Biology

Urinary proteomics was used to investigate the potential effects of e-cigarettes on the human body. In this study, a rat e-cigarette model was constructed by smoking for two weeks and urine samples before, during, and after e-cigarette smoking were collected. Urine proteomes before-after smoking of each rat were compared individually, while the control group was set up to rule out differences caused by rat growth and development. After smoking, the differential proteins produced by rats shows strong individual variation. Fetuin-B, a biomarker of COPD, and annexin A2, which is recognized as a multiple tumor marker, were identified as the differential proteins in five out of six smoking rats on day 3. To our surprise, odorant-binding proteins expressed in the olfactory epithelium were also found and were significantly upregulated, which may help explain olfactory adaptation. Pathways enriched by the differential proteins shows the evidence that smoking e-cigarettes affects the immune system, cardiovascular system, respiratory system, etc., which provides clues for further exploration of the mechanism of e-cigarettes on the human body.

Withdrawal StatementThe authors have withdrawn this manuscript because the authors have come across some concerns of the content and wish to get the data validated by more experts in the field. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

ObjectiveChanges in the immune system in the urine proteome were observed by injecting bovine serum albumin and aluminum hydroxide adjuvant into rats. MethodsIn this study, bovine serum albumin and aluminum hydroxide adjuvant were injected into rat thigh muscle, urine was collected, differential proteins were identified by liquid chromatography--mass spectrometry (LC--MSMS/MS), and biological pathways of differential proteins were analyzed by IPA software to observe the changes in the immune system as evidenced by rat urinary proteins. ResultsFifteen rats were intramuscularly injected with normal saline, aluminum hydroxide adjuvant, bovine serum albumin, aluminum hydroxide adjuvant, and bovine serum albumin (BSA) mixture to construct the models of the control, adjuvant, BSA, and mixed groups. Upon comparing the different proteins between different groups to obtain the relevant biological pathways, it was found that adjuvants can be observed in urine to help bovine serum albumin stimulate the immune system to respond earlier. It was also observed in urine that the mixed group successively stimulated immune-related pathways, such as the inflammatory response, T-cell activation, antigen-presenting cell-related pathways, and B-cell-related pathways. DiscussionWe can observe changes in the immune system from the urine proteome in the early stage, providing some new clues and a basis for future research on the immune system and accelerating vaccine research and development.

The gastric tumor microenvironment is dynamically shaped by the interactions between the local microbiota and the host immune system, although the functional integration of these elements remains incompletely understood. In this study, we characterized microbial diversity, immune cell composition, and immune-related gene expression profiles in samples of gastric adenocarcinoma (GAC) and adjacent peritumoral tissue (PTT), aiming to elucidate their functional organization. A total of 106 samples of 75 patients were analyzed using bulk RNA-Seq expression profiling, immune deconvolution, and bacterial taxonomic reconstruction. While alpha diversity remained preserved between GAC and PTT, distinct compositional differences emerged: GAC was enriched with Pseudomonadota, Enterobacteriaceae, and Escherichia, whereas PTT exhibited a predominance of Helicobacteraceae and Helicobacter. Immune deconvolution revealed an expansion of cancer-associated fibroblasts (CAFs) and mast cells in GAC, correlated with higher expression levels of TGFB1 and FOXP3, while neutrophils and B cells predominated in PTT. Integrated analysis demonstrated that GAC formed dense and cohesive networks connecting pro-inflammatory bacteria, activated immune cells, and inflammatory genes such as IL1B, CXCL8, and IFNG. In contrast, PTT exhibited dispersed networks and negative correlations, suggesting a less structured, tolerogenic environment. Our findings indicate that gastric cancer progression involves not only compositional shifts in microbiota and immune cells but also the active construction of functionally integrated inflammatory networks, providing new insights into potential therapeutic targets at the microbiome-immune interface.

