A pH-/Enzyme-Responsive Nanoparticle Selectively Targets Endosomal Toll-like Receptors to Potentiate Robust Cancer Vaccination.

Toll-like receptor (TLR) agonists are potent immune-stimulators that hold great potential in vaccine adjuvants as well as cancer immunotherapy. However, TLR agonists in free form are prone to be eliminated quickly by the circulatory system and cause systemic inflammation side effects. It remains a challenge to achieve precise release of TLR7/8 agonist in the native form at the receptor site in the endosomal compartments while keeping stable encapsulation and inactive in nontarget environment. Here, we report a pH-/enzyme-responsive TLR7/8 agonist-conjugated nanovaccine (TNV), which responds intelligently to the acidic environment and cathepsin B in the endosome, precisely releases TLR7/8 agonist to activate its receptor signaling at the endosomal membrane, stimulates DCs maturation, and provokes specific cellular immunity. In vivo experiments demonstrate outstanding prophylactic and therapeutic efficacy of TNV in mouse melanoma and colon cancer. The endosome-targeted responsive nanoparticle strategy provides a potential delivery toolbox of adjuvants to advance the development of tumor nanovaccines.

Cooperative Self-Assembled Nanoparticle Induces Sequential Immunogenic Cell Death and Toll-Like Receptor Activation for Synergistic Chemo-immunotherapy.

Anticancer immunotherapy is hampered by poor immunogenicity and a profoundly immunosuppressive microenvironment in solid tumors and lymph nodes. Herein, sequential pH/redox-responsive nanoparticles (SRNs) are engineered to activate the immune microenvironment of tumor sites and lymph nodes. The two-modular SRNs could sequentially respond to the acidic tumor microenvironment and endosome compartments of dendritic cells (DCs) to precisely deliver doxorubicin (DOX) and imidazoquinolines (IMDQs). In the tumor microenvironment, released DOX triggers immunogenic cell death. In sentinel lymph nodes, the IMDQ nanoparticle module is dissociated in the acidic endosome compartment to specifically stimulate toll-like receptor 7/8 for DC maturation. Thus, the orchestrated nanoparticle system could enhance the infiltration of CD8α+ T cells in tumors and provoke a strong antitumor immune response toward primary and abscopal tumors in B16-OVA and CT26 tumor-bearing mice models. The cooperative self-assembled nanoparticle strategy provides a potential candidate of nanomedicine to advance the synergistic cancer chemo-immunotherapy.

A pH-Responsive Nanoparticle Library with Precise pH Tunability by Co-Polymerization with Non-Ionizable Monomers.

Precise monitoring of the subtle pH fluctuation during biological events remains a big challenge. Previously, we reported an ultra-pH-sensitive (UPS) nanoprobe library with a sharp pH response using co-polymerization of two tertiary amine-containing monomers with distinct pKa . Currently, we have generalized the UPS nanoparticle library with tunable pH transitions (pHt ) by copolymerization of a tertiary amine-containing monomer with a series of non-ionizable monomers. The pHt of nanoparticles is fine-tuned by the non-ionizable monomers with different hydrophobicity. Each non-ionizable monomer presents a constant contribution to pH tunability regardless of tertiary amine-containing monomers. Based on this strategy, we produced two libraries of nanoprobes with continuous pHt covering the entire physiological pH range (5.0-7.4) for fluorescent imaging of endosome maturation and cancers. This generalized strategy provides a powerful toolkit for biological studies and cancer theranostics.

pH-Amplified CRET Nanoparticles for In Vivo Imaging of Tumor Metastatic Lymph Nodes.

Noninvasive imaging strategies have been extensively investigated for in vivo mapping of sentinel lymph nodes (SLNs). However, the current imaging strategies fail to accurately assess tumor metastatic status in SLNs with high sensitivity. Here we report pH-amplified self-illuminating near-infrared nanoparticles, which integrate chemiluminescence resonance energy transfer (CRET) and signal amplification strategy, enabling accurate identification of metastatic SLNs. After draining into lymph nodes, the nanoparticles were phagocytosed and dissociated in acidic phagosomes of inflammatory macrophages to emit near-infrared luminescent light. Using these nanoparticles, we successfully differentiated tumor metastatic lymph nodes from benign ones. These nanoparticles also exhibited excellent imaging capability for early detection of metastatic SLNs in diverse animal tumor models with small tumor volume (100-200 mm3 ).

Chicken Intestinal Mycobiome: Initial Characterization and Its Response to Bacitracin Methylene Disalicylate.

