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BackgroundHypervirulent Klebsiella pneumoniae (hvKp) is a critical pathogen that causes highly lethal invasive infections with its virulence linked to that of aerobactin, the core siderophore system of hvKp.MethodsHere, 20 porcine-derived K. pneumoniae strains were subjected to capsular genotyping and the virulence of the strains was evaluated through mouse lethality assays. Whole-genome sequencing and analysis was performed on two K2 serotype strains (including highly and weakly virulent strains), and the differential virulence genes were analyzed. The virulence-related gene iucC, identified in the hypervirulent K2 serotype strain KP10, was knocked out using homologous recombination to preliminarily explore its function in K. pneumoniae.ResultsIn this study, a highly virulent K2-type strain KP10 and a low virulent K2-type strain KP6 were screened from 20 strains of porcine K. pneumoniae. The results of the genome and virulence gene analyses revealed that compared with KP6, the highly virulent strain KP10 specifically encodes the iucC gene. The △ iucC mutant of the high-virulence strain KP10 exhibited significantly reduced biofilm formation, decreased siderophore production, moderate serum sensitivity, and attenuated virulence. Furthermore, the mutant displayed decreased adhesion to IPEC-J2 cells, reduced cytotoxicity, and decreased cell mortality. These results revealed that iucC is a major contributor to virulence in the KP10 hvKp strain. Notably, iucC genes were detected in the genomes of K. pneumoniae from humans and six different animal and environmental sources, and some iucC genes were shared in K. pneumoniae from these sources.ConclusionOverall, this work provides a crucial theoretical foundation for elucidating iucC function and the pathogenesis of K. pneumoniae. Its widespread presence in different hosts and environments considerably increases the risk of cross species transmission and public health events.

IntroductionStaphylococcus aureus (S. aureus) is one of the most important zoonotic pathogens and can be transmitted to humans through the meat diet routes, causing necrotising pneumonia.MethodsThis study investigated the therapeutic effect of bacteriophage lysin LysGH15 on necrotising pneumonia in rabbit model caused by S. aureus.ResultsIn the in vitro experiments, 50 μg/mL LysGH15 not only significantly reduced the viable count (approximately 3.24 × 106 CFU/g) of chicken meat stored at 4°C for 48 h but also effectively reduced the viable count of chicken meat thawed at 4°C and 30°C, with reductions of approximately 1.42 × 106 CFU/g and 2.78 × 106 CFU/g, respectively. In the in vivo experiments, a single intranasal administration of 300 μg/rabbit increased the survival rate of rabbits to 60%. At 72 h postinfection, the number of bacteria in the lung tissues of the rabbits treated with LysGH15 was 7 × 104 CFU/g, which was significantly lower than that in the lung tissues of rabbits treated with PBS (7.76 × 106 CFU/g) or linezolid (6.38 × 105 CFU/g). In addition, LysGH15 treatment alleviated lung tissue damage in infected rabbits and significantly reduced the levels of Panton-Valentine leukocidin (PVL), alpha-toxin (Hla), and the cytokines IFN-γ, TNF-α, and IL-8 in their lung tissues, similar to those in rabbits treated with linezolid.DiscussionThese results suggest that LysGH15 has the potential to be used as a novel antimicrobial agent for the treatment of necrotising pneumonia caused by S. aureus.

Food contamination with Clostridium perfringens (C. perfringens) and Streptococcus suis (S. suis) may lead to severe foodborne illnesses in humans, and new effective strategies are needed to limit foodborne microbial contamination. In this study, a novel C. perfringens phage, XBG01, was isolated, and its lysin, Lys19, was characterized. Lys19 consists of an N-terminal N-acetylmuramoyl-L-alanine amidase catalytic domain and a C-terminal binding domain comprising two 35-residue repeats. H13, E26, H81, R124, and E146 were identified as key sites for maintaining the activity of the catalytic domain. The activity of the catalytic domain is independent of the presence of metal ions and is not affected by the binding domain. Notably, in addition to type A C. perfringens, Lys19 can also efficiently lyse 2, 3, 7, 9 and 12 serotypes of S. suis. This is the first reported C. perfringens phage lysin with a cross-bacterial species lytic function. It maintains stable activity in the temperature range of 4-55 °C and pH range of 3-9, and can effectively prevent and eliminate strong biofilms formed by C. perfringens. The bacterial viability on lettuce was significantly lower in the group treated with lysin Lys19 than in the PBS group, and 88.75 % of C. perfringens were killed within 15 min. In addition, Lys19 treatment significantly reduced the bacterial load in raw pork by about 84.65 % at a temperature of 4 °C within 12 h. These results indicate that Lys19 has potential applications in controlling C. perfringens and S. suis in lettuce and meat.

