MTFR2-dependent mitochondrial fission promotes HCC progression.

BACKGROUND: The role of mitochondrial dynamics, encompassing fission, fusion, and mitophagy, in cancer progression has been extensively studied. However, the specific impact of mitochondrial dynamics on hepatocellular carcinoma (HCC) is still under investigation. METHODS: In this study, mitochondrial dynamic genes were obtained from the MitoCarta 3.0 database, and gene expression data were collected from The Cancer Genome Atlas (TCGA) database. Based on the expression of these dynamic genes and differentially expressed genes (DEGs), patients were stratified into two clusters. Subsequently, a prognostic model was constructed using univariate COX regression and the least absolute shrinkage and selection operator (LASSO) regression, and the prognostic signature was evaluated. We analyzed the interaction between these model genes and dynamic genes to identify hub genes and reveal mitochondrial status. Furthermore, we assessed immune infiltration, tumor mutational burden (TMB), tumor stemness indices (TSI), and the response to immune checkpoint block (ICB) therapy using the TIDE algorithm and risk scores. Additionally, transmission electron microscopy (TEM), hematoxylin-eosin (H&E) staining, immunohistochemistry (IHC), western blotting (WB), and immunofluorescence (IF) were conducted to afford detailed visualization of the morphology of the mitochondria and the expression patterns of fission-associated proteins. RESULTS: Patients in Cluster 2 exhibited heightened mitochondrial fission and had a worse prognosis. The up-regulated dynamic genes in Cluster 2 were identified as fission genes. GO/KEGG analyses reconfirmed the connection of Cluster 2 to augmented mitochondrial fission activities. Subsequently, a ten-gene prognostic signature based on the differentially expressed genes between the two clusters was generated, with all ten genes being up-regulated in the high-risk group. Moreover, the potential links between these ten signature genes and mitochondrial dynamics were explored, suggesting their involvement in mediating mitochondrial fission through interaction with MTFR2. Further investigation revealed that the high-risk group had an unfavorable prognosis, with a higher mutation frequency of TP53, increased immune checkpoint expression, a higher TIS score, and a lower TIDE score. The mitochondrial imbalance characterized by increased fission and upregulated MTFR2 and DNM1L expression was substantiated in both HCC specimens and cell lines. CONCLUSIONS: In conclusion, we developed a novel MTFR2-related prognostic signature comprising ten mitochondrial dynamics genes. These genes play crucial roles in mitochondrial fission and have the potential to serve as important predictors and therapeutic targets for HCC.

Underground Mine Safety and Health: A Hybrid MEREC-CoCoSo System for the Selection of Best Sensor.

This research addresses the paramount issue of enhancing safety and health conditions in underground mines through the selection of optimal sensor technologies. A novel hybrid MEREC-CoCoSo system is proposed, integrating the strengths of the MEREC (Method for Eliciting Relative Weights) and Combined Compromise Solution (CoCoSo) methods. The study involves a three-stage framework: criteria and sensor discernment, criteria weight determination using MEREC, and sensor prioritization through the MEREC-CoCoSo framework. Fifteen criteria and ten sensors were identified, and a comprehensive analysis, including MEREC-based weight determination, led to the prioritization of "Ease of Installation" as the most critical criterion. Proximity sensors were identified as the optimal choice, followed by biometric sensors, gas sensors, and temperature and humidity sensors. To validate the effectiveness of the proposed MEREC-CoCoSo model, a rigorous comparison was conducted with established methods, including VIKOR, TOPSIS, TODIM, ELECTRE, COPRAS, EDAS, and TRUST. The comparison encompassed relevant metrics such as accuracy, sensitivity, and specificity, providing a comprehensive understanding of the proposed model's performance in relation to other established methodologies. The outcomes of this comparative analysis consistently demonstrated the superiority of the MEREC-CoCoSo model in accurately selecting the best sensor for ensuring safety and health in underground mining. Notably, the proposed model exhibited higher accuracy rates, increased sensitivity, and improved specificity compared to alternative methods. These results affirm the robustness and reliability of the MEREC-CoCoSo model, establishing it as a state-of-the-art decision-making framework for sensor selection in underground mine safety. The inclusion of these actual results enhances the clarity and credibility of our research, providing valuable insights into the superior performance of the proposed model compared to existing methodologies. The main objective of this research is to develop a robust decision-making framework for optimal sensor selection in underground mines, with a focus on enhancing safety and health conditions. The study seeks to identify and prioritize critical criteria for sensor selection in the context of underground mine safety. The research strives to contribute to the mining industry by offering a structured and effective approach to sensor selection, prioritizing safety and health in underground mining operations.

