those with low expression. Experiments in vivo and in vitro also verified that NEDD4L suppressed the growth and metastasis of esophageal carcinoma. Based on co-immunoprecipitation and proteome analysis, the NEDD4L ubiquitination-degraded protein ITGB4 was obtained. In terms of the mechanism, the HECT domain of NEDD4L specifically bound to the Galx-β domain of ITGB4, which modified the K915 site of ITGB4 in an ubiquitination manner, and promoted the ubiquitination degradation of ITGB4, thus suppressing the malignant phenotype of esophageal carcinoma.

Glycosyltransferase Slr1064 regulates carbon metabolism by modulating the levels of UDP-GlcNAc in Synechocystis sp. PCC 6803.

Glycosyltransferases (GTs) are enzymes that transfer sugars to various targets. They play important roles in diverse biological processes, including photosynthesis, cell motility, exopolysaccharide biosynthesis, and lipid metabolism; however, their involvement in regulating carbon metabolism in Synechocystis sp. PCC 6803 has not been reported. We identified a novel GT protein, Slr1064, involved in carbon metabolism. The effect of slr1064 deletion on the growth of Synechocystis cells and functional mechanisms of Slr1064 on carbon metabolism were thoroughly investigated through physiological, biochemistry, proteomic, and metabolic analyses. We found that this GT, which is mainly distributed in the membrane compartment, is essential for the growth of Synechocystis under heterotrophic and mixotrophic conditions, but not under autotrophic conditions. The deletion of slr1064 hampers the turnover rate of Gap2 under mixotrophic conditions and disrupts the assembly of the PRK/GAPDH/CP12 complex under dark culture conditions. Additionally, UDP-GlcNAc, the pivotal metabolite responsible for the O-GlcNAc modification of GAPDH, is downregulated in the Δslr1064. Our work provides new insights into the role of GTs in carbon metabolism in Synechocystis and elucidate the mechanism by which carbon metabolism is regulated in this important model organism.

APIR: Aggregating Universal Proteomics Database Search Algorithms for Peptide Identification with FDR Control

Abstract Advances in mass spectrometry (MS) have enabled high-throughput analysis of proteomes in biological systems. The state-of-the-art MS data analysis relies on database search algorithms to quantify proteins by identifying peptide-spectrum matches (PSMs), which convert mass spectra to peptide sequences. Different database search algorithms use distinct search strategies and thus may identify unique PSMs. However, no existing approaches can aggregate all user-specified database search algorithms with a guaranteed increase in the number of identified peptides and control on the false discovery rate (FDR). To fill in this gap, we proposed a statistical framework, Aggregation of Peptide Identification Results (APIR), that is universally compatible with all database search algorithms. Notably, under an FDR threshold, APIR is guaranteed to identify at least as many, if not more, peptides as individual database search algorithms do. Evaluation of APIR on a complex proteomics standard showed that APIR outpowers individual database search algorithms and empirically controls the FDR. Real data studies showed that APIR can identify disease-related proteins and post-translational modifications missed by some individual database search algorithms. The APIR framework is easily extendable to aggregating discoveries made by multiple algorithms in other high-throughput biomedical data analysis, e.g., differential gene expression analysis on RNA sequencing data. The APIR R package is available at https://github.com/yiling0210/APIR.

Exploring the Molecular Therapeutic Mechanisms of Gemcitabine through Quantitative Proteomics.

Gemcitabine (GEM) is widely employed in the treatment of various cancers, including pancreatic cancer. Despite their clinical success, challenges related to GEM resistance and toxicity persist. Therefore, a deeper understanding of its intracellular mechanisms and potential targets is urgently needed. In this study, through mass spectrometry analysis in data-dependent acquisition mode, we carried out quantitative proteomics (three independent replications) and thermal proteome profiling (TPP, two independent replications) on MIA PaCa-2 cells to explore the effects of GEM. Our proteomic analysis revealed that GEM led to the upregulation of the cell cycle and DNA replication proteins. Notably, we observed the upregulation of S-phase kinase-associated protein 2 (SKP2), a cell cycle and chemoresistance regulator. Combining SKP2 inhibition with GEM showed synergistic effects, suggesting SKP2 as a potential target for enhancing the GEM sensitivity. Through TPP, we pinpointed four potential GEM binding targets implicated in tumor development, including in breast and liver cancers, underscoring GEM's broad-spectrum antitumor capabilities. These findings provide valuable insights into GEM's molecular mechanisms and offer potential targets for improving treatment efficacy.