OBJECTIVE:In chronic obstructive pulmonary disease (COPD) patients, Candida colonization in the lower respiratory tract is associated with long-term prognosis and acute exacerbations, though the underlying mechanisms remain unclear. This study explores the impact of Candida.albicans (C.albicans) airway colonization on macrophages and airway inflammation in mice with emphysema. METHODS:A chronic emphysema mouse model was established using cigarette smoke and porcine pancreatic elastase(PPE) exposure. Mice were then instilled with C. albicans spore suspension (5 × 105 CFU/mouse) to induce airway colonization. The fungal burden in lung homogenates and the body weight change of mice were measured at 24h, 72h, and 120h post installation. At 72h, the fungal load, alveolar macrophages(AMs) polarization, reactive oxygen species(ROS) content, and NLRP3 and IL-1β expression in the airways or lungs were assessed. In vitro, THP-1-derived macrophages were treated with cigarette smoke extract (CSE), with or without antioxidant inhibitor NAC or NLRP3 inhibitor MCC950 pretreatment, followed by the stimulation of heat-killed C. albicans (HKCA) spores. The ROS production, NLRP3, and IL-1β protein expression in cells were detected. RESULTS:After instilling C. albicans into the airways, the body weight changes of the mice were less than 20 % at 24h, 72h, and 120h. Fungal spores are mainly concentrated in the airways, and no spores or hyphae were detected in the alveoli or lung interstitium. The fungal load decreased by 2.5 log units at 72h. C. albicans colonization enhanced CD86, CD206, and ROS fluorescence in alveolar macrophages, increased IL-1β levels in bronchoalveolar lavage fluid (BALF), and upregulated NLRP3 and IL-1β transcription in lung tissue. In THP-1-derived macrophages, HKCA following CSE stimulation increased CD86, CD206, ROS, NLRP3, and IL-1β levels. ROS inhibition with NAC or NLRP3 inhibition with MCC950 downregulated NLRP3 and IL-1β production. CONCLUSION:C. albicans airway colonization promotes macrophage polarization and intensifies inflammation, especially in emphysema mice. ROS production in macrophages may activate NLRP3, leading to increased IL-1β release, which could be a potential mechanism for exacerbating inflammation.

B. longum BL300 can protect against colitis by activating IgA production immune network to reshape gut microbiota, which could further maintain the integrity of the intestinal mechanical barrier and mucus layer.

The restrictions on antibiotic growth promoters in poultry production make the exploration of safe and effective alternatives necessary. Astragalus polysaccharides (APS), as bioactive macromolecules, have attracted significant interest from poultry scientists. In the current study, a multilevel meta-analysis was conducted to quantitatively evaluate the effects of APS administration on broiler performance, antioxidant status, and gut health. The results revealed that APS supplementation significantly improved average daily gain (ADG; SMD = 0.64 [0.13, 1.15]) and feed conversion ratio (FCR; SMD = -1.20 [-2.03, -0.35]) of broilers. The observed benefits directly correlated with increased superoxide dismutase activity (SOD; SMD = 1.35 [0.78, 1.92]) and decreased malondialdehyde levels (MDA; SMD = -1.07 [-1.54, -0.60]). Subgroup analysis demonstrated that dietary supplementation was more effective than injection for improving ADG and the villus height to crypt depth ratio (VCratio), indicating the importance of direct gut-level interaction of APS. Nonlinear dose-response modelling identified an optimal dietary APS range of 1000-1500 mg/kg for maximising growth and antioxidant parameters. This meta-analysis suggests that APS shows promise as an antibiotic alternative for improving broiler growth performance, warranting further direct comparative studies with antibiotics. In addition, further large-scale trials are required to confirm its consistent efficacy on gut health and microbial composition due to substantial heterogeneity in current datasets.

