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To manipulate the protein concentration at a certain functional state through chemical stabilizers is crucial for protein-related studies. It not only plays a key role in protein structure analysis and protein folding kinetics, but also affects protein functionality to a large extent and thus has wide applications in medicine, food industry, etc. However, due to concerns about side effects or financial costs of stabilizers, identifying optimal strategies for enhancing protein stability with a minimal amount of stabilizers is of great importance. Here, we prove that either for the fixed terminal time (including both finite and infinite cases) or for the free one, the optimal control strategy for stabilizing the folding intermediates with a linear strategy for stabilizer addition belongs to the class of bang–bang controls. The corresponding optimal switching time is derived analytically, whose phase diagram with respect to several key parameters is explored in detail. The bang–bang control will be broken when nonlinear strategies for stabilizer addition are adopted. Moreover, the above theory is applied to the stabilization of erythropoietin by ten different kinds of chemicals, providing theoretical guidance for the selection and rational usage of stabilizers. Our current study on optimal strategies for protein stabilizers not only offers deep insights into the general picture of protein folding kinetics but also provides valuable theoretical guidance on treatments for protein-related diseases in medicine. [ABSTRACT FROM AUTHOR]

Background and Purpose: Prostate cancer remains a major public health burden worldwide. Polo like kinase 4 (PLK4) has emerged as a promising therapeutic target in prostate cancer due to its key roles in cell cycle regulation and tumour progression. This study aims to develop and characterize the novel curcumin analogue NL13 as a potential therapeutic agent and PLK4 inhibitor against prostate cancer. Experimental Approach: NL13 was synthesized and its effects were evaluated in prostate cancer cells and mouse xenograft models. Kinome screening and molecular modelling identified PLK4 as the primary target. Antiproliferative and proapoptotic mechanisms were explored via cell cycle, apoptosis, gene and protein analyses. Key Results: Compared with curcumin, NL13 exhibited much greater potency in inhibiting PC3 (IC50, 3.51 μM vs. 35.45 μM) and DU145 (IC50, 2.53 μM vs. 29.35 μM) prostate cancer cells viability and PLK4 kinase activity (2.32 μM vs. 246.88 μM). NL13 induced G2/M cell cycle arrest through CCNB1/CDK1 down‐regulation and triggered apoptosis via caspase‐9/caspase‐3 cleavage. These effects were mediated by PLK4 inhibition, which led to the inactivation of the AKT signalling pathway. In mice, NL13 significantly inhibited tumour growth and modulated molecular markers consistent with in vitro findings, including decreased p‐AKT and increased cleaved caspase‐9/3. Conclusion and Implications: NL13, a novel PLK4‐targeted curcumin analogue, exerts promising anticancer properties against prostate cancer by disrupting the PLK4‐AKT‐CCNB1/CDK1 and apoptosis pathways. NL13 represents a promising new agent for prostate cancer therapy. [ABSTRACT FROM AUTHOR]

Prolyl-4-hydroxylase subunit alpha3 (P4HA3) is a triple helical procollagen synthesis protein. The role of P4HA3 in cancer development is not well known and lacks comprehensive analyses among human cancers. This study aimed to investigate the relationship between P4HA3 expression and anti-tumor immunity and its prognostic value in pan-cancer. P4HA3 expression was analyzed from TIMER2.0, GTEx, GEPIA2.0 and TCGA databases. Genetic and DNA methylation alterations, survival analysis and proteins co-expression analysis of P4HA3 in cBio Cancer Genomics Portal, TCGA, GSCA and TIMER2.0. The correlation between P4HA3 expression and immune infiltration was analyzed by TIDE, XCELL, MCPCOUNTER, and EPIC. We performed EdU and transwell experiments to evaluate the influence of P4HA3 on the proliferation, migration and invasion abilities of different tumors. Patients derived xenograft (PDX) and subcutaneous transplantation models were utilized to explore the correlation between P4HA3 and immunotherapy response in triple-negative breast cancer (TNBC). Among 33 types of cancers, P4HA3 had generally different expression between different tumors, further analysis showed that the expression of P4HA3 was correlated with the cells infiltration of the tumor microenvironment (TME). The expression of P4HA3 was positively with the cell proliferation markers and epithelial-mesenchymal transition (EMT) markers. Moreover, P4HA3 deficiency inhibited the proliferation, migration and invasion abilities of tumor cells, and promoted anti-tumor immunotherapy of PD-1/PD-L1 inhibitor. This pan-cancer analysis of P4HA3 provides a comprehensive understanding of its oncogenic and prognosis role in different cancers, P4HA3 abnormal expression could be a useful biomarker for predicting the effectiveness of immunotherapy in cancer patients. Author summary: The results in this study presented the close correlation and the significant prognostic value of P4HA3 aberrant expression among diverse human tumors. Because the expression of P4HA3 was up-regulated in various of tumors and correlated with worse survival prognosis, we thought that P4HA3 possibly become a new therapeutic target for cancers. More importantly, our research provided a significant perspective in the important role of P4HA3 in anti-tumor immunotherapy and CAFs infiltration. The aberrant expression of P4HA3 might modulate tumor metabolic activity, immune cells and EMT processes in cancers. Future prospective research focusing on P4HA3 and TME could be useful for providing an immuno-based and metabolic-based anti-tumor therapeutic strategies. [ABSTRACT FROM AUTHOR]

Background: Fatigue is a prevalent issue that can lead individuals to a sub-health condition, impacting their work efficiency and quality of life. There are limited effective treatment options available for fatigue. Ren-Shen-Bu-Qi decoction (RSBQD) is a proprietary herbal remedy that is designed to address fatigue. However, the specific pharmacological mechanisms and basis of RSBQD are not yet fully understood. Purpose: This study aimed to investigate the pharmacological effects and mechanisms of RSBQD in a mouse model of exercise fatigue. Materials and methods: UPLC-Q-Orbitrap HRMS was used to analyze the chemical composition of RSBQD. The pharmacological basis and molecular mechanism of RSBQD on exercise fatigue were predicted using network pharmacology analysis. Subsequently, an exercise fatigue mouse model was established and used to analysis the effects of RSBQD. The potential mechanisms were verified by hematoxylin–eosin (HE) staining, real-time fluorescence quantitative PCR (RT-qPCR), Western blot (WB) and molecular docking. Results: The results showed that 88 main components of RSBQD were identified, which have mainly belonged to flavonoids and carboxylic acid compounds. The network pharmacology analysis indicated that RSBQD ameliorate fatigue through PI3K/AKT signaling pathway. Notably, RSBQD prolonged the swimming time and diminished body weight loss of exercise fatigue mice (P < 0.05). Meanwhile, RSBQD significantly alleviated the injury of liver and kidney induced by exhaustive exercise, and decreasing the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea and BUN levels (P < 0.05). In addition, RSBQD was found could relieve exercise fatigue by decreasing the content of creatine kinase (CK), lactate dehydrogenase (LDH), and lactic acid (LA), but increasing the blood glucose (GLU) and liver glycogen (HG) levels (P < 0.05). RSBQD also significantly increased the hepatic superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) but decreased hepatic malondialdehyde (MDA) levels. Moreover, RSBQD was able to upregulate protein level of activated Nrf2 and PI3K/AKT signaling pathways. Conclusions: RSBQD mitigates exercise fatigue by reversing metabolic changes and reducing oxidative damage through the PI3K/AKT/Nrf2 signaling pathway. This study offers pharmacological support for the utilization of RSBQD in exercise fatigue treatment. [ABSTRACT FROM AUTHOR]

