Integrative multiomics analysis identifies molecular subtypes and potential targets of hepatocellular carcinoma.

BACKGROUND: The liver is anatomically divided into eight segments based on the distribution of Glisson's triad. However, the molecular mechanisms underlying each segment and its association with hepatocellular carcinoma (HCC) heterogeneity are not well understood. In this study, our objective is to conduct a comprehensive multiomics profiling of the segmentation atlas in order to investigate potential subtypes and therapeutic approaches for HCC. METHODS: A high throughput liquid chromatography-tandem mass spectrometer strategy was employed to comprehensively analyse proteome, lipidome and metabolome data, with a focus on segment-resolved multiomics profiling. To classify HCC subtypes, the obtained data with normal reference profiling were integrated. Additionally, potential therapeutic targets for HCC were identified using immunohistochemistry assays. The effectiveness of these targets were further validated through patient-derived organoid (PDO) assays. RESULTS: A multiomics profiling of 8536 high-confidence proteins, 1029 polar metabolites and 3381 nonredundant lipids was performed to analyse the segmentation atlas of HCC. The analysis of the data revealed that in normal adjacent tissues, the left lobe was primarily involved in energy metabolism, while the right lobe was associated with small molecule metabolism. Based on the normal reference atlas, HCC patients with segment-resolved classification were divided into three subtypes. The C1 subtype showed enrichment in ribosome biogenesis, the C2 subtype exhibited an intermediate phenotype, while the C3 subtype was closely associated with neutrophil degranulation. Furthermore, using the PDO assay, exportin 1 (XPO1) and 5-lipoxygenase (ALOX5) were identified as potential targets for the C1 and C3 subtypes, respectively. CONCLUSION: Our extensive analysis of the segmentation atlas in multiomics profiling defines molecular subtypes of HCC and uncovers potential therapeutic strategies that have the potential to enhance the prognosis of HCC.

Carcinoembryonic antigen potentiates non-small cell lung cancer progression via PKA-PGC-1ɑ axis.

Carcinoembryonic antigen (CEA) is a tumor-associated antigen primarily produced by tumor cells. It has been implicated in various biological processes such as cell adhesion, proliferation, differentiation, and metastasis. Despite this, the precise molecular mechanisms through which CEA enhances tumor cell proliferation remain largely unclear. Our study demonstrates that CEA enhances the proliferation and migration of non-small cell lung cancer (NSCLC) while also inhibiting cisplatin-induced apoptosis in NSCLC cells. Treatment with CEA led to an increase in mitochondrial numbers and accumulation of lipid droplets in A549 and H1299 cells. Additionally, our findings indicate that CEA plays a role in regulating the fatty acid metabolism of NSCLC cells. Inhibiting fatty acid metabolism significantly reduced the CEA-mediated proliferation and migration of NSCLC cells. CEA influences fatty acid metabolism and the proliferation of NSCLC cells by activating the PGC-1α signaling pathway. This regulatory mechanism involves CEA increasing intracellular cAMP levels, which in turn activates PKA and upregulates PGC-1α. In NSCLC, inhibiting the PKA-PGC-1α signaling pathway reduces both fatty acid metabolism and the proliferation and migration induced by CEA, both in vitro and in vivo. These results suggest that CEA contributes to the promotion of proliferation and migration by modulating fatty acid metabolism. Targeting CEA or the PKA-PGC-1ɑ signaling pathway may offer a promising therapeutic approach for treating NSCLC.

Identification and biological characteristics of Enterococcus casseliflavus TN-47 isolated from Monochamus alternatus.