Stoichiometry of Nucleotide Binding to Proteasome AAA+ ATPase Hexamer Established by Native Mass Spectrometry.

AAA+ ATPases constitute a large family of proteins that are involved in a plethora of cellular processes including DNA disassembly, protein degradation and protein complex disassembly. They typically form a hexametric ring-shaped structure with six subunits in a (pseudo) 6-fold symmetry. In a subset of AAA+ ATPases that facilitate protein unfolding and degradation, six subunits cooperate to translocate protein substrates through a central pore in the ring. The number and type of nucleotides in an AAA+ ATPase hexamer is inherently linked to the mechanism that underlies cooperation among subunits and couples ATP hydrolysis with substrate translocation. We conducted a native MS study of a monodispersed form of PAN, an archaeal proteasome AAA+ ATPase, to determine the number of nucleotides bound to each hexamer of the WT protein. We utilized ADP and its analogs (TNP-ADP and mant-ADP), and a nonhydrolyzable ATP analog (AMP-PNP) to study nucleotide site occupancy within the PAN hexamer in ADP- and ATP-binding states, respectively. Throughout all experiments we used a Walker A mutant (PANK217A) that is impaired in nucleotide binding as an internal standard to mitigate the effects of residual solvation on mass measurement accuracy and to serve as a reference protein to control for nonspecific nucleotide binding. This approach led to the unambiguous finding that a WT PAN hexamer carried - from expression host - six tightly bound ADP molecules that could be exchanged for ADP and ATP analogs. Although the Walker A mutant did not bind ADP analogs, it did bind AMP-PNP, albeit at multiple stoichiometries. We observed variable levels of hexamer dissociation and an appearance of multimeric species with the over-charged molecular ion distributions across repeated experiments. We posit that these phenomena originated during ESI process at the final stages of ESI droplet evolution.

Quantitative Tagless Copurification: A Method to Validate and Identify Protein-Protein Interactions.

Identifying protein-protein interactions (PPIs) at an acceptable false discovery rate (FDR) is challenging. Previously we identified several hundred PPIs from affinity purification - mass spectrometry (AP-MS) data for the bacteria Escherichia coli and Desulfovibrio vulgaris These two interactomes have lower FDRs than any of the nine interactomes proposed previously for bacteria and are more enriched in PPIs validated by other data than the nine earlier interactomes. To more thoroughly determine the accuracy of ours or other interactomes and to discover further PPIs de novo, here we present a quantitative tagless method that employs iTRAQ MS to measure the copurification of endogenous proteins through orthogonal chromatography steps. 5273 fractions from a four-step fractionation of a D. vulgaris protein extract were assayed, resulting in the detection of 1242 proteins. Protein partners from our D. vulgaris and E. coli AP-MS interactomes copurify as frequently as pairs belonging to three benchmark data sets of well-characterized PPIs. In contrast, the protein pairs from the nine other bacterial interactomes copurify two- to 20-fold less often. We also identify 200 high confidence D. vulgaris PPIs based on tagless copurification and colocalization in the genome. These PPIs are as strongly validated by other data as our AP-MS interactomes and overlap with our AP-MS interactome for D.vulgaris within 3% of expectation, once FDRs and false negative rates are taken into account. Finally, we reanalyzed data from two quantitative tagless screens of human cell extracts. We estimate that the novel PPIs reported in these studies have an FDR of at least 85% and find that less than 7% of the novel PPIs identified in each screen overlap. Our results establish that a quantitative tagless method can be used to validate and identify PPIs, but that such data must be analyzed carefully to minimize the FDR.