The tumor microenvironment (TME) plays a crucial role in the development and survival of neoplastic cells, with tumor-infiltrating leukocytes (TILs) constituting a significant component. This immune infiltrate exhibits a diverse composition of adaptive and innate immunological cell subtypes, with varying prognostic implications across different cancer types. Recent advancements in immunotherapy underscore the importance of evaluating TILs as potential biological identifiers, particularly in the context of novel treatment strategies. In lung adenocarcinoma, the most prevalent histological subtype of lung cancer, multiple immune cell types have been identified within the TME, influencing tumor classification, clinical outcomes, and patient survival. While prior research has demonstrated a correlation between tumor-infiltrating immune cells and the progression of lung adenocarcinoma, few studies have examined their prognostic implications comprehensively. Building upon our previous work, where we constructed a signature matrix (Verma, 2024b.) and evaluated the fractions of 14 immune cell types in TCGA-LUAD data and performed ESTIMATE analysis to assess immune infiltration, stromal infiltration, and tumor purity (Verma, 2024a.), in this study, we investigate the association between immune cell infiltration patterns and the overall survival and prognosis of TCGA-LUAD patients across different histological subtypes and stages. Our findings aim to elucidate the immune cell types positively or negatively impacting patient outcomes in lung adenocarcinoma and inform future therapeutic approaches.

Do rats have corresponding changes in their urinary proteome when smelling different odors? In this study, urine samples were collected from six rats after smelling sesame oil and essential balm for three days. And samples were collected before and on the third and fourth days. Comparing the urinary protein groups of Day0 and Day4 of the sesame oil group, 143 differential proteins were identified, and the average number of randomly generated differential proteins was 7.3, which means that about 95% of the differential proteins could not be randomly generated. in the sesame oil group, differential proteins such as low-density lipoprotein receptor-related protein 2 and fetuin B, a biomarker of COPD, which are associated with olfaction, were identified. While uteroglobulin, trichothecene factor 3, and visfatin 2 were identified in the essential balm group, which had significant changes and were related to the production of olfactory sensation. It is noteworthy that we identified odor-binding protein 2A in the essential balm group, which was present in the e-cigarette model. This study demonstrates that odor can affect rat urinary proteome, with different odors affecting it differently. This provides a new approach to explore the biological process of olfaction.