Dissecting the MUC5AC/ANXA2 signaling axis: implications for brain metastasis in lung adenocarcinoma.

Non-small cell lung carcinoma (NSCLC) exhibits a heightened propensity for brain metastasis, posing a significant clinical challenge. Mucin 5ac (MUC5AC) plays a pivotal role in the development of lung adenocarcinoma (LUAD); however, its role in causing brain metastases remains unknown. In this study, we aimed to investigate the contribution of MUC5AC to brain metastasis in patients with LUAD utilizing various brain metastasis models. Our findings revealed a substantial increase in the MUC5AC level in LUAD brain metastases (LUAD-BrM) samples and brain-tropic cell lines compared to primary samples or parental control cell lines. Intriguingly, depletion of MUC5AC in brain-tropic cells led to significant reductions in intracranial metastasis and tumor growth, and improved survival following intracardiac injection, in contrast to the observations in the control groups. Proteomic analysis revealed that mechanistically, MUC5AC depletion resulted in decreased expression of metastasis-associated molecules. There were increases in epithelial-to-mesenchymal transition, tumor invasiveness, and metastasis phenotypes in tumors with high MUC5AC expression. Furthermore, immunoprecipitation and proteomic analysis revealed a novel interaction of MUC5AC with Annexin A2 (ANXA2), which activated downstream matrix metalloproteases and facilitated extracellular matrix degradation to promote metastasis. Disrupting MUC5AC-ANXA2 signaling with a peptide inhibitor effectively abrogated the metastatic process. Additionally, treatment of tumor cells with an astrocyte-conditioned medium or the chemokine CCL2 resulted in upregulation of MUC5AC expression and enhanced brain colonization. In summary, our study demonstrates that the MUC5AC/ANXA2 signaling axis promotes brain metastasis, suggesting a potential therapeutic paradigm for LUAD patients with high MUC5AC expression.

Characterization of exosome-mediated propagation of systemic inflammatory responses into the Central Nervous System.

The mechanisms through which systemic inflammation exerts its effect on the CNS are still not completely understood. Exosomes are small (30 to 100 nanometers) membrane-bound extracellular vesicles released by most of the mammalian cells. Exosomes play a vital role in cell-to-cell communication. This includes regulation of inflammatory responses by shuttling mRNAs, miRNAs, and cytokines both locally and systemically to the neighboring as well as distant cells to further modulate their transcriptional and/or translational states and affect the functional phenotype of those cells that have taken up these exosomes. The role of circulating blood exosomes leading to neuroinflammation during systemic inflammatory conditions was further characterized. Serum-derived exosomes from LPS-challenged mice (SDEL) were freshly isolated from the sera of the mice that were earlier treated with LPS and used to study SDEL effects on neuroinflammation. Exosomes isolated from the sera of the mice injected with saline were used as a control. In-vitro studies showed that the SDEL upregulate pro-inflammatory cytokine gene expression in the murine cell lines of microglia (BV-2), astrocytes (C8-D1A), and cerebral microvascular endothelial cells (bEnd.3). To further study their effects in-vivo, SDEL were intravenously injected into normal adult mice. Elevated mRNA expression of pro-inflammatory cytokines was observed in the brains of SDEL recipient mice. Proteomic analysis of the SDEL confirmed the increased expression of inflammatory cytokines in them. Together, these results further demonstrate and strengthen the novel role of peripheral circulating exosomes in causing neuroinflammation during systemic inflammatory conditions.

Proteomic profiling of extracellular vesicles derived from human serum for the discovery of biomarkers in Avascular necrosis