Bacterial Interactomes: Interacting Protein Partners Share Similar Function and Are Validated in Independent Assays More Frequently Than Previously Reported.

Numerous affinity purification-mass spectrometry (AP-MS) and yeast two-hybrid screens have each defined thousands of pairwise protein-protein interactions (PPIs), most of which are between functionally unrelated proteins. The accuracy of these networks, however, is under debate. Here, we present an AP-MS survey of the bacterium Desulfovibrio vulgaris together with a critical reanalysis of nine published bacterial yeast two-hybrid and AP-MS screens. We have identified 459 high confidence PPIs from D. vulgaris and 391 from Escherichia coli Compared with the nine published interactomes, our two networks are smaller, are much less highly connected, and have significantly lower false discovery rates. In addition, our interactomes are much more enriched in protein pairs that are encoded in the same operon, have similar functions, and are reproducibly detected in other physical interaction assays than the pairs reported in prior studies. Our work establishes more stringent benchmarks for the properties of protein interactomes and suggests that bona fide PPIs much more frequently involve protein partners that are annotated with similar functions or that can be validated in independent assays than earlier studies suggested.

Liquefaction of semen generates and later degrades a conserved semenogelin peptide that enhances HIV infection.

UNLABELLED: Semen enhances HIV infection in vitro, but how long it retains this activity has not been carefully examined. Immediately postejaculation, semen exists as a semisolid coagulum, which then converts to a more liquid form in a process termed liquefaction. We demonstrate that early during liquefaction, semen exhibits maximal HIV-enhancing activity that gradually declines upon further incubation. The decline in HIV-enhancing activity parallels the degradation of peptide fragments derived from the semenogelins (SEMs), the major components of the coagulum that are cleaved in a site-specific and progressive manner upon initiation of liquefaction. Because amyloid fibrils generated from SEM fragments were recently demonstrated to enhance HIV infection, we set out to determine whether any of the liquefaction-generated SEM fragments associate with the presence of HIV-enhancing activity. We identify SEM1 from amino acids 86 to 107 [SEM1(86-107)] to be a short, cationic, amyloidogenic SEM peptide that is generated early in the process of liquefaction but that, conversely, is lost during prolonged liquefaction due to the activity of serine proteases. Synthetic SEM1(86-107) amyloids directly bind HIV-1 virions and are sufficient to enhance HIV infection of permissive cells. Furthermore, endogenous seminal levels of SEM1(86-107) correlate with donor-dependent variations in viral enhancement activity, and antibodies generated against SEM1(86-107) recognize endogenous amyloids in human semen. The amyloidogenic potential of SEM1(86-107) and its virus-enhancing properties are conserved among great apes, suggesting an evolutionarily conserved function. These studies identify SEM1(86-107) to be a key, HIV-enhancing amyloid species in human semen and underscore the dynamic nature of semen's HIV-enhancing activity. IMPORTANCE: Semen, the most common vehicle for HIV transmission, enhances HIV infection in vitro, but how long it retains this activity has not been investigated. Semen naturally undergoes physiological changes over time, whereby it converts from a gel-like consistency to a more liquid form. This process, termed liquefaction, is characterized at the molecular level by site-specific and progressive cleavage of SEMs, the major components of the coagulum, by seminal proteases. We demonstrate that the HIV-enhancing activity of semen gradually decreases over the course of extended liquefaction and identify a naturally occurring semenogelin-derived fragment, SEM1(86-107), whose levels correlate with virus-enhancing activity over the course of liquefaction. SEM1(86-107) amyloids are naturally present in semen, and synthetic SEM1(86-107) fibrils bind virions and are sufficient to enhance HIV infection. Therefore, by characterizing dynamic changes in the HIV-enhancing activity of semen during extended liquefaction, we identified SEM1(86-107) to be a key virus-enhancing component of human semen.

