Metagenomic and transcriptomic analysis revealing the impact of oxytetracycline and ciprofloxacin on gut microbiota and gene expression in the Chinese giant salamander (Andrias davidianus).

Excessive antibiotic use has led to the spread of antibiotic resistance genes (ARGs), impacting gut microbiota and host health. However, the effects of antibiotics on amphibian populations remain unclear. We investigated the impact of oxytetracycline (OTC) and ciprofloxacin (CIP) on Chinese giant salamanders (Andrias davidianus), focusing on gut microbiota, ARGs, and gene expression by performing metagenome and transcriptome sequencing. A. davidianus were given OTC (20 or 40 mg/kg) or CIP (50 or 100 mg/kg) orally for 7 days. The results revealed that oral administration of OTC and CIP led to distinct changes in microbial composition and functional potential, with CIP treatment having a greater impact than OTC. Antibiotic treatment also influenced the abundance of ARGs, with an increase in fluoroquinolone and multi-drug resistance genes observed post-treatment. The construction of metagenome-assembled genomes (MAGs) accurately validated that CIP intervention enriched fish-associated potential pathogens Aeromonas hydrophila carrying an increased number of ARGs. Additionally, mobile genetic elements (MGEs), such as phages and plasmids, were implicated in the dissemination of ARGs. Transcriptomic analysis of the gut revealed significant alterations in gene expression, particularly in immune-related pathways, with differential effects observed between OTC and CIP treatments. Integration of metagenomic and transcriptomic data highlighted potential correlations between gut gene expression and microbial composition, suggesting complex interactions between the host gut and its gut microbiota in response to antibiotic exposure. These findings underscore the importance of understanding the impact of antibiotic intervention on the gut microbiome and host health in amphibians, particularly in the context of antibiotic resistance and immune function.

miRNA Expression Analysis of IPEC-J2 Cells Damaged by Soybean 7S Globulin Reveals ssc-miR-221-5p as the Factor Alleviating Cell Damage.

The most significant and sensitive antigen protein that causes diarrhea in weaned pigs is soybean 7S globulin. Therefore, identifying the primary target for minimizing intestinal damage brought on by soybean 7S globulin is crucial. MicroRNA (miRNA) is closely related to intestinal epithelium's homeostasis and integrity. However, the change of miRNAs' expression and the function of miRNAs in Soybean 7S globulin injured-IPEC-J2 cells are still unclear. In this study, the miRNAs' expression profile in soybean 7S globulin-treated IPEC-J2 cells was investigated. Fifteen miRNAs were expressed differently. The differentially expressed miRNA target genes are mainly concentrated in signal release, cell connectivity, transcriptional inhibition, and Hedgehog signaling pathway. Notably, we noticed that the most significantly decreased miRNA was ssc-miR-221-5p after soybean 7S globulin treatment. Therefore, we conducted a preliminary study on the mechanisms of ssc-miR-221-5p in soybean 7S globulin-injured IPEC-J2 cells. Our research indicated that ssc-miR-221-5p may inhibit ROS production to alleviate soybean 7S globulin-induced apoptosis and inflammation in IPEC-J2 cells, thus protecting the cellular mechanical barrier, increasing cell proliferation, and improving cell viability. This study provides a theoretical basis for the prevention and control of diarrhea of weaned piglets.

Formulation design and characterization of silymarin liposomes for enhanced antitumor activity.

Liposomes, a nanoscale carrier, plays an important role in the delivery of drug, affects the in vivo efficacy of drugs. In this paper, silymarin(SM)-loaded liposomes was optimized using the response surface method (RSM), with entrapment efficiency (EE%) as an index. The formulation was optimized as follow: lecithin (7.8mg/mL), SM/lecithin (1/26) and lecithin/cholesterol (10/1). The optimized SM liposomes had a high EE (96.58 ±3.06%), with a particle size of 290.3 ±10.5nm and a zeta potential of +22.98 ±1.73mV. In vitro release tests revealed that SM was released in a sustained-release manner, primarily via diffusion mechanism. In vitro cytotoxicity studies demonstrated that the prepared SM liposomes had stronger inhibitory effects than the model drug. Overall, these results indicate that this liposome system is suitable for intravenous delivery to enhance the antitumor effects of SM.

Chronic kidney disease and cognitive performance: NHANES 2011-2014.