BackgroundAvascular necrosis (AVN) is a medical condition characterized by the destruction of bone tissue due to a diminished blood supply. When the rate of tissue destruction surpasses the rate of regeneration, effective treatment becomes challenging, leading to escalating pain, arthritis, and bone fragility as the disease advances. A timely diagnosis is imperative to prevent and initiate proactive treatment for osteonecrosis. We explored the potential of differentially expressed proteins in serum-derived extracellular vesicles (EVs) as biomarkers for AVN of the femoral head in humans. We analyzed the genetic material contained in serum-derived exosomes from patients for early diagnosis, treatment, and prognosis of avascular necrosis.MethodsEVs were isolated from the serum of both patients with AVN and a control group of healthy individuals. Proteomic analyses were conducted to compare the expression patterns of these proteins by proteomic analysis using LC-MS/MS.ResultsOur results show that the levels of IGHV3-23, FN1, VWF, FGB, PRG4, FCGBP, and ZSWIM9 were upregulated in the EVs of patients with AVN compared with those of healthy controls. ELISA results showed that VWF and PRG4 were significantly upregulated in the patients with AVN.ConclusionsThese findings suggest that these EV proteins could serve as promising biomarkers for the early detection and diagnosis of AVN. Early diagnosis is paramount for effective treatment, and the identification of new osteonecrosis biomarkers is essential to facilitate swift diagnosis and proactive intervention. Our study provides novel insights into the identification of AVN-related biomarkers that can enhance clinical management and treatment outcomes.

Unveiling the complexity: Liquid biopsy-based proteomic exploration of triple-negative breast cancer in women.

e13138 Background: This groundbreaking study places a spotlight on breast cancer in women, with a specific emphasis on the challenging triple-negative breast cancer (TNBC) subtype. Delving into the intricate world of extracellular vesicles (EVs) from TNBC-diagnosed individuals, the research meticulously compares various conditions, including healthy controls, benign tumors, recurrent tumors, and those in remission. Employing cutting-edge techniques like liquid biopsy and LC-MS/MS, the study meticulously explores the proteomic profile of serum exosomes, aiming to pinpoint diagnostic and prognostic markers in breast cancer. Methods: In the initial phase, EVs were expertly isolated and characterized from blood plasma samples exclusively from women, utilizing state-of-the-art methods such as ultracentrifugation and nanoparticle tracking analysis (NTA). Despite applying rigorous statistical criteria, the study found no significant differences in EV concentrations and sizes among the diverse groups. However, the ensuing proteomic analysis uncovered 130 proteins, with Cystatin A and Histone H2A emerging as promising biomarkers for TNBC. To validate them, the study intricately tested tumor cell lines from distinct subtypes, representing luminal, triple-negative, and HER2 overexpressed. The examination of gene and protein expression through RT-qPCR and western blotting added a layer of depth to the research, unraveling the complexity of regulatory processes within cells and demonstrating a remarkable congruence between protein levels found in the proteomics from patients’ serum and in immortalized cell lines. Results: The proteomic analysis brought to light the absence of CSTA and HIST1H2A expression in benign tumor patients, varied levels in malignant tumor patients, and a substantial increase in TNBC patients pre-mastectomy. Validating these findings through western blotting further accentuated the potential of these proteins as distinguishing factors. Conclusions: In conclusion, this pioneering study not only underscores the immense potential of liquid biopsy-based proteomic analysis in identifying crucial diagnostic and prognostic markers in breast cancer but also sheds light on the intricate nuances of the challenging TNBC subtype. Our findings unveil valuable insights that pave the way for future personalized treatment strategies, marking a significant stride forward in breast cancer research.

Unraveling the role of ubiquitin C-terminal hydrolase L1 in high grade serous ovarian cancer pathogenesis.