Pseudouridine is an important modification site, which is widely present in a variety of non-coding RNAs and is involved in a variety of important biological processes. Studies have shown that pseudouridine is important in many biological functions such as gene expression, RNA structural stability, and various diseases. Therefore, accurate identification of pseudouridine sites can effectively explain the functional mechanism of this modification site. Due to the rapid increase of genomics data, traditional biological experimental methods to identify RNA modification sites can no longer meet the practical needs, and it is necessary to accurately identify pseudouridine sites from high-throughput RNA sequence data by computational methods. In this study, we propose a deep learning-based computational method, Definer, to accurately identify RNA pseudouridine loci in three species, Homo sapiens, Saccharomyces cerevisiae and Mus musculus. The method incorporates two sequence coding schemes, including NCP and One-hot, and then feeds the extracted RNA sequence features into a deep learning model constructed from CNN, GRU and Attention. The benchmark dataset contains data from three species, H. sapiens, S. cerevisiae and M. musculus, and the results using 10-fold cross-validation show that Definer significantly outperforms other existing methods. Meanwhile, the data sets of two species, H. sapiens and S. cerevisiae, were tested independently to further demonstrate the predictive ability of the model. In summary, our method, Definer, can accurately identify pseudouridine modification sites in RNA.

IntroductionKlebsiella pneumoniae (K. pneumoniae) is an important opportunistic and zoonotic pathogen which is associated with many diseases in humans and animals. However, the pathogenicity of K. pneumoniae has been neglected and the prevalence of K. pneumoniae is poorly studied due to the lack of rapid and sensitive diagnosis techniques.MethodsIn this study, we infected mice and pigs with K. pneumoniae strain from a human patient. An indirect ELISA was established using the KHE protein as the coating protein for the detection of K. pneumoniae specific antibody in clinical samples. A nested PCR method to detect nuclei acids of K. pneumoniae was also developed.ResultsWe showed that infection with K. pneumoniae strain from a human patient led to mild lung injury of pigs. For the ELISA, the optimal coating concentration of KHE protein was 10 µg/mL. The optimal dilutions of serum samples and secondary antibody were 1:100 and 1:2500, respectively. The analytical sensitivity was 1:800, with no cross-reaction between the coated antigen and porcine serum positive for antibodies against other bacteria. The intra-assay and inter-assay reproducibility coefficients of variation are less than 10%. Detection of 920 clinical porcine serum samples revealed a high K. pneumoniae infection rate by established indirect ELISA (27.28%) and nested PCR (19.13%). Moreover, correlation analysis demonstrated infection rate is positively correlated with gross population, Gross Domestic Product (GDP), and domestic tourists.DiscussionIn conclusion, K. pneumoniae is highly prevalent among pigs in China. Our study highlights the role of K. pneumoniae in pig health, which provides a reference for the prevention and control of diseases associated with K. pneumoniae.

Abstract Background Immunogenic cell death (ICD) is defined as cell death sufficient to activate an adaptive immune response in an immunocompetent host. Cuproptosis is induced by increased intracellular toxicity due to copper ion accumulation. ICD and cuproptosis have great potential in regulating the growth and proliferation of cancer cells. Methods We used transcriptome data from TCGA database to identify two subtypes based on consensus clustering and explored the differences between the two groups. We further combined ICD- and cuproptosis-related genes to develop a signature and predict OS, immune status, and treatment responses in HCC patients. An ICGC cohort was used for external validation. Results Patients in the groups with high expression of ICD- and cuproptosis-related genes had higher levels of immune cell infiltration and a better immunotherapeutic response. Eight related genes (BAX, HSP90AA1, IFNB1, LY96, NT5E, LIPT1, DLAT, and CDKN2A) were screened for the prognostic signature. The survival analysis revealed that patients in the low-risk group were less likely to die at an early stage than were those in the high-risk group. The signature also showed better results in predicting immune cell infiltration and the treatment response. Conclusions Our study revealed that the prognostic signature constructed by combining ICD- and cuproptosis-related genes performs well in predicting the survival of patients with HCC and is closely associated with immune cell infiltration and immunotherapy.

Breast cancer disproportionately affects African American women under the age of 50, leading to higher incidence rates, more aggressive cancer subtypes, and increased mortality compared to other racial and ethnic groups. To enhance the prediction of onset risk and enable timely intervention and treatment, it is crucial to investigate the genetic and molecular factors associated with these disparities. This study introduces COMBINE, an innovative ensemble learning model that combines three types of omics data to improve the accuracy of breast cancer prognosis classification and reduce the model's time complexity. A comparative analysis of the fusion effects for African American and White women reveals a significant improvement in the fusion effect for African American women. Additionally, gene enrichment analysis highlights the importance of considering race when selecting relevant biomarkers. To address the challenges of cancer prognosis classification, a combination of qualitative and quantitative methods, along with ensemble learning, is employed. This comprehensive approach facilitates the exploration of new concepts for the application of multi-omics data, potentially leading to more personalized and effective treatment strategies. The study highlights the potential of ensemble learning as a fusion technique for multi-omics data in cancer prognosis classification. It emphasizes the importance of refining our understanding of the genetic and molecular factors contributing to disparities in breast cancer incidence and outcomes. Ultimately, this research has the potential to improve healthcare outcomes for African American women and alleviate the burden of this formidable disease.