PURPOSE: Previous studies suggest an association between chronic kidney disease (CKD) and cognitive impairment. The purpose of this study was to explore the association between the diverse stages of CKD and the cognitive performance of elderly American adults. METHODS: Data from the National Health and Nutrition Examination Survey (NHANES) 2011-2014 were used. Multivariate adjusted logistic regression, subgroup analysis, and the restricted cubic spline model were used to assess the associations of CKD stage and estimated glomerular filtration rate (eGFR) with cognitive performance. The measures used to evaluate cognitive function included the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) test, the Animal Fluency test, and the Digit Symbol Substitution test (DSST). RESULTS: This study included 2234 participants aged ≥ 60 years. According to the fully adjusted model, stages 3-5 CKD were significantly associated with the CERAD test score (OR = 0.70, 95% CI [0.51, 0.97], p = 0.033), the Animal Fluency test score (OR = 0.64, 95% CI [0.48, 0.85], p = 0.005), and the DSST score (OR = 0.60, 95% CI [0.41, 0.88], p = 0.013). In addition, the incidence of poor cognitive function increased with decreasing eGFR, especially for individuals with low and moderate eGFRs. Both the DSST score (p nonlinearity < 0.0001) and the Animal Fluency test score (p nonlinearity = 0.0001) had nonlinear dose-response relationships with the eGFR. However, a linear relationship was shown between the eGFR and CERAD test score (p nonlinearity = 0.073). CONCLUSIONS: CKD, especially stages3-5 CKD, was significantly associated with poor cognitive performance in terms of executive function, learning, processing speed, concentration, and working memory ability. All adults with CKD should be screened for cognitive impairment.

Subgroups of High-cost Patients and Their Preventable Inpatient Cost in Rural China.

BACKGROUND: High-cost patients account for most healthcare costs and are highly heterogeneous. This study aims to classify high-cost patients into clinically homogeneous subgroups, describe healthcare utilization patterns of subgroups, and identify subgroups with relatively high preventable inpatient cost (PIC) in rural China. METHODS: A population-based retrospective study was performed using claims data in Xi County, Henan Province. 32,108 high-cost patients, representing the top 10% of individuals with the highest total spending, were identified. A density-based clustering algorithm combined with expert opinions were used to group high-cost patients. Healthcare utilization (including admissions, length of stay and outpatient visits) and spending characteristics (including total spending, and the proportion of PIC, inpatient and out-of-pocket spending on total spending) were described among subgroups. PIC was calculated based on potentially preventable hospitalizations which were identified according to the Agency for Healthcare Research and Quality Prevention Quality Indicators algorithm. RESULTS: High-cost patients were more likely to be older (M=51.87, SD=22.28), male (49.03%) and from poverty-stricken families (37.67%) than non-high-cost patients, with 2.49 (SD=2.47) admissions and 3.25 (SD=4.52) outpatient visits annually. Fourteen subgroups of high-cost patients were identified: chronic disease, non-trauma diseases which need surgery, female disease, cancer, eye disease, respiratory infection/inflammation, skin disease, fracture, liver disease, vertigo syndrome and cerebral infarction, mental disease, arthritis, renal failure, other neurological disorders. The annual admissions ranged from 1.83 (SD=1.23, fracture) to 12.21 (SD=9.26, renal failure), and the average length of stay ranged from 6.61 (SD=10.00, eye disease) to 32.11 (SD=28.78, mental disease) days among subgroups. The chronic disease subgroup showed the largest proportion of PIC on total spending (10.57%). CONCLUSION: High-cost patients were classified into 14 clinically distinct subgroups which had different healthcare utilization and spending characteristics. Different targeted strategies may be needed for subgroups to reduce preventable hospitalizations. Priority should be given to high-cost patients with chronic diseases.

Behavioral carry-over effect and power consideration in crossover trials.

A crossover trial is an efficient trial design when there is no carry-over effect. To reduce the impact of the biological carry-over effect, a washout period is often designed. However, the carry-over effect remains an outstanding concern when a washout period is unethical or cannot sufficiently diminish the impact of the carry-over effect. The latter can occur in comparative effectiveness research, where the carry-over effect is often non-biological but behavioral. In this paper, we investigate the crossover design under a potential outcomes framework with and without the carry-over effect. We find that when the carry-over effect exists and satisfies a sign condition, the basic estimator underestimates the treatment effect, which does not inflate the type I error of one-sided tests but negatively impacts the power. This leads to a power trade-off between the crossover design and the parallel-group design, and we derive the condition under which the crossover design does not lead to type I error inflation and is still more powerful than the parallel-group design. We also develop covariate adjustment methods for crossover trials. We evaluate the performance of cross-over design and covariate adjustment using data from the MTN-034/REACH study.