N -terminal acetylation is being recognized as a factor affecting protein lifetime and proteostasis. It is a modification where an acetyl group is added to the N -terminus of proteins, and this occurs in 80 % of the human proteome. N -terminal acetylation is catalyzed by enzymes called N -terminal acetyltransferases (NATs). The various NATs acetylate different N -terminal amino acids, and methionine is a known target for some of the NATs. Currently, the acetylation status of most proteins can only be assessed with a limited number of methods, including mass spectrometry, which although powerful and robust, remains laborious and can only survey a fraction of the proteome. We here present testing of an antibody that was developed to specifically recognize Nt-acetylated methionine-starting proteins. We have used dot blots with synthetic acetylated and non-acetylated peptides in addition to protein analysis of lysates from NAT knockout cell lines to assess the specificity and application of this anti-Nt-acetylated methionine antibody (anti-NtAc-Met). Our results demonstrate that this antibody is indeed NtAc-specific and further show that it has selectivity for some subtypes of methionine-starting N -termini, specifically potential substrates of the NatC, NatE and NatF enzymes. We propose that this antibody may be a powerful tool to identify NAT substrates or to analyse changes in N -terminal acetylation for specific cellular proteins of interest. • The function and regulation of Nt-acetylation is unknown for most proteins. • We evaluated a pan Nt-acetylation antibody as a method to monitor Nt-acetylation. • We used peptide dot blot, WB of NAT KO cell lysates, and MS of mouse tissue. • Met-hydrophobic starting N -termini are most suitable for NtAc-dependent detection with this antibody. • The antibody may be used for various Met-starting proteins, to be validated with peptide. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is a debilitating disease that impacts millions of individuals and has limited therapeutic options. A significant hindrance to therapeutic discovery is the lack of in vitro OA models that translate reliably to in vivo preclinical animal models. An alternative to traditional inflammatory cytokine models is the matrikine stimulation model, in which fragments of matrix proteins naturally found in OA tissues and synovial fluid, are used to stimulate cells of the joint. The objective of this study was to determine if matrikine stimulation of equine synovial fibroblasts and chondrocytes with fibronectin fragments (FN7‐10) would result in an OA phenotype. We hypothesized that FN7‐10 stimulation of equine articular cells would result in an OA phenotype with gene and protein expression changes similar to those previously described for human chondrocytes stimulated with FN7‐10. Synovial fibroblasts and chondrocytes isolated from four horses were stimulated in monolayer culture for 6 or 18 h with 1 µM purified recombinant 42 kD FN7‐10 in serum‐free media. At the conclusion of stimulation, RNA was collected for targeted gene expression analysis and media for targeted protein production analysis. Consistent with our hypothesis, FN7‐10 stimulation resulted in significant alterations to many important genes that are involved in OA pathogenesis including increased expression of IL‐1β, IL‐4, IL‐6, CCL2/MCP‐1, CCL5/RANTES, CXCL6/GCP‐2, MMP‐1, MMP‐3, and MMP13. The results of this study suggest that the equine matrikine stimulation model of OA may prove useful for in vitro experiments leading up to preclinical trials. [ABSTRACT FROM AUTHOR]

Objective: Pigmented villonodular synovitis (PVNS) is a rare benign proliferative disease affecting the soft‐tissue lining the synovial joints and tendons. Its etiology is poorly understood, largely limiting the availability of current therapeutic options. Here, we mapped the synovial gene and protein profiles of patients with PVNS, revealed a link between synovial inflammation and invasion, and elucidated the potential molecular mechanism involved. Methods: The expression of synovial genes from 6 control individuals, 7 patients with osteoarthritis (OA), and 19 patients with PVNS was analyzed via RNA sequencing. Protein profiles from 5 control individuals, 10 patients with OA, and 32 patients with PVNS were analyzed using label‐free proteomics. Microarray and reverse transcription–polymerase chain reaction analyses and immunohistochemical staining were used to evaluate inflammatory cytokine and target gene expression levels in synovial tissue, epithelial cells, and synovial fibroblasts (FLSs) derived from tissue of patients with PVNS. Various signaling pathway inhibitors, small interfering RNAs, and Western blots were used for molecular mechanism studies. Transwell migration and invasion assays were subsequently performed. Results: In total, 522 differentially expressed proteins were identified in the tissues of patients with PVNS. By integrating RNA sequencing and microarray analyses, significant changes in the expression of epithelial–mesenchymal transition (EMT)‐related genes, including transforming growth factor TGF‐b induced, neural cadherin, epithelial cadherin, SNAIL, and TWIST, were confirmed in the tissue of patients with PVNS compared to the control tissue. In vitro, TGFβ induced EMT and increased epithelial cell migration and invasion. Moreover, TGFβ not only promoted interactions between epithelial cells and FLSs but also directly increased the migration and invasion abilities of FLSs by activating the classical Smad2/3 and nonclassical JNK/AKT signaling pathways. Conclusion: This study provides overall protein and gene profiles of PVNS and identifies the crucial role of TGFβ in synovial invasion pathology. Exploring the related molecular mechanism may also reveal a new strategy or target for PVNS therapy. [ABSTRACT FROM AUTHOR]

Monozygotic (MZ) twins cannot be distinguished using conventional forensic STR typing because they present identical STR genotypings. However, MZ twins do not always live in the same environment and often have different dietary and other lifestyle habits. Metabolic profiles are deyermined by individual characteristics and are also influenced by the environment in which they live. Therefore, they are potential markers capable of identifying MZ twins. Moreover, the production of proteins varies from organism to organism and is influenced by both the physiological state of the body and the external environment. Hence, we used metabolomics and proteomics to identify metabolites and proteins in peripheral blood to discriminate MZ twins. We identified 1749 known metabolites and 622 proteins in proteomic analysis. The metabolic profiles of four pairs of MZ twins revealed minor differences in intra-MZ twins and major differences in inter-MZ twins. Each pair of MZ twins exhibited distinct characteristics, and four metabolites—methyl picolinate, acesulfame, paraxanthine, and phenylbenzimidazole sulfonic acid—were observed in all four MZ twin pairs. These four differential exogenous metabolites conincidently show that the different external environments and life styles can be well distinguished by metabolites, considering that twins do not all have the same eating habits and living environments. Moreover, MZ twins showed different protein profiles in serum but not in whole blood. Thus, our results indicate that differential metabolites provide potential biomarkers for the personal identification of MZ twins in forensic medicine. [ABSTRACT FROM AUTHOR]

Traditional risk assessment methodologies in toxicology have relied upon animal testing, despite concerns regarding interspecies consistency, reproducibility, costs, and ethics. New Approach Methodologies (NAMs), including cell culture and multi-level omics analyses, hold promise by providing mechanistic information rather than assessing organ pathology. However, NAMs face limitations, like lacking a whole organism and restricted toxicokinetic interactions. This is an inherent challenge when it comes to the use of omics data from in vitro studies for the prediction of organ toxicity in vivo. One solution in this context are comparative in vitro–in vivo studies as they allow for a more detailed assessment of the transferability of the respective NAM data. Hence, hepatotoxic and nephrotoxic pesticide active substances were tested in human cell lines and the results subsequently related to the biology underlying established effects in vivo. To this end, substances were tested in HepaRG and RPTEC/tERT1 cells at non-cytotoxic concentrations and analyzed for effects on the transcriptome and parts of the proteome using quantitative real-time PCR arrays and multiplexed microsphere-based sandwich immunoassays, respectively. Transcriptomics data were analyzed using three bioinformatics tools. Where possible, in vitro endpoints were connected to in vivo observations. Targeted protein analysis revealed various affected pathways, with generally fewer effects present in RPTEC/tERT1. The strongest transcriptional impact was observed for Chlorotoluron in HepaRG cells (increased CYP1A1 and CYP1A2 expression). A comprehensive comparison of early cellular responses with data from in vivo studies revealed that transcriptomics outperformed targeted protein analysis, correctly predicting up to 50% of in vivo effects. [ABSTRACT FROM AUTHOR]

The purpose of this study is to explore the potential molecular mechanism of Bidentis Bipinnatae Herba against gastric cancer by using network pharmacology methods, molecular docking, and cellular experimental validation. Medicinal plants related to gastric cancer were queried through TCMSP, SymMap, and Herb databases. The TCSMP database (drug-likeness ≥ 0.18) was used to retrieve the bioactive constituents. TCSMP, SwissTargetPrediction, and Herb databases were used to retrieve the target genes, and Cytoscape software was used to construct the "active ingredient-target" network. After protein interaction analysis using String 11.0 platform, the hub genes were screened using CytoHubba. The obtained hub genes were uploaded to the cBioPortal for pathway enrichment. The genes involved in gastric cancer-related RTK-RAS pathway were molecularly docked and experimentally validated. Bidentis Bipinnatae Herba was common to TCMSP, SymMap, and Herb databases. A total of nine active ingredients were obtained in Bidentis Bipinnatae Herba, acting on 192 targets. Seven hub genes were obtained from these target genes and enriched in the RTK-RAS pathway in gastric cancer. MAPK1 and EGFR had good molecular docking results with their corresponding chemicals. Cellular experiments showed that the treatment of luteolin, quercetin, and Okanin reduced the expression of EGFR in AGS cells; the treatment of luteolin and quercetin could reduce the expression of MAPK1. Bidentis Bipinnatae Herba contained active components, which may be anti-gastric cancer in a multi-target (MAPK1 and EGFR) manner. [ABSTRACT FROM AUTHOR]