Abstract Background Prostate cancer (PCa) diagnosis remains challenged by the limited specificity of prostate-specific antigen (PSA) testing, which cannot reliably distinguish malignancy from benign prostatic hyperplasia (BPH). MicroRNAs (miRNAs) are emerging candidates for liquid biopsy-based diagnostics, but most studies assess expression in isolation within a single compartment (biological source - Tissue, blood, serum, urine etc.), overlooking both compartment-specific behavior and the coordinated relationships among miRNAs. Methods We profiled four candidate miRNAs --- miR-19b-3p, miR-21-5p, miR-101-3p and miR-375-3p, across four biological compartments (prostate tumor tissue, urine, serum, and blood) in 179 patients undergoing prostate biopsy for clinical suspicion of PCa (104 PCa, 75 BPH) using qRT-PCR. Urinary exosomal RNA was isolated with a commercial exosome isolation kit so from here onwards this compartment will be referred to as urine. Differential expression was quantified using Cohen's d; inter-miRNA coordination was assessed via Spearman correlation and differential correlation ({delta} r) analysis; and a compartment-level network rewiring score was derived as the sum of {delta} r| across miRNA pairs. Cross-compartment structural alignment was evaluated by comparing correlation patterns at the population level. Diagnostic models combining PSA, age, and urinary exosomal-miRNA features were evaluated using Logistic Regression, Elastic Net Logistic Regression and Naive Bayes classifiers under leave-one-out cross-validation (LOOCV). Results Effect sizes were largest and most consistent in urine, with miR-101-3p showing the strongest separation between PCa and BPH (d = -1.01), followed by miR-21-5p (d {approx}-0.72$) and miR-19b-3p (d {approx}-0.64). Two markers (miR-19b-3p, miR-375-3p) showed directional reversals across compartments, indicating that disease-associated signals are compartment-specific rather than uniformly conserved. In tumor tissue, PCa was associated with substantial reorganization of inter-miRNA coordination (network rewiring score = 2.46), including the emergence of a strong miR-21-5p--miR-375-3p co-regulatory axis ({delta} r = +0.87$) and decoupling of the miR-21-5p--miR-19b-3p relationship ({delta}r = -0.64$). Urine showed a structurally distinct coordination pattern (rewiring score = 1.77), dominated by a miR-101-3p--miR-19b-3p axis (r = +0.56) absent from tissue; cross-compartment comparison showed concordance in only 1 of 5 miRNA pairs, indicating that urine's architecture is largely independent of tissue's. For diagnostic translation, the conventional PSA cutoff (4 ng/mL) achieved 100% sensitivity but only 23.5% specificity. In urine, miR-101-3p performs better than other miRNAs, with AUC of 0.77 (95% CI: 0.62--0.90). Adding PSA and age to the urinary miR-101-3p further improved discrimination to an AUC of 0.91 (95% CI: 0.82--0.99), with 70% specificity at 92% sensitivity; this pattern was consistent across Elastic Net and Logistic Regression classifiers. Expanding the model to include all urinary miRNAs, age, and pair-derived coordination features did not improve on this result (AUC = 0.88), indicating that population-level coordination changes did not translate into additional individual-level diagnostic value in this cohort. Conclusions miRNA signals in extracellular compartments do not represent direct surrogates of tumor-level molecular architecture; each compartment harbors a distinct, transformed coordination structure reflecting its biological context. While these coordination-level changes are mechanistically informative, the most direct translational gain in this study came from a parsimonious model combining PSA, age with a single urinary marker, miR-101-3p, which improved AUC from 0.77 to 0.91, with specificity 70.5% at 90% sensitivity criteria. This combination represents a promising, interpretable candidate for reducing unnecessary prostate biopsies, pending validation in larger, independent cohorts. Keywords: MicroRNA, Compartment-Specific Biomarkers, Urinary Exosomes, Differential Correlation, Liquid Biopsy, Machine learning, PSA, Early diagnosis

BackgroundMetastasis is the primary cause of prostate cancer-related deaths. However, the underlying molecular mechanisms and evolutionary patterns remain largely uncharacterized. MethodsWe evaluate the heterogeneity and genomic evolution of prostate cancer with multi-organ metastases. The samples include 32 primary samples, 23 lymph node metastases, 22 bone metastases, 16 liver metastases, and four pelvic mess metastases. They are analyzed to identify the mutated genes enriched in metastatic samples, selected by metastases, and leading to different long-distance migrations. These metastasis-related alterations constitute a Mscore for evaluating the metastatic risk of primary prostate tumors. ResultsOur analysis discovers 21 metastasis-related mutated genes in total. Of them, 14 genes are finally selected for metastatic risk prognosis, including the mutations of AR and KMT2C with high prediction ability. A Mscore established with these 14 characteristics by the xgboost model displays its ability to classify primary tumors and metastases. This score can further divide primary prostate tumors from the TCGA cohort into two groups. The two subsets present significantly differential survival risks. This score can also identify metastasis-featured primary tumors for breast cancer, bladder cancer, liver cancer, and uterine corpus endometrial carcinoma. ConclusionOur research proposes 14 molecular features potentially driving prostate cancer metastasis. The Mscore established on them can estimate the metastatic risk of primary tumors.