Genus Shewanella: A Potential Intestinal Colonizer Associated With Post-Operative Surgical Site Infections in Coastal Regions.

Background: This study aims to elucidate the clinical characteristics of Shewanella-related surgical site infections (SSIs) and assess the risk of mortality in patients by establishing a predictive model. Patients and Methods: A retrospective analysis of medical history and laboratory data of Shewanella-related SSI patients over the past decade was conducted via the electronic medical record (EMR) system. A predictive model for mortality risk in Shewanella-related SSI patients was established using plasma interleukin-6 (IL-6) levels combined with the Howell-PIRO scoring system. Results: Over the past 10 years, 45 strains of Shewanella were isolated from specimens such as bile, drainage fluid, and whole blood in patients with digestive tract SSIs. Among them, 21 of 45 (46.67%) patients underwent malignant tumor resection of the digestive system, 14 of 45 (31.11%) underwent endoscopic retrograde cholangiopancreatography (ERCP) common bile duct exploration or the stone removal, and seven of 45 (15.56%) were trauma repair patients with fractures and abdominal injuries. Among the 45 Shewanella-related SSI patients, 10 died within 30 days of infection, six cases involved infections with more than two other types of bacteria. The combined use of IL-6 and Howell-PIRO scores for mortality risk assessment yielded an receiver operating characteristic (ROC) curve with an area under the curve (AUC) of 0.9350, a positive predictive value of 92.71%, a negative predictive value of 94.58%, a diagnostic sensitivity of 95.35%, and a diagnostic specificity of 92.14%-all higher than the model using IL-6 or Howell-PIRO scores alone. Conclusions: We found that residents in coastal areas faced an increased risk of Shewanella-related SSI. Moreover, the higher the number of concurrent microbial infections occurring alongside Shewanella-related SSI, the greater the mortality rate among patients. The combined application of plasma IL-6 levels and the Howell-PIRO scoring system is beneficial for assessing patient mortality risk and guiding timely and proactive clinical interventions.

Use of tumor markers in distinguishing lung adenocarcinoma-associated malignant pleural effusion from tuberculous pleural effusion.

BACKGROUND: The distinction between lung adenocarcinoma-associated malignant pleural effusion (MPE) and tuberculous pleural effusion (TPE) continues to pose a challenge. This study sought to assess the supplementary value of tumor markers in enabling a differential diagnosis. METHODS: Data concerning tumor markers, which included carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), cancer antigen 153 (CA153), cancer antigen 724 (CA724), neuron-specific enolase (NSE), cytokeratin19 fragment (Cyfra21-1), and squamous cell carcinoma antigen (SCCA), in both serum and pleural effusion samples, were retrospectively compiled from lung adenocarcinoma-associated MPE and TPE patients. A comparative analysis of tumor marker concentrations between the two groups was performed to assess diagnostic utility, followed by a multiple logistic regression to control for confounding variables. RESULTS: While gender, serum CA125 and SCCA, and pleural effusion SCCA manifested comparability between the groups, distinctions were noted in patient age and the concentration of other tumor markers in serum and pleural effusion, which were notably elevated in the MPE group. Multiple logistic regression demonstrated a positive association between the risk of lung adenocarcinoma-associated MPE and levels of CEA and CA153 in serum and pleural effusion, as well as Cyfra21-1 in serum (P < 0.05). The odds ratio for CEA surpassed that of CA153 and Cyfra21-1. CONCLUSIONS: CEA and CA153 in serum and pleural effusion, and Cyfra21-1 in serum emerge as biomarkers possessing supplementary diagnostic value in distinguishing lung adenocarcinoma-associated MPE from TPE. The diagnostic efficacy of CEA is superior to CA153 and Cyfra21-1. Conversely, the utility of CA125, CA724, NSE, and SCCA appears constrained.

Methylation patterns associated with C-reactive protein in racially and ethnically diverse populations.