Objective: In this study, we examined the effectiveness of hyperbaric oxygen (HBO) therapy in ameliorating cognitive deficits in mice with Alzheimer’s disease (AD), while also assessing its impact on the autophagic pathway within the context of AD. Methods: 20 double-transgenic mice expressing the amyloid precursor protein and presenilin 1 (APP/PS1) were purposefully selected and randomly assigned to groups A and B. Concurrently, 20 C57BL/6 mice were chosen and randomly categorized into groups C and D, each consisting of 10 mice. Mice in groups B and D received HBO treatment. The Morris water maze assay was used to assess changes in mouse behavior. Immunohistochemistry techniques were used to quantify the expression levels of amyloid-beta 42 (Aβ42) and microtubuleassociated protein 1A/1B-light chain 3 (LC3) in hippocampal tissues, while western blot analysis was used to investigate the levels of LC3-II, p62, phosphoinositide 3-kinase (PI3K), and mammalian target of rapamycin (mTOR) proteins within hippocampal tissues. Results: Mice allocated to group B exhibited reduced escape latency and prolonged dwell time in the target quadrant compared to other groups. Histological examination revealed conspicuous plaque-like deposits of Aβ42 in the hippocampal tissues of mice in groups A and B. Group B displayed diminished Aβ42-positive reactants and augmented microtubule-associated protein 1A/1B-LC3-positive reactants compared to group A. LC3-positive reactants were also detected in the hippocampal tissues of mice in groups C and D, surpassing the levels observed in groups A and B. Furthermore, group B demonstrated significantly lower expression of mTOR protein and markedly higher expression of LC3-II protein in mouse hippocampal tissues when compared to group A (P < 0.05). Conversely, there were no significant disparities noted in PI3K and p62 protein expression between groups B and A. Notably, no discernible discrepancies were observed in the expression levels of mTOR, PI3K, LC3-II, and p62 proteins between groups C and D within mouse hippocampal tissues. Conclusion: HBO treatment demonstrates efficacy in enhancing cognitive function in mice with AD and holds promise as a potential therapeutic intervention for AD by facilitating the activation of the mTOR pathwaymediated autophagy. [ABSTRACT FROM AUTHOR]

Methylobacterium sp. XJLW converts formaldehyde into methanol and formic acid via a Cannizzaro reaction in response to environmental formaldehyde stress. Methanol is further assimilated without formaldehyde or formic acid formation, whereas formic acid accumulates without undergoing further metabolism. Synthetic biology-based biotransformation of methanol to generate additional products can potentially achieve carbon neutrality. However, practical applications are hampered by limitations such as formaldehyde tolerance. In this study, we aimed to explore the specific mechanism of strain XJLW in response to formaldehyde stress. Thus, a transcriptomic analysis of XJLW under formaldehyde treatment was performed, revealing changes in the expression of specific genes related to one-carbon metabolism. Central metabolic genes were downregulated, whereas metabolic bypass genes were upregulated to maintain methanol assimilation in XJLW's response to formaldehyde treatment. In total, 100 genes potentially related to methyl transfer were identified. The function of only one gene, RS27765, was similar to that of glyA, which encodes a methyltransferase involved in one-carbon metabolism. The double-mutant strain, lacking RS27765 and glyA, lost its ability to grow in methanol, whereas the single-mutant strain, lacking only one of these genes, still grew in methanol. Co-expression of RS27765 and RS31205 (YscQ/HrcQ type III secretion apparatus protein) enabled Escherichia coli BL21 (DE3) to effectively degrade methanol. Using protein sequence analysis and molecular docking, we proposed a model wherein RS27765 is necessary for cell growth by using methanol generated via formaldehyde cannizzaro reaction. This process enables direct assimilation of methanol without producing formaldehyde and formic acid as intermediate metabolites. The RS27765 gene cluster, in conjunction with metabolic bypass genes, constitutes a novel auxiliary pathway facilitating formaldehyde stress tolerance in the strain. [ABSTRACT FROM AUTHOR]

Benign prostatic hyperplasia (BPH) is a condition commonly observed in aging males. Inflammatory and metabolic factors are pivotal in the development and progression of BPH. The degree to which the effects of 91 inflammation-related proteins on BPH are mediated by 1400 plasma metabolites remains ambiguous. Our research analyzed the impact of these traits utilizing genetic evidence.Two-sample Mendelian randomization (MR) and multivariable MR (MVMR) were utilized in our study to infer the genetic causal effect of inflammation-related proteins on BPH, with metabolites serving as mediators. Increased levels of IL-2 were linked to a heightened incidence of BPH (β = 0.071, OR:1.074, 95% CI [1.002–1.152], p = 0.045), whereas lower concentrations of N6,N6-dimethyllysine were associated with decreased risk (β1=-0.127, p = 0.02; β2=-0.039, p = 0.008). The mediation effect was 0.005 (95% CI [0.0004, 0.012], OR: 1.005, 95% CI [1.000, 1.012]), accounting for 7.04% of the total effect. subsequently, we examined the phenotypic co-localization of the two pairings independently, revealing that the posterior probability of rs145516501 associated with IL-2 and BPH was 80.7%, whereas the posterior likelihood of rs4917820 linked to N6,N6-dimethyllysine levels and BPH was 95.9%. The research indicated that N6,N6-dimethyllysine levels seem to influence the causative relationship between IL-2 and BPH. These results elucidate the complex interplay between inflammation-related proteins and metabolism in the context of BPH, offering novel diagnostic and therapeutic avenues and enhancing our comprehension of the disease's etiology for prospective research. [ABSTRACT FROM AUTHOR]

In the elderly population, coronary heart disease (CHD) often coexists with hypertension. However, excessive blood pressure reduction can paradoxically increase the incidence of adverse events. Understanding the molecular mechanisms underlying hypertension and CHD in aged populations is crucial for developing targeted therapies and improving clinical outcomes. In this study, we constructed myocardial infarction (MI) and transverse aortic constriction (TAC) modelsY in aged mice to simulate the disease states of CHD and hypertension, respectively. Using integrated proteomic and phosphoproteomic analyses, we investigated the molecular signatures associated with MI and TAC in these models. Our aim was to identify key molecules involved in these conditions and to understand their unique and shared characteristics. Through our comprehensive proteomic and phosphoproteomic analysis, we identified a total of 1583 proteins and 232 phosphorylated proteins. We observed significant upregulation of heart disease markers such as Myh7, Xirp2, and Acta1, indicating the successful establishment of the MI and TAC models. The overlapped differentially expressed proteins (DEPs) and differentially phosphorylated proteins (DPPs) in MI and TAC were involved in heart failure‐related processes including cardiac muscle contraction and hypertrophic cardiomyopathy, further supporting the validity of the models. Among the DEPs, Ppme1 was upregulated in the TAC model but downregulated in the MI model, while Sec31a and Gm56451 displayed the opposite expression patterns. Among the DPPs, Ablim1 and Atp2a2 were found to be significantly upregulated in the TAC model, whereas their expression was markedly reduced in the MI model. In addition, five other DPPs, including REV_Q3TAY5, Cbx3, PITPNB, Eif4b, and A0A1Y7VP73, were elevated in the MI model but decreased in the TAC model. In conclusion, these findings suggest that MI and TAC not only share certain molecular features but also retain their unique characteristics, providing potential biomarkers and therapeutic targets. [ABSTRACT FROM AUTHOR]

Background: Type 2 diabetes mellitus (T2DM) has emerged as a global epidemic issue, with high rates of disability and fatality. Traditional diagnostic biomarkers are typically detected once a metabolic imbalance has already occurred, thus the development of early diagnostic biomarkers is crucial for T2DM. Metabolomics studies have identified several predictive biomarkers for T2DM, including miR‐320. Our previous research found that miR‐320b was significantly downregulated in T2DM patients, but the underlying mechanism remains unclear. Therefore, this study was designed to investigate the significance of miR‐320b for T2DM diagnosis and to explore the involved molecular mechanism. Methods: A total of 50 patients with T2DM and 80 sex‐ and age‐matched healthy subjects were selected. The plasma miR‐320b of all participations was detected by qRT‐PCR and its correlations with other biomarkers of T2DM were analyzed. Besides, the expression of miR‐320b in HepG2 cells was suppressed by miRNA inhibitors. Then the glucose consumption of HepG2 cells was measured. The target gene of miR‐320b was predicted by four bioinformatics tools and intersected these prediction results by Venny method. The T2DM relevant target genes were identified by the GeneCards database. To ensure disease relevance, these T2DM relevant target genes were subsequently intersected with the target genes of miR‐320b. Protein–protein analysis (PPI) was used to screening the gene with the most connections in these target genes. Finally, the target gene of miR‐320b specific to T2DM was confirmed directly by luciferase reporter assay. The expression of target gene in HepG2 cell culture supernatant and plasma of all participations was detected. Results: Our results showed that the expression level of miR‐320b was significantly lower in T2DM patients compared to the healthy controls. It was negatively correlated with fasting plasma glucose (FPG), glycated hemoglobin (HbA1C), and homeostasis model assessment of insulin resistance (HOMA‐IR), but positively with HOMA‐β. The glucose consumption of HepG2 cells in the miR‐320b inhibitor group was significantly lower compared to inhibitor‐NC and blank control group. We predicted and confirmed that phosphatase and tensin homolog (PTEN) was the direct target gene of miR‐320b using Bioinformation tools and luciferase reporter assay. Moreover, the concentration of PTEN was significantly higher in the HepG2 cell culture supernatant and plasma of T2DM patients. Conclusions: Our research demonstrated a negative correlation between miR‐320b and FPG, HbA1C, and HOMA‐IR, while exhibiting a positive correlation with HOMA‐β. Suppressing miR‐320b expression would impair glucose consumption of HepG2 cells through PI3K pathway by targeting PTEN. These results suggest that miR‐320b may be a potential biomarker for diagnosing T2DM and a promising target for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Deep single-cell multi-omic profiling offers a promising approach to understand and overcome drug resistance in relapsed or refractory (rr) acute myeloid leukemia (AML). Here, we combine single-cell ex vivo drug profiling (pharmacoscopy) with single-cell and bulk DNA, RNA, and protein analyses, alongside clinical data from 21 rrAML patients. Unsupervised data integration reveals reduced ex vivo response to the Bcl-2 inhibitor venetoclax (VEN) in patients treated with both a hypomethylating agent (HMA) and VEN, compared to those pre-exposed to chemotherapy or HMA alone. Integrative analysis identifies both known and unreported mechanisms of innate and treatment-related VEN resistance and suggests alternative treatments, like targeting increased proliferation with the PLK inhibitor volasertib. Additionally, high CD36 expression in VEN-resistant blasts associates with sensitivity to CD36-targeted antibody treatment ex vivo. This study demonstrates how single-cell multi-omic profiling can uncover drug resistance mechanisms and treatment vulnerabilities, providing a valuable resource for future AML research. The molecular mechanisms underlying drug resistance in relapsed or refractory (rr) acute myeloid leukemia (AML) remain to be explored. Here, the use of bulk and single cell multi-omics and ex vivo drug profiling for 21 rrAML patients reveals mechanisms of resistance to the Bcl-2 inhibitor venetoclax and treatment vulnerabilities. [ABSTRACT FROM AUTHOR]