e15078 Background: High grade serous ovarian cancer (HGSOC) is the most lethal of all gynecologic malignancies, due to the fact that the majority of patients are diagnosed at an advanced stage and eventually develop chemoresistant disease 12-18 months following completion of frontline therapy. Therefore, novel targeted therapies are urgently needed to combat chemoresistance and improve patient clinical outcomes. Ubiquitin C-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme that plays a vital role in protein homeostasis and is highly overexpressed in HGSOC. It was previously uncovered that targeting UCHL1 with small molecule inhibition reduced cell viability in a chemoresistant ovarian cancer cell line. Methods: Comparative-label free proteomic analysis was used to uncover significant changes in HGSOC cell line OVCAR8WT following treatment with UCHL1 small molecule inhibitor, LDN-5744 or corresponding DMSO control. Significant pathway changes were determined by KEGG analysis. Matched chemosensitive and resistant HGSOC cells PEA1 and PEA2 were treated in combination with LDN-5744 and carboplatin, with cell viability was determined by an MTS assay. Furthermore, western blot analysis was employed to compare common cell growth pathway changes as a result of UCHL1 inhibition in HGSOC cells. Results: Proteomic analysis revealed a significant (p<0.05) upregulation in methyl phosphate capping enzyme (MEPCE) (4.9-fold), asparagine synthetase (ASNS) (2.5-fold), Phosphoserine aminotransferase 1 (PSAT1) (1.7-fold), and downregulation in centrosomal protein 55 (CEP55) (-7.2-fold), These identified proteomic changes were confirmed by western blot. KEGG pathways analysis using all significantly (p<0.05) differentially expressed proteins showcased significant (p<0.05) enrichment in amino acid biosynthesis, and metabolism, and chemical carcinogenesis-reactive oxygen species. Chemoresistant PEA2 cells treated in combination with LDN-5744 and carboplatin demonstrated a 59% reduction in cell viability, significantly (p<0.001) lower than the 13% and 22%, that was observed from UCHL1 and carboplatin treatment alone, respectively. Conversely, PEA1, the chemosensitive counterpart to PEA2 demonstrated a significant (p<0.001) 37% increase in cell viability upon combinatorial treatment compared with carboplatin alone, indicating that UCHL1 inhibition rescues the cells from chemotherapy induced cell death. Finally, LDN-5744 treatment in PEA2 led to a reduction in levels of phospho-(p-)AKT, p-STAT3, and p-ERK, while conversely in PEA1 cells levels of these proteins were increase or unchanged. Conclusions: Our findings demonstrate that targeting UCHL1 results in prominent metabolic changes in HGSOC cells and that the efficacy of UCHL1 inhibition is heavily dependent upon platinum status. Overall, this study highlights UCHL1’s novel therapeutic role in the recurrent HGSOC.

Multi-omics study on the molecular mechanism of anlotinib in regulating tumor metabolism.

e15121 Background: Anlotinib, an orally administered small molecule inhibitor of receptor tyrosine kinases (RTKs), exerts significant anti-angiogenic and vascular normalization effects. However, the mechanisms underlying its involvement in tumor metabolic reprogramming are still unclear. This study aims to investigate the distribution and expression levels of metabolites within tumors after anlotinib treatment using spatial metabolomics analysis. Methods: This study involves spatially analyzing the distribution differences of metabolites post-anlotinib administration and integrating transcriptomics and proteomics to explore the internal mechanisms of anlotinib action at a molecular level. Results: By integrating the transcriptomics and proteomics analyses, we identified that anlotinib treatment primarily modulated four metabolic pathways, including taurine and hypotaurine metabolism, steroid synthesis, pentose phosphate pathway, and lipid biosynthesis. This regulation significantly influenced the metabolic levels of compounds such as sulfonic acids, cholesterol, inositol phosphate pyrophosphate, and palmitoyl-CoA in the tumor, thereby impacting tumor initiation and progression. Conclusions: This study provides potential metabolic biomarkers for anlotinib treatment in tumors, and may identify potential biomarkers and pathways associated with the therapeutic efficacy of anlotinib treatment.

Analysis of proteomics based on LC-MS detection on the efficacy of immunocombined therapy for hepatocellular carcinoma.

e14549 Background: Combined immunotherapy has become the preferred treatment for HCC. However, it faces challenges: possible serious adverse reactions, the low objective response rate, and the median overall survival is about 20 months, which is far from meeting the clinical treatment needs. There is still a lack of effective predictive biomarkers in clinical practice to screen the dominant population of combined immunotherapy. The purpose of this study is to use LC-MS to screen and identify differential metabolites between patients with different efficacy to help screen the dominant population of clinical immunotherapy. Methods: 38 HCC patients were included who were treated in the Tumor Center of the First Hospital of Jilin University from June 2020 to March 2022 and received combined immunotherapy. Sample collection and follow· -up, and collect basic clinical data. The enrolled patients underwent enhanced CT examination every 3 treatment cycles, and the efficacy of the patients was evaluated according to the solid tumor efficacy evaluation standard 1.1 (RECIST V1.1). The serum of patients before treatment was detected by LC-MS, and the difference of mass spectrometry data between patients with different curative effects was compared, and the differential metabolites were explored by proteomics analysis. Results: The best of response was partial response in 7 patients (22.6 %), stable disease in 6 patients, and progressive disease in 18 patients (58.1 %). Based on LC-MS proteomics analysis, differential metabolites between patients with different therapeutic effects were screened and identified, namely Q9Y275, Q56UQ5, P13611, and P30530 (P < 0.05). The relative abundance of Q9Y275, Q56UQ5, P13611 and P30530 in the disease control rate group was higher than that in the PD group. Conclusions: Based on LC-MS proteomics analysis, four types of differential metabolites between different populations of PLC patients receiving combined immunotherapy were screened and identified, namely Q9Y275, Q56UQ5, P13611 and P30530.