Interaction of fibronectin with semen amyloids synergistically enhances HIV infection.

Semen harbors amyloids that enhance human immunodeficiency virus type 1 (HIV-1) infection. We set out to identify factors that bind these amyloids and to determine whether these factors modulate amyloid-mediated HIV-enhancing activity. Using biochemical and mass spectrometric approaches, we identified fibronectin as a consistent interaction partner. Although monomeric fibronectin did not enhance HIV infection, it synergistically increased the infectivity enhancement activity of the amyloids. Depletion of fibronectin decreased the enhancing activity of semen, suggesting that interfering with the binding interface between fibronectin and the amyloids could be an approach to developing a novel class of microbicides targeting the viral-enhancing activity of semen.

The proteolytic processing of amelogenin by enamel matrix metalloproteinase (MMP-20) is controlled by mineral ions.

BACKGROUND: Enamel synthesis is a highly dynamic process characterized by simultaneity of matrix secretion, assembly and processing during apatite mineralization. MMP-20 is the first protease to hydrolyze amelogenin, resulting in specific cleavage products that self-assemble into nanostructures at specific mineral compositions and pH. In this investigation, enzyme kinetics of MMP-20 proteolysis of recombinant full-length human amelogenin (rH174) under different mineral compositions is elucidated. METHODS: Recombinant amelogenin was cleaved by MMP-20 under various physicochemical conditions and the products were analyzed by SDS-PAGE and MALDI-TOF MS. RESULTS: It was observed that mineral ions largely affect cleavage pattern, and enzyme kinetics of rH174 hydrolysis. Out of the five selected mineral ion compositions, MMP-20 was most efficient at high calcium concentration, whereas it was slowest at high phosphate, and at high calcium and phosphate concentrations. In most of the compositions, N- and C-termini were cleaved rapidly at several places but the central region of amelogenin was protected up to some extent in solutions with high calcium and phosphate contents. CONCLUSION: These in vitro studies showed that the chemistry of the protein solutions can significantly alter the processing of amelogenin by MMP-20, which may have significant effects in vivo matrix assembly and subsequent calcium phosphate mineralization. GENERAL SIGNIFICANCE: This study elaborates the possibilities of the processing of the organic matrix into mineralized tissue during enamel development.

Reprogramming Exosomes to Escape from Immune Surveillance for Mitochondrial Protection in Hepatic Ischemia-Reperfusion Injury.

Background: Therapeutic interventions such as synthetic drugs and microRNA (miR) modulators have created opportunities for mitigating hepatic ischemia/reperfusion injury (HIRI) by alleviating mitochondrial dysfunction. However, delivering multi-therapeutic ingredients with low toxicity to hepatocytes still lags behind its development. Methods: In this study, we endowed exosomes with delivery function to concentrate on hepatocytes for multidimensionally halting mitochondria dysfunction during HIRI. Concretely, exosomes were reprogrammed with a transmembrane protein CD47, which acted as a "camouflage cloak" to mimic the "don't eat me" mechanism to escape from immune surveillance. Besides, HuR was engineered bridging to the membrane by fusing with CD47 and located in the cytoplasm for miR loading. Results: This strategy successfully delivered dual payloads to hepatocytes and efficiently protected mitochondria by inhibiting the opening of mitochondrial permeability transition pore (mPTP) and upregulating mitochondrial transcription factor A (TFAM), respectively. Conclusions: The reprogramming of exosomes with CD47 and HuR for targeted delivery of CsA and miR inhibitors represents a promising therapeutic strategy for addressing HIRI. This approach shows potential for safe and effective clinical applications in the treatment of HIRI.

Necroptosis-Related Prognostic Model for Pancreatic Carcinoma Reveals Its Invasion and Metastasis Potential through Hybrid EMT and Immune Escape.