Systemic low-grade inflammation is a feature of chronic disease. C-reactive protein (CRP) is a common biomarker of inflammation and used as an indicator of disease risk; however, the role of inflammation in disease is not completely understood. Methylation is an epigenetic modification in the DNA which plays a pivotal role in gene expression. In this study we evaluated differential DNA methylation patterns associated with blood CRP level to elucidate biological pathways and genetic regulatory mechanisms to improve the understanding of chronic inflammation. The racially and ethnically diverse participants in this study were included as 50% White, 41% Black or African American, 7% Hispanic or Latino/a, and 2% Native Hawaiian, Asian American, American Indian, or Alaska Native (total n = 13,433) individuals. We replicated 113 CpG sites from 87 unique loci, of which five were novel (CADM3, NALCN, NLRC5, ZNF792, and cg03282312), across a discovery set of 1,150 CpG sites associated with CRP level (p < 1.2E-7). The downstream pathways affected by DNA methylation included the identification of IFI16 and IRF7 CpG-gene transcript pairs which contributed to the innate immune response gene enrichment pathway along with NLRC5, NOD2, and AIM2. Gene enrichment analysis also identified the nuclear factor-kappaB transcription pathway. Using two-sample Mendelian randomization (MR) we inferred methylation at three CpG sites as causal for CRP levels using both White and Black or African American MR instrument variables. Overall, we identified novel CpG sites and gene transcripts that could be valuable in understanding the specific cellular processes and pathogenic mechanisms involved in inflammation.

Spatiotemporal dual-periodic soliton pulsation in a multimode fiber laser.

Spatiotemporal mode-locked (STML) fiber lasers have become a new platform for investigating nonlinear phenomena. In this work, spatiotemporal dual-periodic soliton pulsation (SDSP) is firstly observed in an STML fiber laser. It is found that in the SDSP, the long-period pulsations (LPPs) of different transverse modes are synchronous, while the short-period pulsations (SPPs) exhibit asynchronous modulations. The numerical simulation confirms the experimental results and further reveals that the proportion of transverse mode components can manipulate the periods of the LPP and SPP but does not affect the synchronous and asynchronous pulsations of different transverse modes. The obtained results bring the study of spatiotemporal dissipative soliton pulsation into the multi-period modulation stage, which helps to understand the complex spatiotemporal dynamics in STML fiber lasers and discover new dynamics in high-dimensional nonlinear systems.

Cognitive and emotional impairment in stroke survivors: insights from a multi-center study on inpatient rehabilitation therapy.

BACKGROUND: Individuals recovering from stroke often experience cognitive and emotional impairments, but rehab programs tend to focus on motor skills. The aim of this investigation is to systematically assess the change of magnitude of cognitive and emotional function subsequent to a conventional rehabilitative protocol administered to stroke survivors within a defined locale in China. METHODS: This is a multicenter study; a total of 1884 stroke survivors who received in-hospital rehabilitation therapy were assessed on admission (T0) and discharge (T1). The tool of InterRAI was used to assess cognitive, emotional, and behavioral abnormality. RESULTS: The patients aged >60 years, with a history of hypertension, and long stroke onset duration were more exposed to functional impairment (all p < 0.05). Both cognitive and emotional sections were significantly improved at T1 compared to T0 (p < 0.001). Initially, 64.97% and 46.55% of patients had cognitive or emotional impairment at T0, respectively; this percentage was 58.55% and 37.15% at T1. CONCLUSION: Many stroke survivors have ongoing cognitive and emotional problems that require attention. It is essential to focus on rehabilitating these areas during the hospital stay, especially for older patients, those with a longer recovery, and those with hypertension history.

Delipid extracorporeal lipoprotein filter from plasma system: a new intensive lipid lowering therapy for patients with acute ischemic stroke.

Objectives: To investigate the safety and efficacy of the delipid extracorporeal lipoprotein filter from plasma (DELP) system, a new low-density lipoprotein cholesterol (LDL-C) adsorption system, in acute ischemic stroke (AIS) patients. Patients and methods: In the present study, a total of 180 AIS patients were enrolled during March 2019 to February 2021. They were divided into DELP group (n1 = 90) and the control group (n2 = 90). The treatment protocol and vascular access of DELP treatment was established and evaluated. For the DELP group, clinical data and laboratory results including plasma lipid and safety parameters before and after the apheresis were collected and analyzed. For all participants, neurological scores were assessed and recorded. Results: For the DELP group, 90 patients including 70 males and 20 females were included. The mean LDL-C was significantly decreased from 3.15 ± 0.80 mmol/L to 2.18 ± 0.63 mmol/L (30.79%, p < 0.001) during a single DELP treatment, and decreased from 3.42 ± 0.87 mmol/L to 1.87 ± 0.48 mmol/L (45.32%, p < 0.001) after two DELP treatments. No clinically relevant changes were observed in hematologic safety parameters and blood pressure levels except for hematocrit and total protein throughout the whole period of DELP treatment. The DELP group showed improvement relative to the control group in National Institute of Health stroke scale scores (NIHSS) on the 14th and 90th day after stroke. Moreover, the DELP group had a significantly higher ratio of mRS 0 to 1 on the 90th day after stroke. Conclusion: The new LDL-C adsorption system, the DELP system, may provide a new option for intensive lipid lowering therapy in AIS patients in view of its safety, efficacy, and operation feasibility.