With the widespread use of antibiotics, the incidence of antibiotic resistance in microorganisms has increased. Monochamus alternatus is a trunk borer of pine trees. This study aimed to investigate the in vitro antimicrobial and biological characteristics of Enterococcus casseliflavus TN-47 (PP411196), isolated from the gastrointestinal tract of M. alternatus in Jilin Province, PR China. Among 13 isolates obtained from the insects, five were preliminarily screened for antimicrobial activity. E. casseliflavus TN-47, which exhibited the strongest antimicrobial activity, was identified. E. casseliflavus TN-47 possessed antimicrobial activity against Staphylococcus aureus USA300 and Salmonella enterica serovar Pullorum ATCC 19945. Furthermore, E. casseliflavus TN-47 was sensitive to tetracyclines, penicillins (ampicillin, carbenicillin, and piperacillin), quinolones and nitrofuran antibiotics, and resistant to certain beta-lactam antibiotics (oxacillin, cefradine and cephalexin), macrolide antibiotics, sulfonamides and aminoglycosides. E. casseliflavus TN-47 could tolerate low pH and pepsin-rich conditions in the stomach and grow in the presence of bile acids. E. casseliflavus TN-47 retained its strong auto-aggregating ability and hydrophobicity. This strain did not exhibit any haemolytic activity. These results indicate that E. casseliflavus TN-47 has potential as a probiotic. This study provides a theoretical foundation for the future applications of E. casseliflavus TN-47 and its secondary metabolites in animal nutrition and feed.

STING-dependent trained immunity contributes to host defense against Clostridium perfringens infection via mTOR signaling.

Clostridium perfringens (C. perfringens) infection is recognized as one of the most challenging issues threatening food safety and perplexing agricultural development. To date, the molecular mechanisms of the interactions between C. perfringens and the host remain poorly understood. Here, we show that stimulator of interferon genes (STING)-dependent trained immunity protected against C. perfringens infection through mTOR signaling. Heat-killed Candida albicans (HKCA) training elicited elevated TNF-α and IL-6 production after LPS restimulation in mouse peritoneal macrophages (PM). Although HKCA-trained PM produced decreased levels of TNF-α and IL-6, the importance of trained immunity was demonstrated by the fact that HKCA training resulted in enhanced bacterial phagocytic ability and clearance in vivo and in vitro during C. perfringens infection. Interestingly, HKCA training resulted in the activation of STING signaling. We further demonstrate that STING agonist DMXAA is a strong inducer of trained immunity and conferred host resistance to C. perfringens infection in PM. Importantly, corresponding to higher bacterial burden, reduction in cytokine secretion, phagocytosis, and bacterial killing were shown in the absence of STING after HKCA training. Meanwhile, the high expression levels of AKT/mTOR/HIF1α were indeed accompanied by an activated STING signaling under HKCA or DMXAA training. Moreover, inhibiting mTOR signaling with rapamycin dampened the trained response to LPS and C. perfringens challenge in wild-type (WT) PM after HKCA training. Furthermore, STING­deficient PM presented decreased levels of mTOR signaling-related proteins. Altogether, these results support STING involvement in trained immunity which protects against C. perfringens infection via mTOR signaling.

Dihydroartemisinin is an inhibitor of trained immunity through Akt/mTOR/HIF1α signaling pathway.

Trained immunity is mechanistically defined as the metabolically and epigenetically mediated long-term functional adaptation of the innate immune system, characterized by a heightened response to a secondary stimulation. Given appropriate activation, trained immunity represents an attractive anti-infective therapeutic target. Nevertheless, excessive immune response and subsequent inflammatory cascades may contribute to pathological tissue damage, indicating that the negative impacts of trained immunity appear to be significant. In this study, we show that innate immune responses such as the production of extracellular traps, pro-inflammatory cytokines, and autophagy-related proteins were markedly augmented in trained BMDMs. Furthermore, heat-killed C. albicans priming promotes the activation of the AIM2 inflammasome, and AIM2-/- mice exhibit impaired memory response induced by heat-killed C. albicans. Therefore, we establish that the AIM2 inflammasome is involved in trained immunity and emerges as a promising therapeutic target for potentially deleterious effects. Dihydroartemisinin can inhibit the memory response induced by heat-killed C. albicans through modulation of mTOR signaling and the AIM2 inflammasome. The findings suggest that dihydroartemisinin can reduce the induction of trained immunity by heat-killed C. albicans in C57BL/6 mice. Dihydroartemisinin is one such therapeutic intervention that has the potential to treat of diseases characterized by excessive trained immunity.

Rare Earth Extraction from Phosphogypsum by Aspergillus niger Culture Broth.