Necroptosis, pro-inflammatory programmed necrosis, has been reported to exert momentous roles in pancreatic cancer (PC). Herein, the objective of this study is to construct a necroptosis-related prognostic model for detecting pancreatic cancer. In this study, the intersection between necroptosis-related genes and differentially expressed genes (DEGs) of pancreatic ductal adenocarcinoma (PDAC) was obtained based on GeneCards database, GEO database (GSE28735 and GSE15471), and verified using The Cancer Genome Atlas (TCGA). Next, a prognostic model with Cox and LASSO regression analysis, and divided the patients into high-risk and low-risk groups. Subsequently, the Kaplan-Meier (KM) survival curve and the receiver operating characteristic (ROC) curves were generated to assess the predictive ability of overall survival (OS) of PC patients. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to predict the potential biofunction and possible mechanical pathways. The EMTome database and an immune analysis were applied to further explore underlying mechanism. Finally, clinical samples of PDAC patients were utilized to verify the expression of model genes via immunohistochemistry (IHC), and the normal human pancreatic ductal cell line, hTERT-HPNE as well as human pancreatic ductal carcinoma cell lines, PANC-1 and PL45, were used to identify the levels of model genes by Western blot (WB) and immunofluorescence (IF) in vitro. The results showed that 13 necroptosis-related DEGs (NRDEGs) were screened based on GEO database, and finally four of five prognostic genes, including KRT7, KRT19, IGF2BP3, CXCL5, were further identified by TCGA to successfully construct a prognostic model. Univariate and multivariate Cox analysis ultimately confirmed that this prognostic model has independent prognostic significance, KM curve suggested that the OS of low-risk group was longer than high-risk group, and the area under receiver (AUC) of ROC for 1, 3, 5 years was 0.733, 0.749 and 0.667, respectively. A GO analysis illustrated that model genes may participate in cell-cell junction, cadherin binding, cell adhesion molecule binding, and neutrophil migration and chemotaxis, while KEGG showed involvement in PI3K-Akt signaling pathway, ECMreceptor interaction, IL-17 signaling pathway, TNF signaling pathway, etc. Moreover, our results showed KRT7 and KRT19 were closely related to EMT markers, and EMTome database manifested that KRT7 and KRT19 are highly expressed in both primary and metastatic pancreatic cancer, declaring that model genes promoted invasion and metastasis potential through EMT. In addition, four model genes were positively correlated with Th2, which has been reported to take part in promoting immune escape, while model genes except CXCL5 were negatively correlated with TFH cells, indicating that model genes may participate in immunity. Additionally, IHC results showed that model genes were higher expressed in PC tissues than that in adjacent tumor tissues, and WB and IF also suggested that model genes were more highly expressed in PANC-1 and PL45 than in hTERT-HPNE. Tracing of a necroptosis-related prognostic model for pancreatic carcinoma reveals its invasion and metastasis potential through EMT and immunity. The construction of this model and the possible mechanism of necroptosis in PDAC was preliminarily explored to provide reliable new biomarkers for the early diagnosis, treatment, and prognosis for pancreatic cancer patients.

High-throughput isolation and characterization of untagged membrane protein complexes: outer membrane complexes of Desulfovibrio vulgaris.

Cell membranes represent the "front line" of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a "tagless" process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein-protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms.

Amelogenin exons 8 and 9 encoded peptide enhances leucine rich amelogenin peptide mediated dental pulp repair.

Amelogenins containing exons 8 and 9 are alternatively spliced variants of amelogenin. Some amelogenin spliced variants have been found to promote pulp regeneration following pulp exposure. The function of the amelogenin spliced variants with the exons 8 and 9 remains unknown. In this study, we synthesized recombinant leucine rich amelogenin peptide (LRAP, A-4), LRAP plus exons 8 and 9 peptide (LRAP 8, 9) or exons 8 and 9 peptide (P89), to determine their effects on odontoblasts. In vivo analyses were completed following the insertion of agarose beads containing LRAP or LRAP 8, 9 into exposed cavity preparations of rat molars. After 8, 15 or 30 days' exposure, the pulp tissues were analyzed for changes in histomorphometry and cell proliferation by PCNA stainings. In vitro analyses included the effects of the addition of the recombinant proteins or peptide on cell proliferation, differentiation and adhesion of postnatal human dental pulp cells (DPCs). These studies showed that in vivo LRAP 8, 9 enhanced the reparative dentin formation as compared to LRAP. In vitro LRAP 8, 9 promoted DPC proliferation and differentiation to a greater extent than LRAP. These data suggest that amelogenin exons 8 and 9 may be useful in amelogenin-mediated pulp repair.