Circulation immune cell landscape in canonical pathogenesis of colorectal adenocarcinoma by CyTOF analysis.

Current studies on the immune microenvironment of colorectal cancer (CRC) were mostly limited to the tissue level, lacking relevant studies in the peripheral blood, and failed to describe its alterations in the whole process of adenocarcinoma formation, especially of adenoma carcinogenesis. Here, we constructed a large-scale population cohort and used the CyTOF to explore the changes of various immune cell subsets in peripheral blood of CRC. We found monocytes and basophils cells were significantly higher in adenocarcinoma patients. Compared with early-stage CRC, effector CD4+T cells and naive B cells were higher in patients with lymph node metastasis, whereas the basophils were lower. We also performed random forest algorithm and found monocytes play the key role in carcinogenesis. Our study draws a peripheral blood immune cell landscape of the occurrence and development of CRC at the single-cell level and provides a reference for other researchers.

Lactiplantibacillus plantarum postbiotic protects against Salmonella infection in broilers via modulating NLRP3 inflammasome and gut microbiota.

Salmonella infection is a major concern in poultry production which poses potential risks to food safety. Our previous study confirmed that Lactiplantibacillus plantarum (LP) postbiotic exhibited a strong antibacterial capacity on Salmonella in vitro. This study aimed to investigate the beneficial effects and underlying mechanism of LP postbiotic on Salmonella-challenged broilers. A total of 240 one-day-old male yellow-feathered broilers were pretreated with 0.8% deMan Rogosa Sharpe (MRS) medium or 0.8% LP postbiotic (LP cell-free culture supernatant, LPC) in drinking water for 28 d, and then challenged with 1×109 CFU Salmonella enterica serovar Enteritidis (SE). Birds were sacrificed 3 d postinfection. Results showed that LPC maintained the growth performance by increasing body weight (BW), average daily gain (ADG), and average daily feed intake (ADFI) in broilers under SE challenge. LPC significantly attenuated SE-induced intestinal mucosal damage. Specifically, it decreased the intestinal injury score, increased villus length and villus/crypt, regulated the expression of intestinal injury-related genes (Villin, matrix metallopeptidase 3 [MMP3], intestinal fatty acid-binding protein [I-FABP]), and enhanced tight junctions (zona occludens-1 [ZO-1] and Claudin-1). SE infection caused a dramatic inflammatory response, as indicated by the up-regulated concentrations of interleukin (IL)-1ß, IL-6, TNF-α, and the downregulation of IL-10, while LPC pretreatment markedly reversed this trend. We then found that LPC inhibited the activation of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome by decreasing the gene expression of Caspase-1, IL-lß, and IL-18. Furthermore, LPC suppressed NLRP3 inflammasome activation by inhibiting nuclear factor-kappa B (NF-κB) signaling pathway (the reduced levels of toll-like receptor 4 [TLR4], myeloid differentiation factor 88 [MyD88], and NF-κB). Finally, our results showed that LPC regulated gut microbiota by enhancing the percentage of Ligilactobacillus and decreasing Alistipes and Barnesiella. In summary, we found that LP postbiotic was effective to protect broilers against Salmonella infection, possibly through suppressing NLRP3 inflammasome and optimizing gut microbiota. Our study provides the potential of postbiotics on prevention of Salmonella infection in poultry.

Klebsiella pneumoniae K2 capsular polysaccharide degradation by a bacteriophage depolymerase does not require trimer formation.