The extraction of rare earth elements (REEs) from phosphogypsum (PG) is of great significance for the effective utilization of rare earth resources and enhancing the resource value of PG waste residues. This study used Aspergillus niger (A. niger) fungal culture filtrate as a leaching agent to investigate the behavior of extracting REEs from PG through direct and indirect contact methods. According to the ICP-MS results, direct leaching at a temperature of 30 °C, shaking speed of 150 rpm, and a solid-liquid ratio of 2:1, achieved an extraction rate of 74% for REEs, with the main elements being yttrium (Y), lanthanum (La), cerium (Ce), and neodymium (Nd). Under the same conditions, the extraction rate of REEs from phosphogypsum using an A. niger culture filtrate was 63.3% higher than that using the simulated organic acid-mixed solution prepared with the main organic acid components in the A. niger leachate. Moreover, the morphological changes observed in A. niger before and after leaching further suggest the direct involvement of A. niger's metabolic process in the extraction of REEs. When compared to using organic acids, A. niger culture filtrate exhibits higher leaching efficiency for extracting REEs from PG. Additionally, using A. niger culture filtrate is a more environmentally friendly method with the potential for industrial-scale applications than using inorganic acids for the leaching of REEs from PG.

IFN-α armed gE elicits superior immunogenicity compared to unmodified antigens and flagellin armed gE in mice.

Herpes zoster (HZ) induces significant pain and discomfort, which can seriously affect the quality of life of patients. At present, there is no specific treatment for HZ, and the mosteffective HZ control is vaccination. The main obstacle to developing an effective HZ vaccine is poorly induced cellular immune response. In this study, the IFN-α-gE-Fc fusion protein induced higher levels of humoral and cellular immunity compared to the unengineered gE antigen and higher levels of cellular immunity compared to the flagellin-gE-Fc fusion protein in a murine model. Compared with the marketed recombinant herpes zoster vaccine (Shingrix), IFN-α-gE-Fc can replace current used MPL adjuvant. At the same time, the immunogenicity of the IFN-α-gE-Fc + AQ was not weaker than that of the marketed recombinant zoster vaccine. The novel fusion protein provides a candidate entity for the development of a safe and effective novel HZ vaccine.

Stromal interaction molecule 1/microtubule-associated protein 1A/1B-light chain 3B complex induces metastasis of hepatocellular carcinoma by promoting autophagy.

Metastasis is the leading cause of death in hepatocellular carcinoma (HCC) patients, and autophagy plays a crucial role in this process by orchestrating epithelial-mesenchymal transition (EMT). Stromal interaction molecule 1 (STIM1), a central regulator of store-operated calcium entry (SOCE) in nonexcitable cells, is involved in the development and spread of HCC. However, the impact of STIM1 on autophagy regulation during HCC metastasis remains unclear. Here, we demonstrate that STIM1 is temporally regulated during autophagy-induced EMT in HCC cells, and knocking out (KO) STIM1 significantly reduces both autophagy and EMT. Interestingly, STIM1 enhances autophagy through both SOCE-dependent and independent pathways. Mechanistically, STIM1 directly interacts with microtubule-associated protein 1A/1B-light chain 3B (LC3B) to form a complex via the sterile-α motif (SAM) domain, which promotes autophagosome formation. Furthermore, deletion of the SAM domain of STIM1 abolishes its binding with LC3B, leading to a decrease in autophagy and EMT in HCC cells. These findings unveil a novel mechanism by which the STIM1/LC3B complex mediates autophagy and EMT in HCC cells, highlighting a potential target for preventing HCC metastasis.

Trained immunity in recurrent Staphylococcus aureus infection promotes bacterial persistence.

Bacterial persister cells, a sub-population of dormant phenotypic variants highly tolerant to antibiotics, present a significant challenge for infection control. Investigating the mechanisms of antibiotic persistence is crucial for developing effective treatment strategies. Here, we found a significant association between tolerance frequency and previous infection history in bovine mastitis. Previous S. aureus infection led to S. aureus tolerance to killing by rifampicin in subsequent infection in vivo and in vitro. Actually, the activation of trained immunity contributed to rifampicin persistence of S. aureus in secondary infection, where it reduced the effectiveness of antibiotic treatment and increased disease severity. Mechanically, we found that S. aureus persistence was mediated by the accumulation of fumarate provoked by trained immunity. Combination therapy with metformin and rifampicin promoted eradication of persisters and improved the severity of recurrent S. aureus infection. These findings provide mechanistic insight into the relationship between trained immunity and S. aureus persistence, while providing proof of concept that trained immunity is a therapeutic target in recurrent bacterial infections involving persistent pathogens.