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Chronic heart failure (CHF) can induce chronic kidney disease (CKD), called type 2 cardio-renal syndrome (CRS2). The mechanism is not completely clear, and there is a lack of early warning biomarkers of CKD in the context of CHF. Two CKD, one CHF-PBMC and four CHF-cardiac tissue expression profile datasets were obtained from GEO database. Differential expression analysis and WGCNA were used to detect CKD key genes and CHF-related secreted proteins. Protein–protein interaction (PPI), functional enrichment, and cMAP analysis reveal potential mechanisms and drugs of CHF-related CKD. Five machine learning algorithms were used to screen candidate biomarkers, construct a diagnostic nomogram for CKD and validate it in two external cohorts. Clinical serum samples were collected in our hospital to evaluate the correlation and diagnostic value of biomarkers and CKD. 225 CKD key genes and 316 CHF-related secreted proteins were identified. Four key subgroups, including 204 genes, were identified as CRS2-related pathogenic genes by PPI analysis. Enrichment analysis revealed that the identified subgroups exhibited significant enrichment in cytokine action, immune responses, and inflammatory processes. The cMAP analysis highlighted metiradone as a drug with greater potential for therapeutic intervention for CRS2. Utilizing five machine learning algorithms, three hub genes (CD48, COL3A1, LOXL1) were pinpointed as potential biomarkers for CKD, and a nomogram model was constructed. Receiver operating characteristic analysis demonstrated that the nomogram's area under the curve (AUC) exceeded 0.80 in both the CKD combined dataset and two external cohorts. In addition, the three biomarkers were significantly correlated with the glomerular filtration rate, and the AUC of the model predicting disease progression was 0.944. Furthermore, analysis of immune cell infiltration indicated a correlation between the three biomarkers and the infiltration fraction of macrophages, neutrophils, and other immune cells in CKD. Our clinical cohort validated the expression patterns of three biomarkers in serum, and the diagnostic model achieved an AUC of 0.876. CHF has the potential to facilitate the progression of CKD via the release of cardiac and PBMC secreted proteins. Furthermore, CD48, COL3A1, and LOXL1 have been identified as potential biomarkers for the detection of CKD. [ABSTRACT FROM AUTHOR]

Klotho is an anti-aging protein whose deletion significantly reduces lifespan in mice, while its over-expression increases lifespan. Klotho is a type-I transmembrane protein that is N-glycosylated at eight positions within its ectodomain. Our study demonstrates that N-glycosylation or mutation at position N614, but not at N161, N285, or N346 in mouse Klotho, is critically involved in the transport of Klotho out of the endoplasmic reticulum (ER). Consequently, while wild-type Klotho-EGFP as well as the N-glycosylation mutants N161Q, N285Q, and N346Q were present at the plasma membrane (PM), only small amounts of the N614Q Klotho-EGFP were present at the PM, with most of the protein accumulating in the ER. Protein interactome analysis of Klotho-EGFP N614Q revealed increased interactions with proteasome-related proteins and proteins involved in ER protein processing, like heat shock proteins and protein disulfide isomerases, indicative of impaired protein folding. Co-immunoprecipitation experiments confirmed the interaction of Klotho-EGFP N614Q with ER chaperons. Interestingly, despite the low amounts of Klotho-EGFP N614Q at the PM, it efficiently induced FGF receptor-mediated ERK activation in the presence of FGF23, highlighting its efficacy in triggering downstream signaling, even in limited quantities at the PM. [ABSTRACT FROM AUTHOR]

Lipid nanoparticles (LNPs), widely used in disease diagnosis and drug delivery, face the challenge of being surrounded by biological macromolecules such as proteins upon entering the human body. These molecules compete for binding sites on the nanoparticle surfaces, forming a protein corona. The impact of different types of protein coronas on LNP delivery remains unclear. In this study, we employed a newly developed, highly sensitive LNP labeling platform and analyzed the endocytosis of HeLa cells under different nutritional conditions using proteomics to address this critical issue. Our research found that under conditions of complete medium and amino acid starvation, most DNA-FITC vesicles in HeLa cells were located in the perinuclear region 4 h after transfection. In contrast, under serum starvation conditions, only a small portion of DNA-FITC vesicles were in the perinuclear region. On the other hand, through proteomics, we discovered that cells that were enriched in amino acids and complete medium contained more proteins, whereas those under serum starvation had relatively fewer enriched proteins. Through KEGG pathway enrichment analysis, we identified the phagosome and endocytosis pathways as particularly important. Lastly, differential analysis of proteins in these pathways revealed that proteins such as F-actin, Coronin, vATPase, TUBA, TUBB, MHCII, and TSP may have significant impacts on cellular endocytosis. Our research findings indicate that it is necessary to regulate cellular endocytosis based on different protein coronas to achieve optimal cytoplasmic release. [ABSTRACT FROM AUTHOR]

Mycoplasma is a kind of pathogenic microorganism, and its survival and replication need to be parasitic inside the host cell. Therefore, studies on the metabolic pathway, protein composition, and biological characteristics of Mycoplasma require the use of protein expression techniques. In this paper, the application of protein expression in Mycoplasma research was reviewed, including commonly used protein expression systems, optimization strategy of protein expression, protein omics analysis, and protein function research, and the future development direction has been prospected. [ABSTRACT FROM AUTHOR]

This document is a correction notice for an article titled "Simulated complexes formed from a set of postsynaptic proteins suggest a localized effect of a hypomorphic Shank mutation." The authors of the original article identified an error in Figure 1, where the incorrect version of the figure was accidentally replaced during proofing. The correct version of Figure 1 has been updated in this correction notice. The correction was made by Marcell Miski, Áron Weber, Krisztina Fekete-Molnár, Bence Márk Keömley-Horváth, Attila Csikász-Nagy, and Zoltán Gáspári. Springer Nature, the publisher, remains neutral with regard to jurisdictional claims and institutional affiliations. [Extracted from the article]

Background: Oral leukoplakia (OL) is the most common potentially malignant disease of the oral cavity. In recent years, studies have identified a correlation between the gut microbiota (GM) and oral cancer, in addition, inflammation-related proteins have been reported to play an important role in the development of OL. However, the causal relationship between gut microbiota and OL, as well as whether cytokines play a mediating role, remain unclear. Methods: In this Mendelian randomization (MR) study, the genome-wide association studies (GWAS) (n=18340) of the MiBioGen consortium microbiome was used as exposure data. Genetic variation data related to OL were extracted from the Finngen R9 project (513 cases of OL and 411668 controls). The 91 inflammation-related proteins obtained in the literature serve as potential mediators. Two-sample Mendelian randomization analysis was applied to infer causality using Inverse Variance Weighted (IVW), MR Egger, weighted media, simple mode and weighted mode method. Subsequently, sensitivity analysis was conducted to ensure the robustness of the MR results. In addition, we conducted reverse MR analysis to alleviate reverse causality. Finally, we used mediation analysis to determine the pathway mediated by inflammation-related proteins from the gut microbiota to OL. Results: The five bacterial taxa in the gut microbiota indicate a potential causal relationship with the development of OL. Notably, family Clostridiaceae1 was negatively correlated with the risk of OL development, while genus Dorea, genus Ruminococcus1, genus Senegalimasilia and genus Veillonella were positively associated with the risk of OL development. In addition, this study identified a potential causal relationship between interleukin-10 receptor subunit alpha (IL-10RA), interleukin-18 receptor 1(IL18-R1) and the occurrence of OL. In addition, intermediary analysis indicates that IL18-R1 mediated the pathway between the gut microbiota genus Senegalimasilia and OL. Conclusions: In summary, our research emphasize the complex relationship between gut microbiota, inflammation-related proteins and OL. The identified associations and mediating effects provide new insights into potential therapeutic approaches for targeting the gut microbiota in the management of OL, and contribute to its prevention, diagnosis and treatment. [ABSTRACT FROM AUTHOR]