Proteomics of human platelet lysates and insight from animal studies on platelet protein diffusion to hippocampus upon intranasal administration.

Human platelet lysates (HPLs) from allogeneic platelet concentrates (PCs) are biomaterials, which are rich in various trophic factors, increasingly used in regenerative medicine and biotherapy. Understanding how preparation methods influence the HPL protein profile, biological function, and clinical outcomes is crucial. Our study sheds light on the proteomes and functionality of different HPLs, with the aim of advancing their scientifically grounded clinical applications. To achieve this, PCs suspended in plasma underwent three distinct processing methods, resulting in seven HPL types. We used three characterization techniques: label-free proteomics and tandem mass tag (TMT)-based quantitative proteomics, both before and after the immunodepletion of abundant plasma proteins. Bioinformatic tools assessed the proteome, and western blotting validated our quantitative proteomics data. Subsequent pre-clinical studies with fluorescent labeling and label-free proteomics were used as a proof of concept for brain diffusion. Our findings revealed 1441 proteins detected using the label-free method, 952 proteins from the TMT experiment before and after depletion, and 1114 proteins from the subsequent TMT experiment on depleted HPLs. Most detected proteins were cytoplasmic, playing key roles in catalysis, hemostasis, and immune responses. Notably, the processing methodologies significantly influenced HPL compositions, their canonical pathways, and, consequently, their functionality. Each HPL exhibited specific abundant proteins, providing valuable insight for tailored clinical applications. Immunoblotting results for selected proteins corroborated our quantitative proteomics data. The diffusion and differential effects to the hippocampus of a neuroprotective HPL administered intranasally to mice were demonstrated. This proteomics study advances our understanding of HPLs, suggesting ways to standardize and customize their production for better clinical efficacy in regenerative medicine and biotherapy. Proteomic analyses also offered objective evidence that HPPL, upon intranasal delivery, not only effectively diffuses to the hippocampus but also alters protein expression in mice, bolstering its potential as a treatment for memory impairments.

A Peritrophin-44 gene in Litopenaeus vannamei is involved in disease resistance

Peritrophic membrane (PM) is a pellicle structure present in the midgut of some invertebrates, such as insects and crustaceans. It could isolate harmful components and pathogens in food from intestinal epithelial cells; and it also plays a role in improving digestion and absorption efficiency. So PM is important for survival of its owner. In current study, 44 PM proteins were identified in Litopenaeus vannamei by PM proteome analysis. Among these PM proteins, the Peritrophin-44 homologous protein (LvPT44) was further studied. Chitin-binding assay indicated that LvPT44 could bind to colloidal chitin, and immunoeletron microscopy analysis shown that it was located to PM of L. vannamei. Furthermore, LvPT44 promoter was found to be activated by L. vannamei STAT and c-Jun. Besides, LvPT44 was induced by ER-stress as well as white spot syndrome virus infection. Knocked-down expression of LvPT44 by RNA inference increased the cumulative mortality of shrimp that caused by ER-stress or white spot syndrome virus. These results suggested that LvPT44 has an important role in disease resistance.

Proteomics analysis of coronary atherosclerotic heart disease with different Traditional Chinese Medicine syndrome types before and after percutaneous coronary intervention.