X-ray, digital tomographic fusion, CT, and MRI in early ischemic necrosis of the femoral head.

To investigate the imaging performance of radiography, digital tomographic fusion (DTS), computed tomography (CT), and magnetic resonance imaging (MRI) in the diagnosis of early avascular necrosis of the femoral head (ANFH). A total of 220 patients with ANFH who visited our hospital from January 2020 to January 2022 were included in the study. X-ray, DTS, CT, and MRI examinations of both hips were performed for all patients. The trabecular structure, bone density changes, femoral head morphology, and joint space changes were observed using the aforementioned imaging modalities. The staging was performed according to the Association Research Circulation Osseous (ARCO) criteria. The diagnostic detection rate of each imaging modality, and the sensitivity, specificity, positive predictive value, and negative predictive value of each examination for diagnosing early ANFH were calculated and compared. Patients were diagnosed with stage I (n = 65), stage II (n = 85), stage III (n = 32), and stage IV (n = 38) ANFH. For MRI, the detection rate (97.7%), sensitivity (94.7%), specificity (88.6%), positive predictive value (95.9%), and negative predictive value (92.5%), for diagnosing early ANFH, were significantly higher than those of other imaging methods (P < .05). MRI is the most accurate and sensitive imaging method for diagnosing early ANFH and has important clinical applications.

Retention time prediction and MRM validation reinforce the biomarker identification of LC-MS based phospholipidomics.

Dysfunctional lipid metabolism plays a crucial role in the development and progression of various diseases. Accurate measurement of lipidomes can help uncover the complex interactions between genes, proteins, and lipids in health and diseases. The prediction of retention time (RT) has become increasingly important in both targeted and untargeted metabolomics. However, the potential impact of RT prediction on targeted LC-MS based lipidomics is still not fully understood. Herein, we propose a simplified workflow for predicting RT in phospholipidomics. Our approach involves utilizing the fatty acyl chain length or carbon-carbon double bond (DB) number in combination with multiple reaction monitoring (MRM) validation. We found that our model's predictive capacity for RT was comparable to that of a publicly accessible program (QSRR Automator). Additionally, MRM validation helped in further mitigating the interference in signal recognition. Using this developed workflow, we conducted phospholipidomics of sorafenib resistant hepatocellular carcinoma (HCC) cell lines, namely MHCC97H and Hep3B. Our findings revealed an abundance of monounsaturated fatty acyl (MUFA) or polyunsaturated fatty acyl (PUFA) phospholipids in these cell lines after developing drug resistance. In both cell lines, a total of 29 lipids were found to be co-upregulated and 5 lipids were co-downregulated. Further validation was conducted on seven of the upregulated lipids using an independent dataset, which demonstrates the potential for translation of the established workflow or the lipid biomarkers.

Clinico-biological-radiomics (CBR) based machine learning for improving the diagnostic accuracy of FDG-PET false-positive lymph nodes in lung cancer.