The gastrointestinal (GI) tract harbors a diverse population of microorganisms. While much work has been focused on the characterization of the bacterial community, very little is known about the fungal community, or mycobiota, in different animal species and chickens in particular. Here, we characterized the biogeography of the mycobiota along the GI tract of day 28 broiler chicks and further examined its possible shift in response to bacitracin methylene disalicylate (BMD), a commonly used in-feed antibiotic, through Illumina sequencing of the internal transcribed spacer 2 (ITS2) region of fungal rRNA genes. Out of 124 samples sequenced, we identified a total of 468 unique fungal features that belong to four phyla and 125 genera in the GI tract. Ascomycota and Basidiomycota represented 90% to 99% of the intestinal mycobiota, with three genera, i.e., Microascus, Trichosporon, and Aspergillus, accounting for over 80% of the total fungal population in most GI segments. Furthermore, these fungal genera were dominated by Scopulariopsis brevicaulis (Scopulariopsis is the anamorph form of Microascus), Trichosporon asahii, and two Aspergillus species. We also revealed that the mycobiota are more diverse in the upper than lower GI tract. The cecal mycobiota transitioned from being S. brevicaulis dominant on day 14 to T. asahii dominant on day 28. Furthermore, 2-week feeding of 55 mg/kg BMD tended to reduce the cecal mycobiota α-diversity. Taken together, we provided a comprehensive biogeographic view and succession pattern of the chicken intestinal mycobiota and its influence by BMD. A better understanding of intestinal mycobiota may lead to the development of novel strategies to improve animal health and productivity.IMPORTANCE The intestinal microbiota is critical to host physiology, metabolism, and health. However, the fungal community has been often overlooked. Recent studies in humans have highlighted the importance of the mycobiota in obesity and disease, making it imperative that we increase our understanding of the fungal community. The significance of this study is that we revealed the spatial and temporal changes of the mycobiota in the GI tract of the chicken, a nonmammalian species. To our surprise, the chicken intestinal mycobiota is dominated by a limited number of fungal species, in contrast to the presence of hundreds of bacterial taxa in the bacteriome. Additionally, the chicken intestinal fungal community is more diverse in the upper than the lower GI tract, while the bacterial community shows an opposite pattern. Collectively, this study lays an important foundation for future work on the chicken intestinal mycobiome and its possible manipulation to enhance animal performance and disease resistance.

Redox-Activated Porphyrin-Based Liposome Remote-Loaded with Indoleamine 2,3-Dioxygenase (IDO) Inhibitor for Synergistic Photoimmunotherapy through Induction of Immunogenic Cell Death and Blockage of IDO Pathway.

Immunotherapy through stimulating the host immune system has emerged as a powerful therapeutic strategy for various malignant and metastatic tumors in the clinic. However, harnessing the immune system for cancer treatment often fails to obtain a durable response rate due to the poor immunogenicity and the strong immunosuppressive milieu in the tumor site. Herein, a redox-activated liposome was developed from the self-assembly of the porphyrin-phospholipid conjugate and coencapsulation of indoleamine 2,3-dioxygenase (IDO) inhibitor into the interior lumen via remote-loading for simultaneous induction of immunogenic cell death (ICD) and reversing of suppressive tumor microenvironment. The nanoparticle exhibited prolonged blood circulation and enhanced tumor accumulation in the 4T1 tumor-bearing mice after intravenous injection. The nanovesicle could render exponential activation of fluorescence signal and photodynamic therapy (PDT) activity (>100-fold) in response to the high level of intracellular glutathione after endocytosed by tumor cells, thereby achieving effective inhibition of tumor growth and reduced phototoxicity to normal tissues owing to the activatable design of the nanoparticle. More importantly, redox-activated PDT induced intratumoral infiltration of cytotoxic T lymphocytes by induction of ICD of tumor cells. After combining with the IDO inhibitor, the systemic antitumor immune response was further augmented. Hence, we believe that the present nanovesicle strategy has the potential for the synergistic immunotherapy of the metastatic cancers.

Population genomics identifies patterns of genetic diversity and selection in chicken.