SHP-1, a nonreceptor protein tyrosine phosphatase encoded by ptpn6, has been regarded as a regulatory protein of hematopoietic cell biology for years. However, there is now increasing evidence to support its role in tumors. Thus, the role of ptpn6 for prognosis and immune regulation across 33 tumors was investigated, aiming to explore its functional heterogeneity and clinical significance in pan-cancer. Differential expression of ptpn6 was found between cancer and adjacent normal tissues, and its expression was significantly correlated with the prognosis of tumor patients. In most cancers, ptpn6 expression was significantly associated with immune infiltration. This was further confirmed by ptpn6-related genes/proteins enrichment analysis. Additionally, genetic alterations in ptpn6 was observed in most cancers. As for epigenetic changes, it's phosphorylation levels significantly altered in 6 tumors, while methylation levels significantly altered in 12 tumors. Notably, the methylation levels of ptpn6 were significantly decreased in 11 tumors, accompanied by its increased expression in 8 of them, suggesting that the hypomethylation may be related to its increased expression. Our results show that ptpn6 plays a specific role in tumor immunity and exerts a pleiotropic effect in a variety of tumors. It can serve as a prognostic factor for some cancers. Especially in LGG, KIRC, UCS and TGCT, the increased expression of ptpn6 is associated with poor prognosis and high immune infiltration. This aids in understanding the role of ptpn6 in tumor biology, and can provide insight into presenting a potential biomarker for poor prognosis and immune infiltration in cancers. [ABSTRACT FROM AUTHOR]

Triple-negative breast cancer (TNBC) is known to have the worst outcome compared to the other forms of breast cancer. Moreover, molecular markers identified basal-like breast cancer (BLBC) phenotypes to be also related to a worse prognosis. In this study, we evaluated by immunohistochemistry (IHC) the prognostic significance of combining Cytokeratin-19 (CK19), E-cadherin, and Ki-67 tissue expression in triple-negative breast cancer (TNBC) cases presenting a basal-like (BLBC) or a non-basal-like (n-BLBC) phenotype to improve the selection and the monitoring of BC patients with a more aggressive outcome. Herein, when compared to n-BLBC, patients with BLBC showed a positive correlation with lymph node metastasis occurrence and lower survival rates. Immunohistochemistry analysis revealed significantly lower E-cadherin prevalence and higher prevalence of both CK19 and Ki-67 in BLBC when compared to n-BLBC. Spearman correlation showed that E-cadherin is negatively and significantly correlated to CK19 and Ki-67 expressions. Moreover, in BLBC, expressing both CK19 and Ki-67 combined with E-cadherin loss was associated with the worst relapse-free and overall survival. In conclusion, TNBC/BLBC phenotypes simultaneously losing E-cadherin and overexpressing CK19 and Ki-67 markers are the most aggressive forms. This combined analysis could be a predictive marker of poor prognosis. [ABSTRACT FROM AUTHOR]

The purpose of this study was to determine the impact of feeding time on NPY/AgRP protein and mRNA expression in the brain during prenatal and postnatal periods. Feeding at different times of the day during pregnancy may have lasting effects on the hypothalamic circuitry in offspring energy homeostasis. In the experimental design, adult female hamsters were randomly assigned to receive three different feeding conditions: ad libitum, night-time feeding and day-time feeding groups. After the lactation period, feeding regimens were continued for the offspring born from mothers who were given nutritional regimens, until they were 30 days old. RT-PCR for NPY/AgRP mRNA expression and ELISA analysis for protein levels were performed on hypothalamus tissues of hamsters at 10, 20 and 30 days of postnatal stage. There was no difference between the groups in terms of the daily measured food consumption of the offspring's. Body weights were significantly decreased in both night-time and day-time feeding groups compared to the ad libitum group (p<0.05). The highest increase in mRNA expression of NPY/AgRP was seen in the samples taken at 10. day of lactation in the night-time and day-time feeding groups of the offspring treated with three different feeding regimens. When the feeding regimes were compared, it was observed that the protein and mRNA expression of both NPY and AGRP increased the most in the offspring groups in which the feeding was restricted night -time only. Early stages of development have shown that maternal factors have significantly affected the offspring NPY and AgRP mRNA expression and protein levels. These results show that the metabolic regulation of energy balance may change with maternal factors during the very early stages of development. [ABSTRACT FROM AUTHOR]

In vitro embryo production (IVP) in cattle is crucial for advancing genetic enhancement and preserving valuable genetic lineages, enabling precise genetic modifications and gene studies through modern techniques. Successful genetic manipulation in cattle embryos requires efficient delivery of exogenous DNA/RNA molecules. This research investigates the efficacy of a single embryo culture system for developing genetically modified zona‐free (ZF) embryos and examines the use of liposome‐based SAMTOR target siRNA transfer in these individually cultured ZF embryos. The findings indicated that the individual culture system resulted in increased cleavage rates, and blastocyst rates were minimally impacted. The new culture system effectively achieved SAMTOR silencing, with 8–16 cell embryos exhibiting reduction in transcript levels compared to control. Measurement of total protein content in the spent culture media was performed to validate the single‐culture approach for further analytical applications. Total protein content analysis demonstrated the system's suitability for comprehensive evaluation of the embryo‐media interaction, enhancing the scope for in‐depth genetic research and applications. This research sheds light into an innovative method to improve genetic editing techniques in reproduction research. [ABSTRACT FROM AUTHOR]

Renowned for their role in haemostasis and thrombosis, platelets are also increasingly recognized for their contribution in innate immunity, immunothrombosis and inflammatory diseases. Platelets express a wide range of receptors, which allows them to reach a variety of activation endpoints and grants them immunomodulatory functions. Activated platelets release extracellular vesicles (PEVs), whose formation and molecular cargo has been shown to depend on receptor‐mediated activation and environmental cues. This study compared the immunomodulatory profiles of PEVs generated via activation of platelets by different receptors, glycoprotein VI, C‐type lectin‐like receptor 2 and combining all thrombin‐collagen receptors. Functional assays in vivo in zebrafish and in vitro in human macrophages highlighted distinct homing and secretory responses triggered by the PEVs. In contrast, omics analyses of protein and miRNA cargo combined with physicochemical particle characterization found only subtle differences between the activated PEV types, which were insufficient to predict their different immunomodulatory functions. In contrast, constitutively released PEVs, formed in the absence of an exogenous activator, displayed a distinct immunomodulatory profile from the receptor‐induced PEVs. Our findings underscore that PEVs are tunable through receptor‐mediated activation. To truly comprehend their role(s) in mediating platelet functions among immune cells, conducting functional assays is imperative. [ABSTRACT FROM AUTHOR]

Differences between orbital and subcutaneous abdominal fat in the same patient have been noted but not formally investigated, previously. The objective of this research was to compare the differential expression of protein profiles in subcutaneous abdominal and orbital adipose tissues. In this cross-sectional, observational study, orbital fat tissue was sampled from 10 patients who underwent blepharoplasty and agreed to provide a small sample of subcutaneous abdominal fat. Shotgun mass spectrometry was performed on the extracted proteome. Data were analyzed using protein appearance patterns, differential expression and statistical enrichment. Protein analysis revealed significant differences in proteomics and differential expression between the orbital and subcutaneous abdominal adipose tissues, which presented five proteins that were uniquely expressed in the orbital fat and 18 in the subcutaneous abdominal fat. Gene Ontology analysis identified significantly different cellular processes and components related to the extracellular matrix or basement membrane components. This analysis shows the differences between orbital and subcutaneous abdominal fat found in proteomics differential expression, uniquely expressed proteins, and cellular processes. Further research is needed to correlate specific proteins and cellular processes to the mechanism of fat accumulation and obesity. [ABSTRACT FROM AUTHOR]

Calcium is an important second messenger that is involved in almost all cellular processes. Disruptions in the regulation of intracellular Ca2+ levels ([Ca2+]i) adversely impact normal physiological function and can contribute to various diseased conditions. STIM and Orai proteins play important roles in maintaining [Ca2+]i through store-operated Ca2+ entry (SOCE), with STIM being the primary regulatory protein that governs the function of Orai channels. STIM1 and STIM2 are single-pass ER-transmembrane proteins with their N- and C-termini located in the ER lumen and cytoplasm, respectively. The N-terminal EF-SAM domain of STIMs senses [Ca2+]ER changes, while the C-terminus mediates clustering in ER-PM junctions and gating of Orai1. ER-Ca2+ store depletion triggers activation of the STIM proteins, which involves their multimerization and clustering in ER-PM junctions, where they recruit and activate Orai1 channels. In this review, we will discuss the structure, organization, and function of EF-hand motifs and the SAM domain of STIM proteins in relation to those of other eukaryotic proteins. [ABSTRACT FROM AUTHOR]