BACKGROUND: The main problem of positron emission tomography/computed tomography (PET/CT) for lymph node (LN) staging is the high false positive rate (FPR). Thus, we aimed to explore a clinico-biological-radiomics (CBR) model via machine learning (ML) to reduce FPR and improve the accuracy for predicting the hypermetabolic mediastinal-hilar LNs status in lung cancer than conventional PET/CT. METHODS: A total of 260 lung cancer patients with hypermetabolic mediastinal-hilar LNs (SUVmax ≥ 2.5) were retrospectively reviewed. Patients were treated with surgery with systematic LN resection and pathologically divided into the LN negative (LN-) and positive (LN +) groups, and randomly assigned into the training (n = 182) and test (n = 78) sets. Preoperative CBR dataset containing 1738 multi-scale features was constructed for all patients. Prediction models for hypermetabolic LNs status were developed using the features selected by the supervised ML algorithms, and evaluated using the classical diagnostic indicators. Then, a nomogram was developed based on the model with the highest area under the curve (AUC) and the lowest FPR, and validated by the calibration plots. RESULTS: In total, 109 LN- and 151 LN + patients were enrolled in this study. 6 independent prediction models were developed to differentiate LN- from LN + patients using the selected features from clinico-biological-image dataset, radiomics dataset, and their combined CBR dataset, respectively. The DeLong test showed that the CBR Model containing all-scale features held the highest predictive efficiency and the lowest FPR among all of established models (p < 0.05) in both the training and test sets (AUCs of 0.90 and 0.89, FPRs of 12.82% and 6.45%, respectively) (p < 0.05). The quantitative nomogram based on CBR Model was validated to have a good consistency with actual observations. CONCLUSION: This study presents an integrated CBR nomogram that can further reduce the FPR and improve the accuracy of hypermetabolic mediastinal-hilar LNs evaluation than conventional PET/CT in lung cancer, thereby greatly reducing the risk of overestimation and assisting for precision treatment.

Fibrinogen-like protein 2 promotes tumor immune suppression by regulating cholesterol metabolism in myeloid-derived suppressor cells.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) are crucial mediators of tumor-associated immune suppression. Targeting the accumulation and activation of MDSCs has been recognized as a promising approach to enhance the effectiveness of immunotherapies for different types of cancer. METHODS: The MC38 and B16 tumor-bearing mouse models were established to investigate the role of Fgl2 during tumor progression. Fgl2 and FcγRIIB-deficient mice, adoptive cell transfer, RNA-sequencing and flow cytometry analysis were used to assess the role of Fgl2 on immunosuppressive activity and differentiation of MDSCs. RESULTS: Here, we show that fibrinogen-like protein 2 (Fgl2) regulates the differentiation and immunosuppressive functions of MDSCs. The absence of Fgl2 leads to an increase in antitumor CD8+ T-cell responses and a decrease in granulocytic MDSC accumulation. The regulation mechanism involves Fgl2 modulating cholesterol metabolism, which promotes the accumulation of MDSCs and immunosuppression through the production of reactive oxygen species and activation of XBP1 signaling. Inhibition of Fgl2 or cholesterol metabolism in MDSCs reduces their immunosuppressive activity and enhances differentiation. Targeting Fgl2 could potentially enhance the therapeutic efficacy of anti-PD-1 antibody in immunotherapy. CONCLUSION: These results suggest that Fgl2 plays a role in promoting immune suppression by modulating cholesterol metabolism and targeting Fgl2 combined with PD-1 checkpoint blockade provides a promising therapeutic strategy for antitumor therapy.

One-pot cascade synthesis of dibenzothiophene-based heterobiaryls from dibenzothiophene-5-oxide.

A sulfoxide directed C-H metalation/boration/B2Pin2 mediated reduction/Suzuki coupling process to synthesize 4-substituted dibenzothiophene (DBT) in one-pot from dibenzothiophene-5-oxide (DBTO) was developed. A variety of DBT-based heterobiaryls were prepared in satisfactory to good yields. A mechanism was proposed. The application of this methodology was demonstrated by synthesizing a luminescent material.

18F-FES and 18F-FDG PET/CT imaging as a predictive biomarkers for metastatic breast cancer patients undergoing cyclin-dependent 4/6 kinase inhibitors with endocrine treatment.