BACKGROUND: There are hundreds of phenotypically distinguishable domestic chicken breeds or lines with highly specialized traits worldwide, which provide a unique opportunity to illustrate how selection shapes patterns of genetic variation. There are many local chicken breeds in China. RESULTS: Here, we provide a population genome landscape of genetic variations in 86 domestic chickens representing 10 phenotypically diverse breeds. Genome-wide analysis indicated that sex chromosomes have less genetic diversity and are under stronger selection than autosomes during domestication and local adaptation. We found an evidence of admixture between Tibetan chickens and other domestic population. We further identified strong signatures of selection affecting genomic regions that harbor genes underlying economic traits (typically related to feathers, skin color, growth, reproduction and aggressiveness) and local adaptation (to high altitude). By comparing the genomes of the Tibetan and lowland fowls, we identified genes associated with high-altitude adaptation in Tibetan chickens were mainly involved in energy metabolism, body size maintenance and available food sources. CONCLUSIONS: The work provides crucial insights into the distinct evolutionary scenarios occurring under artificial selection for agricultural production and under natural selection for success at high altitudes in chicken. Several genes were identified as candidates for chicken economic traits and other phenotypic traits.

Ultra-pH-sensitive indocyanine green-conjugated nanoprobes for fluorescence imaging-guided photothermal cancer therapy.

Photothermal therapy (PTT) has been recognized as a promising approach for cancer treatment due to its minimal invasiveness and low systemic side effects. However, developing a photothermal agent with accurate tumor imaging capability is a prerequisite for the efficient PTT. Here, we developed a series of ultra-pH-sensitive indocyanine green (ICG)-conjugated nanoparticles for fluorescence imaging-guided tumor PTT. These nanoparticles exhibited high fluorescence activation ratio (~100-fold) with sharp pH transition (ΔpHon/off <0.25), and superior temperature response than free ICG. The in vivo imaging experiments demonstrated that the nanoparticles generated excellent tumor-to-normal tissue contrast through pH-triggered fluorescence activation in tumor sites, which provided information on tumor mass location, boundaries, and shape. Moreover, comparing to free ICG, the nanosystem had significantly longer blood circulation time and more accurate tumor targeting, providing efficient photothermal therapeutic effect against A549 tumor in living animals. In conclusion, this nanoplatform offers a potential strategy for imaging-guided cancer PTT.

High-throughput sequencing of pituitary and hypothalamic microRNA transcriptome associated with high rate of egg production.

BACKGROUND: MicroRNAs exist widely in viruses, plants and animals. As endogenous small non-coding RNAs, miRNAs regulate a variety of biological processes. Tissue miRNA expression studies have discovered numerous functions for miRNAs in various tissues of chicken, but the regulation of miRNAs in chicken pituitary and hypothalamic development related to high and low egg-laying performance has remained unclear. RESULTS: In this study, using high-throughput sequencing technology, we sequenced two tissues (pituitary and hypothalamus) in 3 high- and 3 low-rate egg production Luhua chickens at the age of 300 days. By comparing low- and high-rate egg production chickens, 46 known miRNAs and 27 novel miRNAs were identified as differentially expressed (P < 0.05). Six differentially expressed known miRNAs, which are expressed in both tissues, were used in RT-qPCR validation and SNP detection. Among them, seven SNPs in two miRNA precursors (gga-miR-1684a and gga-miR-1434) were found that might enhance or reduce the production of the mature miRNAs. In addition, 124 and 30 reciprocally expressed miRNA-target pairs were identified by RNA-seq in pituitary and hypothalamic tissues, respectively and randomly selected candidate miRNA and miRNA-target pairs were validated by RT-qPCR in Jiuyuan black fowl. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation illustrated that a large number of egg laying-related pathways were enriched in the high-rate egg production chickens, including ovarian steroidogenesis and steroid hormone biosynthesis. CONCLUSIONS: These differentially expressed miRNAs and their predicted target genes, especially identified reciprocally expressed miRNA-target pairs, advance the study of miRNA function and egg production associated miRNA identification. The analysis of the miRNA-related SNPs and their effects provided insights into the effects of SNPs on miRNA biogenesis and function. The data generated in this study will further our understanding of miRNA regulation mechanisms in the chicken egg-laying process.

Monoclonal antibodies: formulations of marketed products and recent advances in novel delivery system.

Monoclonal antibodies (mAbs) are extensively employed for disease diagnosis and treatment because of their high homogeneity and antigen specificity. In recent years, important outcomes have been achieved with mAbs due to their admirable therapeutic efficacy and relatively rare side effects. In clinical practice, several mAb products have been approved by regulatory entities, but their formulations have been highly specific given the complex structure and proteinaceous nature of mAbs. Thus, more attention has been given on formulations. An increasing number of novel delivery systems have been exploited to optimize the application of mAbs. In this article, the formulations, dosages, origins and administration routes of available mAbs approved by the Food and Drug Administration (FDA) are summarized and categorized. Key issues involved in formulation, processing and storage are addressed as well as other challenges in achieving effective mAb delivery. Finally, recent advances in delivering mAbs in their most bioavailable forms are also briefly reviewed.