Extracellular vesicles (EVs) are small particles with phospholipid bilayers that carry a diverse range of cargoes including nucleic acids, proteins and metabolites. EVs have important roles in various cellular processes and are increasingly recognized for their ubiquitous role in cell-cell communications and potential applications in therapeutics and diagnostics. Although many methods have been developed for the characterization and measurement of EVs, analyzing them from biofluids remains a challenge with regard to throughput and sensitivity. Recently, we introduced an approach to facilitate high-throughput analysis of EVs from trace amounts of sample. In this method, an amphiphile-dendrimer supramolecular probe (ADSP) is coated onto a nitrocellulose membrane for array-based capture and to enable an in situ immunoblotting assay. Here, we describe the protocol for our array-based method of EV profiling. We describe an enhanced version of the method that incorporates an automated printing workstation, ensuring high throughput and reproducibility. We further demonstrate the use of our array to profile specific glycosylations on the EV surface using click chemistry of an azide group introduced by metabolic labeling. In this protocol, the synthesis of ADSP and the fabrication of ADSP nitrocellulose membrane array can be completed on the same day. EVs are efficiently captured from biological or clinical samples through a 30-min incubation, followed by an immunoblotting assay within a 3-h window, thus providing a high-throughput platform for EV isolation and in situ targeted analysis of EV proteins and their modifications.

The knowledge of gut microbial information helps evaluate the nutrition and health of the host and can be used as an important tool for improving wildlife conservation and management. Guizhou golden monkey ( Rhinopithecus brelichi) ) is on the International Union for Conservation of Nature (IUCN 2023) Red List. The purpose of the current study was to investigate the difference of gut microbiome and resistome among three different groups of Guizhou golden monkeys using a metagenome approach, thus providing valuable insights and tools for management of captive golden monkeys. Fecal samples of Guizhou golden monkeys were collected from three places including Fanjingshan National Nature Reserve in Guizhou (n n =3, Wild), Panxi wildlife rescue center in Guizhou (n=3, n =3, CapGZ), and Beijing Zoo (n=3, n =3, CapBJ). Fecal DNA was extracted and libraries were prepared and then were sequenced on Illumina PE150 platform with 2 x 150 bp paired reads. The results showed that wild Guizhou golden monkeys had lower abundance of Firmicutes , , Uroviricota , , Spirochaetes , , Fibrobacterota , , Campylobacterota , , and Candidatus Melainabacteria and higher abundance Proteobacteria , , Bacteroidetes and Actinobacteria than the two groups of captive monkeys. PCoA analysis showed that fecal samples from three groups could be distinctly separated. Wild monkeys had significantly higher abundance of genes associated with metabolic function, including carbohydrate metabolism, amino acid metabolism, energy metabolism, and metabolism of cofactors and vitamins. Among the top 20 ARGs analyzed, more ARGs were detected in the wild monkeys than the rest two groups. Overall, this study provides a comprehensive analysis of microbiomes and resistome in wild and captive Guizhou golden monkeys. The data is important for improving the health, management, and conservation of Guizhou golden monkeys based on the deviations in gut microbiome. It also establishes the scientific foundation for future comparative studies on the gut microbiome and resistome of Guizhou golden monkeys and other closely related Rhinopithecus species.

As one of the research hotspots in recent years, gut microbiota have been proven to be closely related to host metabolism, nutrient absorption, and immune regulation. However, there are still many urgent issues in the research of gut microbiota, such as the localization and tracking of gut microbiota. In this research, two new fluorescent probes, EF and 6F, were developed by optimizing the structure of the positron salt small molecule probe F16. In vitro labeling experiments showed that EF and 6F can quickly label Gram-positive bacteria, Staphylococcus aureus and Lactobacillus reuteri, as well as Gram-negative bacteria, Escherichia coli and Salmonella pullorum. Meanwhile, EF and 6F have little bacterial toxicity and are used at a maximum concentration of 200 mu M. Compared with EF, 6F has better hydrophilicity and stronger fluorescence characteristics in aqueous solutions, making it more suitable for imaging within gut microbiota populations. The results of in vivo imaging experiments indicate that EF and 6F can label and image the intestinal microbiota colonized by the mouse intestinal mucosal epithelium without causing any damage to intestinal tissue. Compared with commercially available MitoTracker dyes and fluorescein 5-isothiocyanate (FITC) dyes, EF and 6F exhibit better biocompatibility. Therefore, the compounds EF and 6F synthesized in this study are novel small molecule probes suitable for imaging gut microbiota, providing a better probe selection for exploring complex gut microbiota.