OBJECTIVE: The aim of this study was to investigate the potential value of dual tracers 18F-FDG and 18F-FES PET/CT in predicting response to Cyclin-Dependent 4/6 Kinase (CDK4/6) inhibitors combined with endocrine therapy for metastatic estrogen receptor (ER)-positive breast cancer patients. METHODS: This retrospective study enrolled 38 ER-positive metastatic breast cancer patients from our center who underwent both 18F-FDG and 18F-FES PET/CT scans within 1 month before CDK4/6 inhibitors combined with endocrine therapy. The extracted parameters comprised the maximum standardized uptake value (SUVmax) for both FDG and FES PET, as well as the ratio between FES and FDG SUVmax. Each parameter was dichotomized based on its median threshold. The primary endpoint was progression-free survival (PFS), which was estimated using the Kaplan-Meier method and compared by the log-rank test. RESULTS: After a median follow-up of 15.6 months, progressive disease was observed in 23 out of 38 patients, and the median PFS for the whole cohort was 21.0 months [95% confidence interval (CI) 12.7-29.3]. FES and FDG PET identified 6 patients (15.8%) with FES-negative lesions, suggesting ER heterogeneity in metastatic lesions. The median PFS of these patients was only 5.3 months (95% CI 1.7-8.9), which was substantially shorter than that of patients with 100% FES-positive lesions (median PFS 22.9 months, 95% CI 17.1-28.7, P < 0.001). Patients with 100% FES-positive lesions who had high FES/FDG showed significantly shorter PFS compared to those with low FES/FDG (14.9 vs. 30.5 months, P = 0.003). CONCLUSIONS: This study shows that FDG and FES PET imaging may serve as valuable tools for patient selection in the context of CDK4/6 inhibitor therapy combined with endocrine treatment, and have the potential to function as prognostic biomarkers.

Increased OIT3 in macrophages promotes PD-L1 expression and hepatocellular carcinogenesis via NF-κB signaling.

Oncoprotein-induced transcript 3 (OIT3) facilitates macrophage M2 polarization and hepatocellular carcinoma (HCC) progression, however, whether OIT3 regulates tumor immunity remains largely unknown. Here we found that OIT3 was upregulated in HCC-associated macrophages, which inhibited CD4+ and CD8+ T-cell infiltration in the tumor microenvironment (TME). Mechanistically, OIT3 increased the expression of PD-L1 on tumor-associated macrophages (TAMs) by activating NF-κB signaling, blockade of NF-κB reversed the immunosuppressive activity of TAMs and dampens HCC tumorigenesis. Our findings provide the molecular basis for OIT3 enhancing tumor immunosuppression and highlighted a potential therapeutic strategy for targeting the TAMs of HCC.

Equivalent carbon number-based targeted odd-chain fatty acyl lipidomics reveals triacylglycerol profiling in clinical colon cancer.

Odd-chain FAs (OCFAs) are present in very low level at nearly 1% of total FAs in human plasma, and thus, their functions were usually ignored. Recent epidemiological studies have shown that OCFAs are inversely associated with a variety of disease risks. However, the contribution of OCFAs incorporated into complex lipids remains elusive. Here, we developed a targeted odd-chain fatty acyl-containing lipidomics method based on equivalent carbon number and retention time prediction. The method displayed good reproducibility and robustness as shown by peak width at half height within 0.7 min and coefficient of variation under 20%. A total number of 776 lipid species with odd-chain fatty acyl residues could be detected in the ESI mode of reverse-phase LC-MS, of which 309 lipids were further validated using multiple reaction monitoring transitions. Using this method, we quantified odd-chain fatty acyl-containing lipidome in tissues from 12 colon cancer patients, revealing the remodeling of triacylglycerol. The dynamics of odd-chain fatty acyl lipids were further consolidated by the association with genomic and proteomic features of altered catabolism of branched-chain amino acids and triacylglycerol endogenous synthesis in colon cancer. This lipidomics approach will be applicable for screening of dysregulated odd-chain fatty acyl lipids, which enriches and improves the methods for diagnosis and prognosis evaluation of cancer using lipidomics.

Myeloid-derived suppressor cells deficient in cholesterol biosynthesis promote tumor immune evasion.

Cancer immunotherapy targeting myeloid-derived suppressor cells (MDSCs) is one of the most promising anticancer strategies. Metabolic reprogramming is vital for MDSC activation, however, the regulatory mechanisms of cholesterol metabolic reprogramming in MDSCs remains largely unexplored. Using the receptor-interacting protein kinase 3 (RIPK3)-deficient MDSC model, a previously established tumor-infiltrating MDSC-like model, we found that the cholesterol accumulation was significantly decreased in these cells. Moreover, the phosphorylated AKT-mTORC1 signaling was reduced, and downstream SREBP2-HMGCR-mediated cholesterol synthesis was blunted. Interestingly, cholesterol deficiency profoundly elevated the immunosuppressive activity of MDSCs. Mechanistically, cholesterol elimination induced nuclear accumulation of LXRß, thereby promoting LXRß-RXRα heterodimer binding of a novel composite element in the promoter of Arg1. Furthermore, itraconazole enhanced the immunosuppressive activity of MDSCs to boost tumor growth by suppressing the RIPK3-AKT-mTORC1 pathway and impeding cholesterol synthesis. Our findings demonstrate that RIPK3 deficiency leads to cholesterol abrogation in MDSCs, which facilitates tumor-infiltrating MDSC activation, and highlight the therapeutic potential of targeting cholesterol synthesis to overcome tumor immune evasion.