Survey of large protein complexes in D. vulgaris reveals great structural diversity.

An unbiased survey has been made of the stable, most abundant multi-protein complexes in Desulfovibrio vulgaris Hildenborough (DvH) that are larger than Mr approximately 400 k. The quaternary structures for 8 of the 16 complexes purified during this work were determined by single-particle reconstruction of negatively stained specimens, a success rate approximately 10 times greater than that of previous "proteomic" screens. In addition, the subunit compositions and stoichiometries of the remaining complexes were determined by biochemical methods. Our data show that the structures of only two of these large complexes, out of the 13 in this set that have recognizable functions, can be modeled with confidence based on the structures of known homologs. These results indicate that there is significantly greater variability in the way that homologous prokaryotic macromolecular complexes are assembled than has generally been appreciated. As a consequence, we suggest that relying solely on previously determined quaternary structures for homologous proteins may not be sufficient to properly understand their role in another cell of interest.

Evidence-based annotation of transcripts and proteins in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough.

We used high-resolution tiling microarrays and 5' RNA sequencing to identify transcripts in Desulfovibrio vulgaris Hildenborough, a model sulfate-reducing bacterium. We identified the first nucleotide position for 1,124 transcripts, including 54 proteins with leaderless transcripts and another 72 genes for which a major transcript initiates within the upstream protein-coding gene, which confounds measurements of the upstream gene's expression. Sequence analysis of these promoters showed that D. vulgaris prefers -10 and -35 boxes different from those preferred by Escherichia coli. A total of 549 transcripts ended at intrinsic (rho-independent) terminators, but most of the other transcripts seemed to have variable ends. We found low-level antisense expression of most genes, and the 5' ends of these transcripts mapped to promoter-like sequences. Because antisense expression was reduced for highly expressed genes, we suspect that elongation of nonspecific antisense transcripts is suppressed by transcription of the sense strand. Finally, we combined the transcript results with comparative analysis and proteomics data to make 505 revisions to the original annotation of 3,531 proteins: we removed 255 (7.5%) proteins, changed 123 (3.6%) start codons, and added 127 (3.7%) proteins that had been missed. Tiling data had higher coverage than shotgun proteomics and hence led to most of the corrections, but many errors probably remain. Our data are available at http://genomics.lbl.gov/supplemental/DvHtranscripts2011/.

Automated iterative MS/MS acquisition: a tool for improving efficiency of protein identification using a LC-MALDI MS workflow.

We have developed an information-dependent, iterative MS/MS acquisition (IMMA) tool for improving MS/MS efficiency, increasing proteome coverage, and shortening analysis time for high-throughput proteomics applications based on the LC-MALDI MS/MS platform. The underlying principle of IMMA is to limit MS/MS analyses to a subset of molecular ions that are likely to identify a maximum number of proteins. IMMA reduces redundancy of MS/MS analyses by excluding from the precursor ion peak lists proteotypic peptides derived from the already identified proteins and uses a retention time prediction algorithm to limit the degree of false exclusions. It also increases the utilization rate of MS/MS spectra by removing "low value" unidentifiable targets like nonpeptides and peptides carrying large loads of modifications, which are flagged by their "nonpeptide" excess-to-nominal mass ratios. For some samples, IMMA increases the number of identified proteins by ∼20-40% when compared to the data dependent methods. IMMA terminates an MS/MS run at the operator-defined point when "costs" (e.g., time of analysis) start to overrun "benefits" (e.g., number of identified proteins), without prior knowledge of sample contents and complexity. To facilitate analysis of closely related samples, IMMA's inclusion list functionality is currently under development.