To investigate the underlying protein molecular mechanisms of "Qi stagnation and blood stasis syndrome" (QS) and "Qi deficiency and blood stasis syndrome" (QD), as two subtypes of coronary artery disease (CAD) in Traditional Chinese Medicine (TCM), following percutaneous coronary intervention (PCI). In this study, a total of 227 CAD patients with QS and 211 CAD patients with QD were enrolled; all participants underwent PCI. Label-free quantification proteomics were employed to analyze the changes in serum in two subtypes of CAD patients before and 6 months after PCI, aiming to elucidate the intervention mechanism of PCI in treating CAD characterized by two different TCM syndromes. Biochemical analysis revealed significant changes in tumor necrosis factor-α, high density lipoprotein cholesterol, blood stasis clinical symptoms observation, and Gensini levels in both patient groups post-PCI; Proteomic analysis identified 79 and 95 differentially expressed proteins in the QS and QD patient groups, respectively, compared to their control groups. complement C8 alpha chain, complement factor H, apolipoprotein H, apolipoprotein B, plasminogen, carbonic anhydrase 2, and complement factor I were altered in both comparison groups. Furthermore, enrichment analysis demonstrated that cell adhesion and connectivity-related processes underwent changes in QS patients post-PCI, whereas lipid metabolism-related pathways, including the peroxisome proliferator-activated receptor signaling pathway and extracellular matrix receptor interaction, underwent changes in the QD group. The protein-protein interaction network analysis further enriched 52 node proteins, including apolipoprotein B, lipoprotein (a), complement C5, apolipoprotein A4, complement C8 alpha chain, complement C8 beta chain, complement C8 gamma chain, apolipoprotein H, apolipoprotein A-Ⅱ, albumin, complement C4-B, apolipoprotein C3, among others. The functional network of these proteins is posited to contribute to the pathophysiology of CAD characterized by TCM syndromes. The current quantitative proteomic study has preliminarily identified biomarkers of CAD in different TCM subtypes treated with PCI, potentially laying the groundwork for understanding the protein profiles associated with the treatment of various TCM subtypes of CAD.

Protease S of entomopathogenic bacterium Photorhabdus laumondii: expression, purification and effect on greater wax moth Galleria mellonella.

Protease S (PrtS) from Photorhabdus laumondii belongs to the group of protealysin-like proteases (PLPs), which are understudied factors thought to play a role in the interaction of bacteria with other organisms. Since P. laumondii is an insect pathogen and a nematode symbiont, the analysis of the biological functions of PLPs using the PrtS model provides novel data on diverse types of interactions between bacteria and hosts. Recombinant PrtS was produced in Escherichia coli. Efficient inhibition of PrtS activity by photorin, a recently discovered emfourin-like protein inhibitor from P. laumondii, was demonstrated. The Galleria mellonella was utilized to examine the insect toxicity of PrtS and the impact of PrtS on hemolymph proteins in vitro. The insect toxicity of PrtS is reduced compared to protease homologues from non-pathogenic bacteria and is likely not essential for the infection process. However, using proteomic analysis, potential PrtS targets have been identified in the hemolymph. The spectrum of identified proteins indicates that the function of PrtS is to modulate the insect immune response. Further studies of PLPs' biological role in the PrtS and P. laumondii model must clarify the details of PrtS interaction with the insect immune system during bacterial infection.

Alleviation of H2O2 toxicity by extracellular catalases in the phycosphere of Microcystis aeruginosa

High levels of environmental H2O2 represent a threat to many freshwater bacterial species, including toxic-bloom-forming Microcystis aeruginosa, particularly under high-intensity light conditions. The highest extracellular catalase activity-possessing Pseudoduganella aquatica HC52 was chosen among 36 culturable symbiotic isolates from the phycosphere in freshly collected M. aeruginosa cells. A zymogram for catalase activity revealed the presence of only one extracellular catalase despite the four putative catalase genes (katA1, katA2, katE, and srpA) identified in the newly sequenced genome (∼6.8 Mb) of P. aquatica HC52. Analysis of secreted catalase using liquid chromatography–tandem mass spectrometry was identified as KatA1, which lacks a typical signal peptide, although the underlying mechanism for its secretion is unknown. The expression of secreted KatA1 appeared to be induced in the presence of H2O2. Proteomic analysis also confirmed the presence of KatA1 inside the outer membrane vesicles secreted by P. aquatica HC52 following exposure to H2O2. High light intensities (> 100 µmol m−2 s−1) are known to kill catalase-less axenic M. aeruginosa cells, but the present study found that the presence of P. aquatica cells supported the growth of M. aeruginosa, while the extracellular catalases in supernatant or purified form also sustained the growth of M. aeruginosa under the same conditions. Our results suggest that the extracellular catalase secreted by P. aquatica HC52 enhances the tolerance of M. aeruginosa to H2O2, thus promoting the formation of M. aeruginosa blooms under high light intensities.