Aim: To identify new biomarkers to detect untreated and treated periodontitis in gingival crevicular fluid (GCF) using sequential window acquisition of all theoretical mass spectra (SWATH‐MS). Materials and Methods: GCF samples were collected from 44 periodontally healthy subjects and 40 with periodontitis (Stages III–IV). In the latter, 25 improved clinically 2 months after treatment. Samples were analysed using SWATH‐MS, and proteins were identified by the UniProt human‐specific database. The diagnostic capability of the proteins was determined with generalized additive models to distinguish the three clinical conditions. Results: In the untreated periodontitis vs. periodontal health modelling, five proteins showed excellent or good bias‐corrected (bc)‐sensitivity/bc‐specificity values of >80%. These were GAPDH, ZG16B, carbonic anhydrase 1, plasma protease inhibitor C1 and haemoglobin subunit beta. GAPDH with MMP‐9, MMP‐8, zinc‐α‐2‐glycoprotein and neutrophil gelatinase‐associated lipocalin and ZG16B with cornulin provided increased bc‐sensitivity/bc‐specificity of >95%. For distinguishing treated periodontitis vs. periodontal health, most of these proteins and their combinations revealed a predictive ability similar to previous modelling. No model obtained relevant results to differentiate between periodontitis conditions. Conclusions: New single and dual GCF protein biomarkers showed outstanding results in discriminating untreated and treated periodontitis from periodontal health. Periodontitis conditions were indistinguishable. Future research must validate these findings. [ABSTRACT FROM AUTHOR]

The dynamic evolution of SARS‐CoV‐2 variants necessitates ongoing advancements in therapeutic strategies. Despite the promise of monoclonal antibody (mAb) therapies like bebtelovimab, concerns persist regarding resistance mutations, particularly single‐to‐multipoint mutations in the receptor‐binding domain (RBD). Our study addresses this by employing interface‐guided computational protein design to predict potential bebtelovimab‐resistance mutations. Through extensive physicochemical analysis, mutational preferences, precision‐recall metrics, protein–protein docking, and energetic analyses, combined with all‐atom, and coarse‐grained molecular dynamics (MD) simulations, we elucidated the structural‐dynamics‐binding features of the bebtelovimab–RBD complexes. Identification of susceptible RBD residues under positive selection pressure, coupled with validation against bebtelovimab‐escape mutations, clinically reported resistance mutations, and viral genomic sequences enhances the translational significance of our findings and contributes to a better understanding of the resistance mechanisms of SARS‐CoV‐2. [ABSTRACT FROM AUTHOR]

Phage φ29 and related bacteriophages are currently the smallest known tailed viruses infecting various representatives of both Gram-positive and Gram-negative bacteria. They are characterised by genomic content features and distinctive properties that are unique among known tailed phages; their characteristics include protein primer-driven replication and a packaging process characteristic of this group. Searches conducted using public genomic databases revealed in excess of 2000 entries, including bacteriophages, phage plasmids and sequences identified as being archaeal that share the characteristic features of phage φ29. An analysis of predicted proteins, however, indicated that the metagenomic sequences attributed as archaeal appear to be misclassified and belong to bacteriophages. An analysis of the translated polypeptides of major capsid proteins (MCPs) of φ29-related phages indicated the dissimilarity of MCP sequences to those of almost all other known Caudoviricetes groups and a possible distant relationship to MCPs of T7-like (Autographiviridae) phages. Sequence searches conducted using HMM revealed the relatedness between the main structural proteins of φ29-like phages and an unusual lactococcal phage, KSY1 (Chopinvirus KSY1), whose genome contains two genes of RNA polymerase that are similar to the RNA polymerases of phages of the Autographiviridae and Schitoviridae (N4-like) families. An analysis of the tail tube proteins of φ29-like phages indicated their dissimilarity of the lower collar protein to tail proteins of all other viral groups, but revealed its possible distant relatedness with proteins of toxin translocation complexes. The combination of the unique features and distinctive origin of φ29-related phages suggests the categorisation of this vast group in a new order or as a new taxon of a higher rank. [ABSTRACT FROM AUTHOR]

The vitreous humor (VH) is a transparent gelatin-like substance that occupies two-thirds of the eyeball and undergoes the most significant changes during eye elongation. Quantitative proteomics on the normal growth period in the VH could provide new insights into understanding its progression mechanism in the early stages of myopia. In this study, a data-independent acquisition (SWATH-MS) was combined with targeted LC-ESI-MS/MS to identify and quantify the relative protein changes in the vitreous during the normal growth period (4, 7, 14, 21 and 28 days old) in the chick model. Chicks were raised under normal growing conditions (12/12 h Dark/light cycle) for 28 days, where ocular measurements, including refractive and biometric measurements, were performed on days 4 (baseline), 7, 14, 21 and 28 (n = 6 chicks at each time point). Extracted vitreous proteins from individual animals were digested and pooled into a left eye pool and a right pool at each time point for protein analysis. The vitreous proteome for chicks was generated using an information-dependent acquisition (IDA) method by combining injections from individual time points. Using individual pool samples, SWATH-MS was employed to quantify proteins between each time point. DEPs were subsequently confirmed in separate batches of animals individually on random eyes (n = 4) using MRMHR between day 7 and day 14. Refraction and vitreous chamber depth (VCD) were found to be significantly changed (p < 0.05, n = 6 at each time point) during the period. A comprehensive vitreous protein ion library was built with 1576 non-redundant proteins (22987 distinct peptides) identified at a 1% false discovery rate (FDR). A total of 12 up-regulated and 26 down-regulated proteins were found across all time points compared to day 7 using SWATH-MS. Several DEPs, such as alpha-fetoprotein, the cadherin family group, neurocan, and reelin, involved in structural and growth-related pathways, were validated for the first time using MRMHR under this experimental condition. This study provided the first comprehensive spectral library of the vitreous for chicks during normal growth as well as a list of potential growth-related protein biomarker candidates using SWATH-MS and MRMHR during the emmetropization period. [ABSTRACT FROM AUTHOR]

Objective: To explore the biological function and mechanisms of CEBPB and NAT10‐mediated N4‐acetylcytidine (ac4c) modification in salivary adenoid cystic carcinoma (SACC). Materials and Methods: CEBPB and NAT10 were knocked down in SACC–LM cells by siRNA transfection and overexpressed in SACC‐83 cells by plasmid transfection. Malignant phenotypes were evaluated using CCK‐8, Transwell migration and colony formation assays. Real‐time PCR, western blotting, ChIP and acRIP were used to investigate the molecular mechanisms involved. Results: We found that CEBPB was highly expressed in SACC tissues and correlated with lung metastasis and unfavourable prognosis. Gain‐ and loss‐of‐function experiments revealed that CEBPB promoted SACC malignant phenotypes. Mechanistically, CEBPB exerted its oncogenic effect by binding to the vimentin gene promoter region to enhance its expression. Moreover, NAT10‐mediated ac4c modification led to stabilization and overexpression of CEBPB in SACC cells. We also found that NAT10, the only known human enzyme responsible for ac4C modification, promoted SACC cell migration, proliferation and colony formation. Moreover, CEBPB overexpression restored the inhibitory effect of NAT10 knockdown on malignant phenotypes. Conclusions: Our study reveals the critical role of the newly identified NAT10/CEBPB/vimentin axis in SACC malignant progression, and the findings may be applied to improve treatment for SACC. [ABSTRACT FROM AUTHOR]

One of the central issues in the understanding of early cellular evolution is the characterisation of the cenancestor. This includes the description of the chemical nature of its genome. The disagreements on this question comprise several proposals, including the possibility that AlkB-mediated methylation repair of alkylated RNA molecules may be interpreted as evidence of a cenancestral RNA genome. We present here an evolutionary analysis of the cupin-like protein superfamily based on tertiary structure-based phylogenies that includes the oxygen-dependent AlkB and its homologs. Our results suggest that the repair of methylated RNA molecules is the outcome of the enzyme substrate ambiguity, and doesn´t necessarily indicates that the last common ancestor was endowed with an RNA genome. [ABSTRACT FROM AUTHOR]

A group of three deep‐learning tools, referred to collectively as CHiMP (Crystal Hits in My Plate), were created for analysis of micrographs of protein crystallization experiments at the Diamond Light Source (DLS) synchrotron, UK. The first tool, a classification network, assigns images into categories relating to experimental outcomes. The other two tools are networks that perform both object detection and instance segmentation, resulting in masks of individual crystals in the first case and masks of crystallization droplets in addition to crystals in the second case, allowing the positions and sizes of these entities to be recorded. The creation of these tools used transfer learning, where weights from a pre‐trained deep‐learning network were used as a starting point and repurposed by further training on a relatively small set of data. Two of the tools are now integrated at the VMXi macromolecular crystallography beamline at DLS, where they have the potential to absolve the need for any user input, both for monitoring crystallization experiments and for triggering in situ data collections. The third is being integrated into the XChem fragment‐based drug‐discovery screening platform, also at DLS, to allow the automatic targeting of acoustic compound dispensing into crystallization droplets. [ABSTRACT FROM AUTHOR]