A lectin affinity workflow targeting glycosite-specific, cancer-related carbohydrate structures in trypsin-digested human plasma.

Glycans are cell-type-specific, posttranslational protein modifications that are modulated during developmental and disease processes. As such, glycoproteins are attractive biomarker candidates. Here, we describe a mass spectrometry-based workflow that incorporates lectin affinity chromatography to enrich for proteins that carry specific glycan structures. As increases in sialylation and fucosylation are prominent among cancer-associated modifications, we focused on Sambucus nigra agglutinin (SNA) and Aleuria aurantia lectin (AAL), lectins which bind sialic acid- and fucose-containing structures, respectively. Fucosylated and sialylated glycopeptides from human lactoferrin served as positive controls, and high-mannose structures from yeast invertase served as negative controls. The standards were spiked into Multiple Affinity Removal System (MARS) 14-depleted, trypsin-digested human plasma from healthy donors. Samples were loaded onto lectin columns, separated by HPLC into flow-through and bound fractions, and treated with peptide: N-glycosidase F to remove N-linked glycans. The deglycosylated peptide fractions were interrogated by ESI HPLC-MS/MS. We identified a total of 122 human plasma glycoproteins containing 247 unique glycosites. Importantly, several of the observed glycoproteins (e.g., cadherin 5 and neutrophil gelatinase-associated lipocalin) typically circulate in plasma at low nanogram per milliliter levels. Together, these results provide mass spectrometry-based evidence of the utility of incorporating lectin-separation platforms into cancer biomarker discovery pipelines.

Fluoride incorporation into apatite crystals delays amelogenin hydrolysis.

Enamel fluorosis has been related to an increase in the amount of amelogenin in fluorosed enamel compared with normal enamel in the maturation stage. In this study we tested the hypothesis that fluoride incorporated into carbonated apatite alters amelogenin hydrolysis. Recombinant human amelogenin (rh174) was allowed to bind to 0.15 mg of carbonated hydroxyapatite (CAP) or to fluoride-containing carbonated hydroxyapatite (F-CAP) synthesized to contain 100, 1,000, or 4,000 ppm F(-). After 3 h of digestion with recombinant human matrix metalloproteinase 20 (MMP20) or kallikrein-related peptidase 4 (KLK4), bound protein was characterized by reverse-phase high-performance liquid chromatography (HPLC). Proteolytic fragments of amelogenin formed after 24h of digestion with MMP20 of KLK 4 were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The hydrolysis, by both MMP20 and KLK4, of amelogenin bound to F100-CAP was significantly reduced in a dose-dependent manner compared with the hydrolysis of amelogenin bound to CAP. After 24 h of hydrolysis, a similar number of MMP20 cleavage sites was found for amelogenin bound to CAP and amelogenin bound to F100-CAP; however, 24 fewer KLK4 cleavage sites were identified for amelogenin bound to F100-CAP than for amelogenin bound to CAP. These results suggest that the reduced hydrolysis of amelogenins in fluorosed enamel may be partially caused by the increased fluoride content in fluoride-containing apatite, contributing to the hypomineralized enamel matrix phenotype observed in fluorosed enamel.

A Review on Cement Asphalt Emulsion Mortar Composites, Structural Development, and Performance.