Bacteriophages (phages) can significantly influence the composition and functions of their host communities, and enhance host pathogenicity via the transport of phage-encoded virulence genes. Phages are the main component of animal gut viruses, however, there are few reports on the piglet gut phageome and its contribution to virulence genes. Here, a total of 185 virulence genes from 59,955 predicted genes of gut phages in weaned piglets were identified, with 0.688 % of the phage contigs coding for at least one virulence gene. The virulence gene pblA was the most abundant, with various virulence genes significantly correlated with gut phages and their encoded mobile gene element (MGE) genes. Importantly, multiple virulence genes and MGE genes coexist in some phage sequences, and up to 12 virulence genes were detected in a single phage sequence, greatly increasing the risk of phage-mediated transmission of virulence genes into the bacterial genome. In addition, diarrhoea has driven changes in the composition and structure of phage and bacterial communities in the intestinal tract of weaned piglets, significantly increasing the abundance of phage contigs encoding both virulence genes and MGE genes in faecal samples, which potentially increases the risk of phage-mediated virulence genes being transfected into the gut bacterial genome. In summary, this study expands our understanding of the gut microbiome of piglets, advances our understanding of the potential role of phages in driving host pathogenesis in the gut system, and provides new insights into the sources of virulence genes and genetic evolution of bacteria in pig farm environments.

Background Ribosomal protein SA (RPSA) of human brain microvascular endothelial cells (HBMECs) can transfer from the cytosol to the cell surface and act as a receptor for some pathogens, including Streptococcus suis serotype 2 (SS2), a zoonotic pathogen causing meningitis in pigs and humans. We previously reported that SS2 virulence factor enolase (ENO) binds to RPSA on the cell surface of HBMECs and induces apoptosis. However, the mechanism that activates RPSA translocation to the cell surface and induces ENO-mediated HBMEC apoptosis is unclear. Results Here, we show that RPSA localization and condensation on the host cell surface depend on its internally disordered region (IDR). ENO binds to the IDR of RPSA and promotes its interaction with RPSA and vimentin (VIM), which is significantly suppressed after 1,6-Hexanediol (1,6-Hex, a widely used tool to disrupt phase separation) treatment, indicating that ENO incorporation and thus the concentration of RPSA/VIM complexes via co-condensation. Furthermore, increasing intracellular calcium ions (Ca 2+ ) in response to SS2 infection further facilitates the liquid-like condensation of RPSA and aggravates ENO-induced HBMEC cell apoptosis. Conclusions Together, our study provides a previously underappreciated molecular mechanism illuminating that ENO-induced RPSA condensation activates the migration of RPSA to the bacterial cell surface and stimulates SS2-infected HBMEC death and, potentially, disease progression. This study offers a fresh avenue for investigation into the mechanism by which other harmful bacteria infect hosts via cell surfaces’ RPSA.

Clostridium perfringens is one of the critical causative agents causing diarrhea in piglets, with significant economic losses to the pig industry. Under normal gut microbiota homeostasis and well-managed barns, diarrhea caused by C. perfringens could be controlled. Some reports show that probiotics, such as Bacillus subtilis, are beneficial in preventing necrotic enteritis (NE) in chickens, but few reports on piglets. Clostridium perfringens was found in the piglets’ diarrhea with intestinal microbiota dysbiosis in our survey. Bacillus subtilis G2B9-Q, which was isolated from the feces of healthy pigs, was found to have anti-Clostridium activity after screening. Clostridium perfringens was used to challenge mice by intraperitoneal injection for modeling to evaluate the anti-infective activity of cell-free supernatant (CFS) of B. subtilis G2B9-Q and different concentrations of B. subtilis G2B9-Q by oral administration. The results showed that G2B9-Q can mitigate intestinal lesions caused by C. perfringens infection, reduce inflammatory reactions, and modulate intestinal microbiota. The CFS of G2B9-Q can alleviate the pathological damage of intestinal tissues caused by C. perfringens infection, reduce the concentration of TNF-α and IL-10 in the sera of mice, as well as the relative expression levels of alpha toxin (CPA), perfringolysin O (PFO) toxin, IL-10, IL-22, and TNF-α in the jejunum and colon tissues, and alleviate the changes in gut microbiota structure caused by C. perfringens infection, which showed better therapeutic effects and indicated that the metabolites of G2B9-Q are essential mediators for their beneficial effects. Therefore, the CFS of G2B9-Q could potentially replace antibiotics in treating C. perfringens infection.