Seasonal breeding is a remarkable adaptive trait, but it constrains efficient production in the sheep industry. Recent studies have shown that seasonal breeding is associated with endogenous circannual rhythms, which are regulated in part by the circadian clock system. FBXL3, a pivotal component of the SCF (SKP1 - CUL1 - F-box) E3 ubiquitin ligase complex, is a known determinant of the mammalian circadian period. In this study, we identified a missense mutation, T183M, in FBXL3 through selective sweep analysis. The allele frequency of this mutation differed significantly between sheep breeds exhibiting year-round estrus and those showing seasonal breeding patterns. The association between the T183M mutation and seasonal breeding was further validated using an ovariectomized, estradiol-implanted sheep model. We then generated mice carrying the homologous T183M mutation and found that they exhibited significantly lengthened circadian periods, accompanied by reduced CRY1 expression and increased CLOCK expression. Co-immunoprecipitation assays confirmed that the mutation reduced the interaction between FBXL3 and CRY1. These findings demonstrate an evolutionarily conserved role of FBXL3 in the circadian clock system. We propose that the T183M mutation disrupts day-length recognition, thereby influencing seasonal estrus in sheep. Author summarySeasonal breeding limits sheep productivity and is regulated by circadian rhythms, yet the key genetic determinants remain poorly understood. Here, we identified an FBXL3 T183M missense mutation whose allele frequency differed markedly between year-round-estrous and seasonally breeding sheep, and validated its association with seasonal reproduction in a sheep population. Functional analyses in mutant mice showed that this variant lengthened the circadian period, disrupted the expression of core clock genes, and weakened the interaction between FBXL3 and CRY1. These findings suggest that the FBXL3 T183M variant impairs day-length perception, thereby modulating seasonal estrus in sheep. Our study reveals a conserved circadian mechanism underlying seasonal breeding and highlights FBXL3 T183M as a promising genetic target for improving reproductive performance in sheep.

BackgroundProstate cancer diagnosis and prognosis is currently limited by the availability of sensitive and specific biomarkers. There is an urgent need to develop molecular biomarkers that allow for the distinction of indolent from aggressive disease, the sensitive detection of heterogeneous tumors, or the evaluation of micro-metastases. The availability of multi-omics datasets in publicly accessible databases provides a valuable foundation to develop computational workflows for the identification of suitable biomarkers for clinical management of cancer patients. ResultsWe combined transcriptomic data of primary localized and advanced prostate cancer from two cancer databases. Transcriptomic analysis of metastatic tumors unveiled a distinct overexpression pattern of genes encoding cell surface proteins intricately associated with cell-matrix components and chemokine signaling pathways. Utilizing an integrated approach combining machine learning and weighted gene correlation network modules, we identified the EZH2-TROAP axis as the main trajectory from initial tumor development to lethal metastatic disease. In addition, we identified and independently validated 58 promising biomarkers that were specifically upregulated in primary localized or metastatic disease. Among those biomarkers, 22 were highly significant for predicting biochemical recurrence. Notably, we confirmed TPX2 upregulation at the protein level in an independent cohort of primary prostate cancer and matched lymph node metastases. ConclusionsThis study demonstrates the effectiveness of using advanced bioinformatics approaches to identify the biological factors that drive prostate cancer progression. Furthermore, the targets identified show promise as prognostic biomarkers in clinical settings. Thus, integrative bioinformatics methods provide both deeper understanding of disease dynamics and open the doors for future personalized interventions.

[Objective]This study focuses on the most important concern of surgeons - whether they resected all of the tumor. Urine can reflect early changes associated with physiological or pathophysiological processes. Based on the above ideas, we conducted experiments to explore changes in the urine proteome between tumor-bearing mice and tumor-resected mice. [Method]The tumor-bearing mouse model was established with MC38 mouse colon cancer cells, and the mice were divided into the healthy control group, complete resection group, and nonresection group. Urine was collected 7 days and 30 days after resection. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to identify the urine proteome and then analyze differentially expressed proteins and biological pathways. [Results](1) Seven days after tumor resection, there were 20 differentially expressed proteins that could distinguish between the complete resection group and the nonresection group. The biological process includes circadian rhythm, Notch signaling pathway, leukocyte cell-cell adhesion, and heterophilic cell-cell adhesion via plasma membrane cell adhesion molecules. (2) Thirty days after tumor resection, there were 33 differentially expressed proteins that could distinguish between the complete resection group and the nonresection group. The biological process includes cell adhesion, complement activation, the alternative pathway, the immune system process, and angiogenesis. (3) There was no significant difference between the two groups at 30 days after tumor resection between the complete resection group and the healthy control group. [Conclusion]Changes in the urine proteome can reflect tumors with or without complete resection.