Impact of Sample Processing and Storage Conditions on RNA Quality of Fresh-Frozen Cancer Tissues.

Background: A biobank is a central resource that supports basic and clinical research. RNA quality of fresh-frozen tissue specimens in the biobank is highly associated with the success of downstream applications. Therefore, it is very important to evaluate the impact of tissue processing and storage conditions on RNA quality. Methods: A total of 238 surgically removed tissue specimens, including esophagus, lung, liver, stomach, colon, and rectal cancer, were used to evaluate RNA quality. Two tissue homogenization methods, manual and TissueLyser, were compared and the impacts of temperature fluctuation, tissue types, storage period, and clinicopathological parameters on RNA quality were analyzed. Results: RNA integrity was not influenced by tissue homogenization methods and tissue types. However, RNA integrity number (RIN) values were significantly correlated with temperature fluctuation. When the power of a -80°C freezer was cut off, RNA integrity of frozen tissues was not significantly affected until the temperature increased to 0°C. When the temperature rose to room temperature and remained for 4 hours, RNA integrity was almost completely destroyed. In addition, various cancer tissues with short-term storage at -80°C (<5 years) or high tumor differentiation had higher RINs. Conclusions: Tissue processing and storage conditions affected RNA quality of fresh-frozen cancer tissues. It is necessary to keep storage temperature stable and keep specimens at ultralow temperatures during homogenization. Also, for a biobank containing multiple types of cancer tissue samples, it is better to store them in liquid nitrogen if the storage duration is more than 5 years.

Culturing the Chicken Intestinal Microbiota and Potential Application as Probiotics Development.

Pure cultures of chicken intestinal microbial species may still be crucial and imperative to expound on the function of gut microbiota, and also contribute to the development of potential probiotics and novel bioactive metabolites from gut microbiota. In this study, we isolated and identified 507 chicken intestinal bacterial isolates, including 89 previously uncultured isolates. Among these, a total of 63 Lactobacillus strains, belonging to L. vaginalis, L. crispatus, L. gallinarum, L. reuteri, L. salivarius, and L. saerimneri, exhibited antibacterial activity against S. Pullorum. Acid tolerance tests showed Limosilactobacillus reuteri strain YPG14 (L. reuteri strain YPG14) has a particularly strong tolerance to acid. We further characterized other probiotic properties of L. reuteri strain YPG14. In simulated intestinal fluid, the growth of L. reuteri strain YPG14 remained stable after incubation for 4 h. The auto-aggregation test showed the auto-aggregation percentage of L. reuteri strain YPG14 was recorded as 15.0 ± 0.38%, 48.3 ± 2.51%, and 75.1 ± 4.44% at 3, 12, and 24 h, respectively. In addition, the mucin binding assay showed L. reuteri strain YPG14 exhibited 12.07 ± 0.02% adhesion to mucin. Antibiotic sensitivity testing showed that L. reuteri strain YPG14 was sensitive to the majority of the tested antibiotics. The anti-Salmonella Pullorum (S. Pullorum) infection effect in vivo revealed that the consumption of L. reuteri strain YPG14 could significantly improve body weight loss and survival rate of chicks infected by S. Pullorum; reduce the loads of S. Pullorum in the jejunum, liver, spleen, and feces; and alleviate the jejunum villi morphological structure damage, crypt loss, and inflammatory cell infiltration caused by S. Pullorum. Overall, this study may help us to understand the diversity of chicken intestinal microflora and provide some insights for potential probiotic development from gut microbiota and may find application in the poultry industry.