K2-capsular Klebsiella pneumoniae is a hypervirulent pathogen that causes fatal infections. Here, we describe a phage tailspike protein, named K2-2, that specifically depolymerizes the K2 capsular polysaccharide (CPS) of K. pneumoniae into tetrasaccharide repeating units. Nearly half of the products contained O-acetylation, which was thought crucial to the immunogenicity of CPS. The product-bound structures of this trimeric enzyme revealed intersubunit carbohydrate-binding grooves, each accommodating three tetrasaccharide units of K2 CPS. The catalytic residues and the key interactions responsible for K2 CPS recognition were identified and verified by site-directed mutagenesis. Further biophysical and functional characterization, along with the structure of a tetrameric form of K2-2, demonstrated that the formation of intersubunit catalytic center does not require trimerization, which could be nearly completely disrupted by a single-residue mutation in the C-terminal domain. Our findings regarding the assembly and catalysis of K2-2 provide cues for the development of glycoconjugate vaccines against K. pneumoniae infection. IMPORTANCE: Generating fragments of capsular polysaccharides from pathogenic bacteria with crucial antigenic determinants for vaccine development continues to pose challenges. The significance of the C-terminal region of phage tailspike protein (TSP) in relation to its folding and trimer formation remains largely unexplored. The polysaccharide depolymerase described here demonstrates the ability to depolymerize the K2 CPS of K. pneumoniae into tetrasaccharide fragments while retaining the vital O-acetylation modification crucial for immunogenicity. By carefully characterizing the enzyme, elucidating its three-dimensional structures, conducting site-directed mutagenesis, and assessing the antimicrobial efficacy of the mutant enzymes against K2 K. pneumoniae, we offer valuable insights into the mechanism by which this enzyme recognizes and depolymerizes the K2 CPS. Our findings, particularly the discovery that trimer formation is not required for depolymerizing activity, challenge the current understanding of trimer-dependent TSP activity and highlight the catalytic mechanism of the TSP with an intersubunit catalytic center.

Prediction of HER2 Expression in Gastric Adenocarcinoma Based On Preoperative Noninvasive Multimodal 18F-FDG PET/CT Imaging.

RATIONALE AND OBJECTIVES: This study aims to investigate the role of a flourine-18 fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) multimodal radiomics model in predicting the status of human epidermal growth factor receptor 2 (HER2) expression preoperatively in cases of gastric adenocarcinoma. MATERIALS AND METHODS: This retrospective study included 133 patients with gastric adenocarcinoma who were classified into training (n = 93) and validation (n = 40) cohorts in a ratio of 7:3. Features were selected using Least Absolute Shrinkage and Selection Operator and Extreme Gradient Boosting (XGBoost) methods; further, prediction models were constructed using logistic regression and XGBoost. These models were evaluated and validated using area under the curve (AUC), decision curves, and calibration curves to select the best-performing model. RESULTS: Six different models were established to predict HER2 expression. Among these, the comprehensive model, which integrates seven clinical features, one CT feature, and five PET features, demonstrated AUC values of 0.95 (95% confidence interval [CI]: 0.89-1.00) and 0.76 (95% CI: 0.52-1.00) in the training and validation cohorts, respectively. Compared with other models, this model exhibited a superior net benefit on the decision curve and demonstrated good alignment agreement with the observed values on the calibration curve. Based on these findings, we constructed a nomogram for visualizing the model, providing a noninvasive preoperative method for predicting HER2 expression. CONCLUSION: The preoperative 18F-FDG PET/CT multimodal radiomics model can effectively predict HER2 expression in patients with gastric adenocarcinoma, thereby guiding clinical decision-making and advancing the field of precision medicine.

Optimization of FFPE preparation and identification of gene attributes associated with RNA degradation.

Formalin-fixed paraffin-embedded (FFPE) tissues are widely available specimens for clinical studies. However, RNA degradation in FFPE tissues often restricts their utility. In this study, we determined optimal FFPE preparation conditions, including tissue ischemia at 4°C (<48 h) or 25°C for a short time (0.5 h), 48-h fixation at 25°C and sampling from FFPE scrolls instead of sections. Notably, we observed an increase in intronic reads and a significant change in gene rank based on expression level in the FFPE as opposed to fresh-frozen (FF) samples. Additionally, we found that more reads were mapped to genes associated with chemical stimulus in FFPE samples. Furthermore, we demonstrated that more degraded genes in FFPE samples were enriched in genes with short transcripts and high free energy. Besides, we found 40 housekeeping genes exhibited stable expression in FF and FFPE samples across various tissues. Moreover, our study showed that FFPE samples yielded comparable results to FF samples in dimensionality reduction and pathway analyses between case and control samples. Our study established the optimal conditions for FFPE preparation and identified gene attributes associated with degradation, which would provide useful clues for the utility of FFPE tissues in clinical practice and research.