Although various tools provide proteomic information, each tool has limitations related to execution platforms, libraries, versions, and data output format. Integrating data generated from different software is a laborious process that can prolong analysis time. Here, we present FastProtein, a protein analysis pipeline that is user-friendly, easily installable, and outputs important information about subcellular location, transmembrane domains, signal peptide, molecular weight, isoelectric point, hydropathy, aromaticity, gene ontology, endoplasmic reticulum retention domains, and N-glycosylation domains. It also helps determine the presence of glycosylphosphatidylinositol and obtain functional information from InterProScan, PANTHER, Pfam, and alignment-based annotation searches. FastProtein provides the scientific community with an easy-to-use computational tool for proteomic data analysis. It is applicable to both small datasets and proteome-wide studies. It can be used through the command line interface mode or a web interface installed on a local server. FastProtein significantly enhances proteomics analysis workflows by producing multiple results in a single-step process, thereby streamlining and accelerating the overall analysis. The software is open-source and freely available. Installation and execution instructions, as well as the source code and test files generated for tool validation, are available at . [ABSTRACT FROM AUTHOR]

Sterile inflammation is involved in the lung pathogenesis induced by respirable particles, including micro- and nanoplastics. Their increasing amounts in the ambient and in indoor air pose a risk to human health. In two human cell lines (A549 and THP-1) we assessed the proinflammatory behavior of polystyrene nanoplastics (nPS) and microplastics (mPS) (Ø 0.1 and 1 μm). Reproducing environmental aging, in addition to virgin, the cells were exposed to oxidized nPS/mPS. To study the response of the monocytes to the inflammatory signal transmitted by the A549 through the release of soluble factors (e.g. alarmins and cytokines), THP-1 cells were also exposed to the supernatants of previously nPS/mPS-treated A549. After dynamic-light-scattering (DLS) analysis and protein measurements for the assessment of protein corona in nPS/mPS, real-time PCR and enzyme-linked-immunosorbent (ELISA) assays were performed in exposed cells. The pro-inflammatory effects of v- and ox-nPS/mPS were attested by the imbalance of the Bax/Bcl-2 ratio in A549, which was able to trigger the inflammatory cascade, inhibiting the immunologically silent apoptosis. The involvement of NFkB was confirmed by the overexpression of p65 after exposure to ox-nPS and v- and ox-mPS. The fast and higher levels of IL-1β, only in THP-1 cells, underlined the NLPR3 inflammasome activation. [ABSTRACT FROM AUTHOR]

Nuclear envelope repair is a fundamental cellular response to stress, especially for cells experiencing frequent nuclear ruptures, such as cancer cells. Moreover, for chromosomally unstable cancer cells, characterized by the presence of micronuclei, the irreversible rupture of these structures constitutes a fundamental step toward cancer progression and therapy resistance. For these reasons, the study of nuclear envelope rupture and repair is of paramount importance. Nonetheless, due to the constraint imposed by the stochastic nature of rupture events, a precise characterization of the initial stage of nuclear repair remains elusive. In this study, we overcame this limitation by developing a new imaging pipeline that deterministically induces rupture while simultaneously imaging fluorescently tagged repair proteins. We provide a detailed step-by-step protocol to implement this method on any confocal microscope and applied it to study the major nuclear repair protein, barrier-to-autointegration factor (BAF). As a proof of principle, we demonstrated two different downstream analysis methods and showed how BAF is differentially recruited at sites of primary and micronuclear rupture. Additionally, we applied this method to study the recruitment at primary nuclei of the inner nuclear membrane protein LEM-domain 2 (LEMD2) and Charged Multivesicular Protein 7 (CHMP7), the scaffolding protein of the endosomal sorting complex required for transport III (ESCRT-III) membrane remodeling complex. The CHMP7-LEMD2 binding is the fundamental step allowing the recruitment of ESCRT-III, which represents the other major nuclear repair mechanism. This demonstrates the method's applicability for investigating protein dynamics at sites of nuclear and micronuclear envelope rupture and paves the way to more time-resolved studies of nuclear envelope repair. [ABSTRACT FROM AUTHOR]

Objectives: In order to investigate the impact of L-cysteine (L-Cys) on starch and protein degradation during barley germination. The amylase activities, degradation of macromolecules during germination were determined in this study. Methods: Barley was germinated in petri dish for 0 to 5 days with different levels of L-Cys (0 mM, 2.5 mM, 5 mM, 10 mM). Results: L-Cys addition increased the total limit dextrinase (LD) activities and decreased the LD inhibitor activities during whole germination stage. The activities of α-amylase, β-amylase and free LD were increased with the addition of 2.5, 5 mM L-Cys at germination days 1 to 4. Due to higher amylase in malt with the addition of L-Cys, the non-fermentable sugars were reduced and the glucose, maltotriose were improved. Furthermore, the protein degradation analysis showed that low molecular weight protein increased and middle molecular weight protein decreased obviously in wort from the malt germinated with L-Cys, demonstrating that the L-Cys promote the protein degradation. Lastly, the filtration performance of malt with the addition of L-Cys during malting was better than the control. Conclusion: In conclusion, L-Cys can promote the degradation of storage material (starch, protein) during barley germination, leading to a better green malt quality. [ABSTRACT FROM AUTHOR]

Active polysaccharides from plants are broadly applied in the food and health industry. The purpose of this study is to identify a new plant active polysaccharide and to investigate its role in modulating spatial memory. Ultrasonics and DEAE-52 chromatography were used to separate and purify the plant active polysaccharide (PAP). Mice were exposed to 100 ppm of lead acetate from birth to 7 weeks old to establish the memory impairment model. PAPs with concentrations of 200 or 400 ppm were fed to the subject mice each day after weaning in a spatiotemporally separated fashion. At the end of the intervention, mice were examined using the Morris water maze test, microbiome sequencing, cytokine profiling and protein analysis. The derived active polysaccharide was constituted by β-anomeric carbon, indicating a new form of PAP. The PAP significantly ameliorates the memory impairment caused by postnatal lead exposure, as evidenced by the preferred coverage of the test mouse in the hidden platform, demonstrating salient neuroregulatory activity. In terms of the gut microbiome in response to PAP treatment, it was found that the 400 ppm PAP reversed the gut dysbiosis, producing a comparable structure to the intact animals, represented by the relative abundance of Firmicutes and Muribaculum, Desulfovibrio, etc. For cytokines, the PAP reversed the plasma levels of IL-6, suggesting an anti-inflammatory trend in the context of proinflammation caused by lead invasion. By injecting an IL-6 antagonist, Tocilizumab, into the deficient mice, the spatial memory was significantly repaired, which demonstrates the central roles of IL-6 in mediating the positive effect of the PAP. Finally, a histone modification mark, H3K27me3, was found to be potent in responding to the signals conveyed by the PAP. The PAP could improve the memory deficits by remodeling the gut–brain axis centered at the microbiota and IL-6, which is regarded as an important cytokine-modulating brain activity. This is an intriguing instance linking neuromodulation with the active polysaccharide, shedding light on the innovative applications of plant polysaccharides due to the scarcity of similar phenotypic connections. [ABSTRACT FROM AUTHOR]

Background: We examined the physicochemical properties of IgD-type M protein from 10 patients with IgD-type M protein and those with other types of M protein. Methods: Identification of the L-chain type by routine methods (Immunoelectrophoresis: IEP, Immunofixation electrophoresis; IFE), detection of IgD-IgG complexes and the changes in the mobility by treatment with acid. Results: Identification of the L-chain type by both IEP and IFE was impossible. Only one patient revealed the possibility of IgD-IgG complex bands in 6 of the 10 samples. Changes in mobility by treatment with acid were observed in 8 of the 10 samples of IgD-type M protein. Conclusions: These abnormalities are caused by the primary structure of IgD, which may be related to the wellknown weak reactivity with L-chain antibody. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes (LM) is a bacterium widely known for contaminating food products and causing infections in humans, leading to severe gastroenteritis. Phages are viruses that infect host-specific bacteria and can serve as alternative therapeutics to combat these infections. The purpose of the present study is to evaluate the MVB phage activity against Listeria monocytogenes (LM) isolates from contaminated foods. L. monocytogenes was isolated from contaminated food in the Bagalkot district using specific media. Bacteriophages against L. monocytogenes was isolated from municipal sewage wastewater in Bagalkot city using the double-layer plate method. The propagation and purification of the phage were carried out. The spot test was performed against L. monocytogenes and the characterization of the phage was conducted by using electron microscopy, phage DNA extraction, restriction digestion and SDS-PAGE. Three bacteriophages were isolated from wastewater sources and assessed for their ability to lyse L. monocytogenes. The phages showed optimal proliferation at pH 7 and 37°C after 36 hours of incubation. The most effective lytic activity was observed at a bacterial cell to phage ratio of 10:1 with a phage concentration of 9 × 109 PFU/mL. The SDS-PAGE phage protein analysis showed major head proteins of 74 kDa and 37 kDa. The size of the phage DNA was 21 kb and restriction digestion with HindIII showed 5 fragments, while EcoRII showed 4 fragments. Study with transmission electron microscopy (TEM) revealed that the isolated phage belongs to the Inoviridae family. These findings suggesting that the isolated phages are from L. monocytogenes could potentially be utilized to combat this pathogen in various contexts, including dairy based food, agriculture based food, wastewater and related infections. [ABSTRACT FROM AUTHOR]