The use of cement emulsified asphalt mortar (CA mortar) in the track structure of high-speed speed railways has been gaining considerations by many researchers due to its coupled merits of the strength of cement as well as the flexibility of asphalt material. The asphalt to cement ratio (A/C) and the compatibility among constituent materials are crucial to the properties of CA mortar. To improve the performance properties and application of CA mortar, it is imperative to have a broad understanding of the composition mechanisms and compatibility between constituent materials. This paper summarizes interesting research outcomes related to the composition and properties of CA mortar. The consumption of water by cement promotes the breakdown of emulsified asphalt, likewise, the adsorption of asphalt droplets on the surface of cement grains retards the hydration process of cement. An appropriate A/C is required for the cement hydration rate to match the speed of demulsification of asphalt emulsion. Depending on the type and properties for which the CA mortar is designed to possess, the A/C ranges from 0.2 to 0.6 for type 1 (CAM I), and 0.6 to 1.2 for type 2 (CAM II). This paper also discusses measures taken to improve performance properties, compatibility, the interaction between constituent materials of CA mortar, and the use of additives as a partial replacement of cement in CA mortar production. The current review also suggests areas of interest for future research studies. This paper is useful to those who aim to understand or study the composition mechanisms and performance properties of CA mortar.

H/ACA snoRNP Gene Family as Diagnostic and Prognostic Biomarkers for Hepatocellular Carcinoma.

BACKGROUND: The H/ACA small nucleolar ribonucleoprotein (snoRNP) gene family, including GAR1 ribonucleoprotein (GAR1), NHP2 ribonucleoprotein (NHP2), NOP10 ribonucleoprotein (NOP10), and dyskerin pseudouridine synthase 1 (DKC1), play important roles in ribosome biogenesis. However, the potential clinical value of the H/ACA snoRNP gene family in hepatocellular carcinoma (HCC) has not yet been reported. METHODS: Bioinformation databases were used to analyze the expression and roles of the H/ACA snoRNP gene family in HCC. Survival analysis, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes enrichment pathway (KEGG) analyses were performed using R software. Tumor Immune Estimation Resource (TIMER) was used to analyze the correlation between the expression of the H/ACA snoRNP gene family and immune infiltration in HCC. Finally, immunohistochemistry and Western blotting were performed to verify the protein expression of the H/ACA snoRNP gene family in HCC tissues and adjacent tissues. RESULTS: The expression of the H/ACA snoRNP gene family was significantly increased in HCC samples compared to normal tissues, and the area under the curve (AUC) of GAR1, NHP2, NOP10, and DKC1 was 0.898, 0.962, 0.884, and 0.911, respectively. Increased expression of the H/ACA snoRNP gene family was associated with poor prognosis in HCC patients (Hazard Ratio, HR = 1.44 [1.02-2.04], 1.70 [1.20-2.40], 1.53 [1.09-2.17], and 1.43 [1.02-2.03], respectively; log-rank P = 0.036, 0.003, 0.014, 0.039, respectively). GO and KEGG analyses showed that co-expressed genes were primarily enriched in ribosome biogenesis. In addition, upregulated expression of H/ACA snoRNP gene family was related to the infiltration of various immune cells and multiple T cell exhaustion markers in HCC patients. Immunohistochemical analysis and Western blotting showed that the protein expression of H/ACA snoRNP gene family was higher in HCC tissues than in adjacent tissues of clinical samples. CONCLUSION: H/ACA snoRNP gene family expression was higher in HCC tissues than in normal or adjacent tissues and was highly associated with poor prognosis of HCC patients and, therefore, has the potential to serve as diagnostic and prognostic biomarkers for HCC.

A Review on Bubble Stability in Fresh Concrete: Mechanisms and Main Factors.

In order to improve the stability of air bubbles in fresh concrete, it is of great significance to have a better understanding of the mechanisms and main influencing factors of bubble stability. In the present review, the formation and collapse process of air bubbles in fresh concrete are essentially detailed; and the advances of major influencing factors of bubble stability are summarized. The results show that the surface tension of air-liquid interface exerts a huge impact on bubble stability by reducing surface free energy and Plateau drainage, as well as increasing the Gibbs surface elasticity. However, surface tension may not be the only determinant of bubble stability. Both the strength of bubble film and the diffusion rate of air through the membrane may also dominate bubble stability. The application of nano-silica is a current trend and plays a key role in ameliorating bubble stability. The foam stability could be increased by 6 times when the mass fraction of nano-particle reached 1.5%.