AimTo investigate if the artificial delivery of microRNAs naturally present in the breastmilk can impact the gut and brain of young rats according to weaning. MethodsAnimals from a new transgenic rat line expressing green-fluorescent protein in the endocrine lineage (cholecystokinin expressing cells) received at Day-12, near neural diversification, a single oral bolus of mir-320-3p or miR-375-3p, embedded in DiOleyl-Succinyl-Paromomycin (DOSP), and were further early (Day-15) or regularly (Day-30) weaned. Relevant miRNA (miR-320-3p, miR-375-3p, miR-375-5p, miR-16-5p, miR-132-3p, miR-504), polr3d, hspb6, inflammation, enteroendocrine, and circadian clock-related mRNAs, chromatin complexes, and duodenal cell density were assayed at 8h post-inoculation and at Day-45. ResultsThe miR-320-3p/DOSP induced immediate effects on H3K4me3 chromatin complexes with polr3d promoter (p<0.05) but no long-term effects. On regular weaning, at Day-45, both miR-320-3p and 375-3p were down-regulated in the stomach, up-regulated in the hypothalamus (p<0.001) but only miR-320-3p was up-regulated in the duodenum. After early weaning, the miR-320-3p and miR-375-3p levels were down-regulated in the stomach and the duodenum, but up-regulated in the hypothalamus and the hippocampus. Combining miR-320-3p/DOSP with early weaning enhanced miR-320-3p and chromogranin A expression in the duodenum. In the hippocampus, the miR-504 was down-regulated for both sexes, but in the brain stem, up regulated only for females, along with miR-320-3p and miR-16-5p levels. In the hypothalamus, clock levels were up regulated for both sexes. In the miR-375-3p/DOSP group, the density of enteroendocrine duodenal cells increased. The long-term effect of miR-375-3p/DOSP was more limited, according to the fourfold lower number of predicted targets than with miR-320-3p. ConclusionAddressing oral miRNA-320-3p loads to duodenal cell lineage is paving the way for the design of new therapeutics, manipulating long term consequences of early life stress.

Bladder cancer shows distinct sex-related patterns, with male patients experiencing significantly higher incidence and female patients facing worse survival outcomes. In this paper, we aimed to address the lack of understanding of the biological mechanisms responsible for this sex-based divergence through an integrative analysis using bladder cancer data from TCGA and GTEx. Our study analyzed various bladder cancer data types, including genomic mutation data, gene expression data, and clinical data. We conducted an in-depth study of protein-protein interactions, pathway analysis, survival analysis, and immune cell correlations. Notably, we discovered that the androgen receptor (AR) related pathways were unique to male hub genes, while the Wnt signaling pathway was unique to female hub genes. Additionally, we identified 14 hub genes with significant sex-biased survival rates, including known DLGAP5, SOX2, LAMA2, and COL5A2, as well as new discoveries of male-specific markers ERCC5, NID1, and ANK2, and female-specific RAD51C, COL22A1 and COL5A2. Furthermore, we identified four male hub genes--DAXX, IKBKB, PDGFRA, and PPARG--that overlapped with immune-related genes. The expression of these genes exhibited differential interactions with immune cells between males and females. These insights could pave the way for more personalized and effective therapeutic interventions tailored to male and female patients.