This study investigates the biochemical complexity of Hammerschmidtiella diesingi, a parasitic nematode inhabiting the alimentary canal of Periplaneta americana (American cockroach). The protein composition was analyzed using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), revealing eight distinct protein bands ranging from 10 to 75 kDa. These bands represent a variety of proteins involved in critical physiological functions, including nutrient absorption, immune evasion and reproduction, enabling the nematode to survive in the host's digestive environment. The 75 kDa band likely corresponds to structural or enzymatic proteins, while the smaller bands (10-25 kDa) may be enzymes involved in digestive processes. The consistency in protein profiles across different sample weights indicates stable protein expression, which is crucial for the nematode's parasitic strategy. The study highlights the evolutionary adaptations of H. diesingi that allow it to grow well in a hostile environment. Future research should focus on functional assays to characterize these proteins and explore genetic mechanisms underlying their expression, which could lead to the development of novel control strategies for parasitic nematode infections in pest species. This research enhances our understanding of the biochemical adaptations of parasitic nematodes and their host interactions. [ABSTRACT FROM AUTHOR]

Mycelium, an innovative and sustainable protein source rich in nutrients, exhibits versatile and functional properties that extend beyond the realm of food applications. This study examined the effect of alkaline extraction pH levels ranging from 10.5 to 12.5 on the yield, functionality, structural attributes, and digestibility of mycelium protein isolate (MPI). The findings reveal a positive correlation between elevated extraction pH levels and increased protein yield, extraction yield, recovery yield, and solubility, with the most substantial yields achieved at pH 12.5. Notably, increased alkaline pH levels corresponded to enhanced protein yield (43.74 ± 0.97%), oil absorption capacity (755.51 ± 5.66%), water solubility index (93.58 ± 4.54%), foaming capacity (41.10 ± 1.27%), and emulsifying activity index (192.92 ± 6.02 m2/g). Moreover, the research underscores the exceptional in-vitro protein digestibility of MPI, registering at 80.69 ± 1.02% at pH 12.0. The analysis of the secondary protein structure indicates a positive relationship between heightened α-helix content at elevated pH levels and improved protein digestibility and solubility. Furthermore, multivariate analyses underscore the intricate connections between techno-functional properties and extraction yields, crucial for tailoring MPI's utility within the food sector. By emphasizing the pivotal role of alkaline extraction pH in influencing the functional and structural attributes of MPI, this study provided potential application of MPI in different food products with variation in alkaline extraction pH. [ABSTRACT FROM AUTHOR]

Bufotalin (BT), a major active constituent of Chansu, has been found to possess multiple pharmacological activities. Although previous studies have shown that BT could inhibit the growth of glioblastoma (GBM), the safety of BT in vivo and the potential mechanism are still unclear. We conducted a systematic assessment to investigate the impact of BT on GBM cell viability, migration, invasion, and colony formation. Furthermore, in vivo results were obtained to evaluate the effect of BT on tumor growth. The preliminary findings of our study demonstrate the effective inhibition of GBM cell growth and subcutaneous tumor development in mice by BT, with tolerable levels of tolerance observed. Mechanistically, BT treatment induced mitochondrial dysfunction, bursts of reactive oxygen species (ROS), and subsequent cell apoptosis. More importantly, proteomic-based differentially expressed proteins analysis revealed a significant downregulation of integrin β4 (ITGB4) following BT treatment. Furthermore, our evidence suggested that the ITGB4/focal adhesion kinase (FAK)/extracellular signal-related kinase (ERK) pathway involved BT-induced apoptosis. Overall, our study demonstrates the anti-GBM effects of BT and elucidates the underlying mechanism, highlighting BT as a potential therapeutic option for GBM. [ABSTRACT FROM AUTHOR]

The transport of biopolymers across nanopores is an important biological process currently under investigation for the rapid analysis of DNA and proteins. While the transport of DNA is generally understood, methods to induce unfolded protein translocation have only recently been discovered (Yu et al., 2023, Sauciuc et al., 2023). Here, we found that during electroosmotically driven translocation of polypeptides, blob-like structures typically form inside nanopores, often obstructing their transport and preventing addressing individual amino acids. This is in contrast with the electrophoretic transport of DNA, where the formation of such structures has not been reported. Comparisons between different nanopore sizes and shapes and modifications by different surface chemistries allowed formulating a mechanism for blob formation. We also show that single-file transport can be achieved by using 1) nanopores that have an entry and an internal diameter smaller than the persistence length of the polymer, 2) nanopores with a nonsticky (i.e., nonaromatic) inner surface, and 3) moderate translocation velocities. These experiments provide a basis for understanding polypeptide transport under confinement and for improving the design and engineering of nanopores for protein analysis. [ABSTRACT FROM AUTHOR]

Our study investigates the impact of FGF23 overexpression on SaOS-2 cells to elucidate its role in cellular stress and morphology, contributing to the understanding of skeletal pathologies like X-linked hypophosphatemia (XLH). Using transmission electron microscopy and protein analysis (Western blot), we analyzed the rough endoplasmic reticulum (rER) and mitochondria in SaOS-2 cells with FGF23 overexpression compared to controls. We found significant morphological changes, including enlarged and elongated rER and mitochondria, with increased contact zones, suggesting enhanced interaction and adaptation to elevated protein synthesis and secretion demands. Additionally, we observed higher apoptosis rates of the cells after 24–72 h in vitro and upregulated proteins associated with ER stress and apoptosis, such as CHOP, XBP1 (spliced and unspliced), GRP94, eIF2α, and BAX. These findings indicate a robust activation of the unfolded protein response (UPR) and apoptotic pathways due to FGF23 overexpression. Our results highlight the critical role of ER and mitochondrial interactions in cellular stress responses and provide new insights into the mechanistic link between FGF23 signaling and cellular homeostasis. In conclusion, our study underscores the importance of analyzing UPR-related pathways in the development of therapeutic strategies for skeletal and systemic diseases and contributes to a broader understanding of diseases like XLH. [ABSTRACT FROM AUTHOR]

Simple Summary: Non-small cell lung cancer (NSCLC) is marked by low survival and resistance to platinum-based chemotherapy. Recent studies have emphasized the critical role of DNA repair mechanisms in NSCLC tumorigenesis and response to treatment. XPG endonuclease, a crucial component of the Nucleotide Excision Repair (NER) pathway, has emerged as a promising biomarker of clinical outcome in advanced NSCLC and its downregulation improved cisplatin efficacy by increasing DNA damage. However, so far, no study has been carried out with the purpose of identifying XPG inhibitors. This work thus aims to discover potential small-molecule inhibitors of XPG to be used in combination with cisplatin therapy to enhance its efficacy in NSCLC patients. NSCLC is marked by low survival and resistance to platinum-based chemotherapy. The XPG endonuclease has emerged as a promising biomarker for predicting the prognosis of cisplatin-treated patients and its downregulation having been reported to increase cisplatin efficacy. This study presents an integrated strategy for identifying small molecule inhibitors of XPG to improve cisplatin therapy in NSCLC. A structure-based virtual screening approach was adopted, including a structural and physicochemical analysis of the protein, and a library of small molecules with reported inhibitory activities was retrieved. This analysis identified Lys84 as a crucial residue for XPG activity by targeting its interaction with DNA. After molecular docking and virtual screening calculations, 61 small molecules were selected as potential XPG inhibitors, acquired from the ChemBridge database and then validated in H1299 cells, a NSCLC cell line exhibiting the highest ERCC5 expression. The MTS assay was performed as a first screening approach to determine whether these potential inhibitors could enhance cisplatin-induced cytotoxicity. Overall, among the eight compounds identified as the most promising, three of them revealed to significantly increase the impact of cisplatin. The inherent cytotoxicity of these compounds was further investigated in a non-tumoral lung cell line (BEAS-2B cells), which resulted in the identification of two non-cytotoxic candidates to be used in combination with cisplatin in order to improve its efficacy in NSCLC therapy. [ABSTRACT FROM AUTHOR]