Salmonella pullorum causes typical "Bacillary White Diarrhea" and loss of appetite in chicks, which leads to the death of chicks in severe cases; thus, it is still a critical issue in China. Antibiotics are conventional medicines used for Salmonella infections; however, due to the extensive long-term use and even abuse of antibiotics, drug resistance becomes increasingly severe, making treating pullorum disease more difficult. Most of the endolysins are hydrolytic enzymes produced by bacteriophages to cleave the host's cell wall during the final stage of the lytic cycle. A virulent bacteriophage, YSP2, of Salmonella was isolated in a previous study. A Pichia pastoris expression strain that can express the Salmonella bacteriophage endolysin was constructed efficiently, and the Gram-negative bacteriophage endolysin, LySP2, was obtained in this study. Compared with the parental phage YSP2, which can only lyse Salmonella, LySP2 can lyse Salmonella and Escherichia. The survival rate of Salmonella-infected chicks treated with LySP2 can reach up to 70% and reduce Salmonella abundance in the liver and intestine. The treatment group showed that LySP2 significantly improved the health of infected chicks and alleviated organ damage caused by Salmonella infection. In this study, the Salmonella bacteriophage endolysin was expressed efficiently by Pichia pastoris, and the endolysin LySP2 showed good potential for the treatment of pullorum disease caused by Salmonella pullorum.

The growing prevalence of antibiotic-resistant pathogens has led to a better understanding of the underlying processes that lead to this expansion. Intensive pig farms are considered one of the hotspots for antibiotic resistance gene (ARG) transmission. Phages, as important mobile carriers of ARGs, are widespread in the animal intestine. However, our understanding of phage-associated ARGs in the pig intestine and their underlying drivers is limited. Here, metagenomic sequencing and analysis of viral DNA and total DNA of different intestinal (ileum, cecum and feces) contents in healthy piglets and piglets with diarrhea were separately conducted. We found that phages in piglet ceca are the main repository for ARGs and mobile genetic element (MGE) genes. Phage-associated MGEs are important factors affecting the maintenance and transfer of ARGs. Interestingly, the colocalization of ARGs and MGE genes in piglet gut phages does not appear to be randomly selected but rather related to a specific phage host (Streptococcus). In addition, in the feces of piglets with diarrhea, the abundance of phages carrying ARGs and MGE genes was significantly increased, as was the diversity of polyvalent phages (phages with broad host ranges), which would facilitate the transfection and wider distribution of ARGs in the bacterial community. Moreover, the predicted host spectrum of polyvalent phages in diarrheal feces tended to be potential enteropathogenic genera, which greatly increased the risk of enteropathogens acquiring ARGs. Notably, we also found ARG-homologous genes in the sequences of piglet intestinal mimiviruses, suggesting that the piglet intestinal mimiviruses are a potential repository of ARGs. In conclusion, this study greatly expands our knowledge of the piglet gut microbiome, revealing the underlying mechanisms of maintenance and dissemination of piglet gut ARGs and providing a reference for the prevention and control of ARG pollution in animal husbandry.

Aim: Transcriptional regulation is actively involved in the onset and progression of various diseases. This study used the feature-engineering approach model-based quantitative transcription regulation to quantitatively measure the correlation between mRNA and transcription factors in a reference dataset of chronic lymphocytic leukemia (CLL) transcriptomes. Methods: A comprehensive investigation of transcriptional regulation changes in CLL was conducted using 973 samples in six independent datasets. Results & conclusion: Seven mRNAs were detected to have significantly differential model-based quantitative transcription regulation values but no differential expression between CLL patients and controls. We called these genes 'dark biomarkers' because their original expression levels did not show differential changes in the CLL patients. The overlapping lncRNAs might have contributed their transcripts to the expression miscalculations of these dark biomarkers.