BackgroundThe C-MYC oncogene is a well-established driver of gastric carcinogenesis, yet the integrated expression pattern of its complex regulatory network and its clinical implications in gastric adenocarcinoma (GAC) remain to be fully elucidated. This study aimed to perform an integrated bioinformatic analysis of C-MYC and its associated pathways in a cohort of GAC patients to delineate its expression profile, assess its potential as a biomarker, and correlate its patterns with clinicopathological factors such as Lauren classification and neoadjuvant treatment status. MethodsThe study included transcriptome data from 74 GAC tumor samples and 68 normal gastric tissue samples. Following RNA sequencing, a comprehensive bioinformatic analysis was conducted on a curated list of 21 C-MYC-associated genes. The methodology included differential expression analysis (DESeq2), unsupervised hierarchical clustering, Principal Component Analysis (PCA), and Receiver Operating Characteristic (ROC) curve analysis to evaluate diagnostic performance. Gene expression levels were also statistically correlated with Lauren histological subtypes and neoadjuvant therapy status using the Wilcoxon rank-sum test. ResultsThe analysis revealed a profound dysregulation of the C-MYC network in GAC. While MYC itself was significantly upregulated, its transcriptional antagonists, particularly MXD4 and MXD3, were the most significantly downregulated genes. This gene signature robustly separated tumor from normal tissues in both hierarchical clustering and PCA. ROC analysis demonstrated the outstanding diagnostic potential of several genes, with MXD4 achieving a perfect Area Under the Curve (AUC) of 1.00, surpassing the diagnostic value of MYC (AUC=0.86). Stratification by Lauren classification showed that MYC and its stability regulator PTBP1 were significantly more expressed in the intestinal subtype, whereas the repressors MXD3 and MXI1 were higher in the diffuse subtype. No significant expression differences were observed based on neoadjuvant treatment status. ConclusionGastric adenocarcinoma is characterized by a coordinated dysregulation of the C-MYC network, marked by both oncogene activation and a concurrent loss of its key transcriptional repressors. The profound downregulation of antagonists like MXD4 serves as an exceptionally accurate molecular signature for GAC, suggesting its potential as a diagnostic biomarker superior to MYC alone. The divergent expression patterns between Lauren subtypes highlight distinct molecular pathobiology and may have implications for targeted therapies

Urine proteomics was applied to explore whether the effect of startle can be detected in urine. A combination of natural enemy odor and sound stimulation was used to establish the rat startle model. Urine samples were collected before and after startle, and urine proteomes before-after startle of each rat were compared individually. Regulatory subunits of glutamate-cysteine ligase was identified as the sole differential protein among all five startled rats. To our surprise, its functional partner catalytic subunits of glutamate-cysteine ligase was also identified in four out of five rats as differential protein. When comparing before-after startle as two groups, 22 differential proteins were identified which represent biological pathways including neurotransmitter transport and glucose transmembrane transport.

Withdrawal StatementThis manuscript has been withdrawn as it was submitted and made public without the full consent of all the authors. Therefore, this work should not be cited as reference for the project. If you have any questions, please contact the corresponding author.

Yakuts are one of the indigenous populations of the subarctic and arctic territories of Siberia characterized by a continental subarctic climate with severe winters, with the regular January average temperature in the regional capital city of Yakutsk dipping below -40{degrees}C. The epigenetic mechanisms of adaptation to such ecologies and environments and, in particular, epigenetic age acceleration in the local population have not been studied before. This work reports the first epigenetic study of the Yakutian population using whole blood DNA methylation data, supplemented with the comparison to the residents of Central Russia. Gene set enrichment analysis revealed, among others, geographic region-specific differentially methylated regions associated with adaptation to climatic conditions (water consumption, digestive system regulation), aging processes (actin filament activity, cell fate), and both of them (channel activity, regulation of steroid and corticosteroid hormone secretion). Further, it is demonstrated that the epigenetic age acceleration of the Yakutian representatives is significantly higher than that of Central Russia counterparts. For both geographic regions, we showed that epigenetically males age faster than females, whereas no significant sex differences were found between the regions.

Plant-derived microRNAs (miRNAs) have been implicated as functional regulators in human diseases, although conclusive evidence of this effect remains to be reported. To examine their potential functional role, we profiled the plant-derived miRNAs in 139 blood exosome samples from renal transplantation patients and were able to identify 331 plant-derived miRNAs representing 149 families. According to their miRBase annotation, these miRNAs can be traced back to 76 plant species, most of which are foods common to the human diet (e.g., tomato, soybean, potato and rice). We also profiled 41 blood exosome samples from 22 patients with acute immune rejection (AR) of renal transplants and compared them to 21 samples from 11 patients with stable allograft function to explore possible roles of the functional plant miRNAs. We identified three plant-derived miRNAs (miR4995, miR2118/2218 and miR167) associated with allograft AR whose regulatory targets are mRNAs controlling immune response, T cell activation, and other cellular functions. miR4995 mimics were generated, transfected into HEK293T cells, and their function verified. Our findings not only demonstrate the presence of functional plant-derived miRNAs in human cells, but also provide initial evidence that these miRNAs may be involved in malfunction of renal transplantation.

Biomarkers are measurable changes associated with physiological or pathophysiological processes in the body. Urine, not strictly regulated by homeostatic mechanisms, accumulates extensive variable information and reflects changes in the body earlier and more sensitively, making it an excellent source for biomarker discovery. However, long-distance transport of urine samples usually requires a strictly maintained cold chain throughout the process, resulting in high costs and operational challenges. Here, we propose a urine sample processing method that uses brief heating to inhibit bacterial proliferation and protein degradation. A single urine sample was equally divided into three groups: a pre-heated transport group, a non-heated transport group, and a -80{degrees}C frozen storage control group, with five replicates in each group. Both the pre-heated and non-heated transport groups were transported simultaneously across northern and southern China for five days under ambient temperature conditions. Samples were analyzed using liquid chromatography coupled with tandem mass spectrometry. The results showed that the intra-group overlap rates of identified proteins within the groups treated with 65{degrees}C water bath heating for 15 minutes and -80{degrees}C frozen storage were 97.1% and 97.5%, respectively, demonstrating high reproducibility. There were no significant differences in the number or types of urinary proteins identified between these two groups, with an inter-group overlap rate of 99.9%. Additionally, the pre-heated transport group and frozen storage control group showed no significant difference in the number of bacterial-derived peptides and proteins identified. In contrast, significantly more bacterial-derived peptides and proteins were identified in the non-heated transport group than in both the pre-heated transport and frozen storage control groups. This indicates that heating effectively suppresses bacterial counts in urine samples during five days of transport at ambient temperatures. This approach provides a more economical and convenient solution for long-distance transport of urine samples, eliminating the need for cold chain transportation or preservatives. We also recommend using consistent urine processing methods within the same study to minimize technical variability.

ObjectiveTo explore the possible effects on the organism by analysing changes in the urinary proteome of mice after autonomous consumption of different sweet substances. MethodsUrine samples were collected from C57BL/6l mice before and after active consumption of sweet substances. The sweet substances included sucrose, stevia glycosides, acesulfame, and sucralose, which are more widely used worldwide and can elicit a preference response in mice, and the concentrations of the non-nutritive sweeteners were chosen to be those that have been shown by existing studies to have the strongest preference response in mice. The analyses were performed by the non-labelled quantitative proteomics technique of high performance liquid chromatography tandem mass spectrometry (LC-MS/MS), and groups were screened for differential proteins in the urinary proteome for the analysis of protein functions and biological processes; comparisons of urinary proteomes before and after consumption of sweeteners by a single mouse were carried out; and side-by-side comparisons of different sweeteners were made. Results and ConclusionsUrine proteome could reflect the changes in the organism of mice after voluntary consumption of sweeteners, and the effects of different sweeteners on the urine proteome were not the same. Among the four sweeteners, sucralose and sucrose induced the most similar changes in the organism, and steviol glycosides induced the furthest changes in the organism; the changes induced by sucrose, acesulfame, and sucralose were similar, and steviol glycosides induced changes different from those of the other sweeteners. Stevia glycosides induced changes that were different from the other sweeteners. The urinary proteomic proteins that differed among the four sweeteners consumed autonomously by mice included proteins that have been reported to be associated with brain reward circuits, whereas only the urinary proteomic proteins that differed among the four sweeteners consumed autonomously by mice were associated with metabolic processes in large quantities after the voluntary consumption of sucrose, acesulfame, and sucralose, and the urinary proteomic proteins that differed among the four sweeteners consumed autonomously by mice were associated with the assembly of nucleosomes, gene expression, and other processes after the voluntary consumption of stevia glycosides.