A tenascin C targeted nanoliposome with navitoclax for specifically eradicating of cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs) play a vitally important role during tumor progression. Navitoclax (Nav) can specifically induce apoptosis in CAFs. The present study aims to develop a novel CAF-targeted nanoliposome for cancer therapy. Nav-loaded nanoliposomes modified with peptide FH (FH-SSL-Nav), which specifically binds to tenascin C, a protein mainly expressed by CAFs, were formulated and characterized. Several experiments were performed to evaluate CAFs selective apoptosis, targeting and eradicating. Compared with SSL-Nav, FH-SSL-Nav achieved higher cellular uptake, and exhibited stronger cytotoxicity in vitro. The in vivo tumor stroma targeting effect was further confirmed by near infrared imaging. Accordingly, FH-SSL-Nav displayed improved tumor growth inhibition by eradicating CAFs in Hep G2 tumor-bearing nude mice model. In conclusion, FH-SSL-Nav could achieve targeting delivery of Nav to CAFs in vitro and in vivo, and may offer a potential strategy for cancer therapy based on tumor stroma. From the Clinical Editor: The progression of cancer cells often depends on the underlying tumor microenvironment, in which cancer-associated fibroblasts play an important role. In this article, the authors developed targeted therapy against CAFs using liposomes as carriers. This new modality was shown to be more effective in tumor killing both in vitro and in vivo. The finding may open a new era in cancer therapy.

Effects of Transportation on Blood Indices, Oxidative Stress, Rumen Fermentation Parameters and Rumen Microbiota in Goats.

The objective of this experiment was to delve into the impacts of transportation on goats. Sixteen healthy goats were selected as experimental animals; these goats were transported at a speed ranging from 35 to 45 km/h for 20 h. The changes in the physiological indexes, blood physiological indexes, biochemical indexes, rumen fermentation indexes, and rumen microbial structure composition of goats before and after transportation were measured. The results showed that after transportation, the contents of IgM, IgA, IgG, and Thyroxine decreased very significantly, while the contents of propionic acid, Hemoglobin and Epinephrine significantly increased, and the contents of VFA, acetic acid, butyric acid, isobutyric acid, isovaleric acid, LPS, IL-1ß, IL-6, TNF-α, Major Acute Phase Protein, protein carbonyl, and cortisol increased very significantly. There was no significant difference in α-diversity and ß-diversity, and the relative abundance of rumen microorganisms was not significantly different at either phylum or genus levels. The experimental findings revealed that continuous transportation for a duration of 20 h can induce a severe stress response in goats, leading to compromised immune function, diminished antioxidant capacity, escalated inflammatory response, and altered rumen fermentation indices. However, the experiment did not reveal any significant impact on the structure and composition of the rumen microbiota.

Bile Acid Composition and Transcriptome Analysis of the Liver and Small Intestine in Different Species.

Bile, a crucial fluid produced continuously by the liver, plays an essential role in digestion within the small intestine. Beyond its primary function in lipid digestion, bile also acts as a pathway for the elimination of various endogenous and exogenous substances. There have been limited studies focusing on interspecies differences. This study offers a comprehensive analysis of bile acid (BA) composition and its correlation with gene expression patterns across six different species, including mammals and poultry, through combining Liquid Chromatography-Mass Spectrometry (LC-MS) and transcriptome sequencing. The BA profiles revealed distinct metabolite clusters: D-glucuronic acid (GLCA) and glycochenodeoxycholic acid (GCDCA) were predominant in mammals, while taurolithocholic acid (TLCA) and T-alpha-MCA were prevalent in poultry, highlighting species-specific BA compositions. Differentially abundant metabolites, particularly GDCA, glycohyodeoxycholic acid (GHDCA) and taurodeoxycholic acid (TDCA) showed significant variations across species, with pigs showing the highest BA content. Transcriptome analysis of the liver and small intestine tissues of 56 cDNA libraries across the six species revealed distinct mRNA expression patterns. These patterns clustered samples into broad categories based on tissue type and phylogenetic relationships. Furthermore, the correlation between gene expression and BA content was examined, identifying the top 20 genes with significant associations. These genes potentially serve as biomarkers for BA regulation.

Insights into left-right asymmetric development of chicken ovary at the single-cell level.

Avian ovaries develop asymmetrically apart from prey birds, with only the left ovary growing more towards functional organs. Here, we analyze over 135,000 cells from chick's left and right ovaries at six distinct embryonic developmental stages utilizing single-cell transcriptome sequencing. We delineate gene expression patterns across 15 cell types within these embryo ovaries, revealing side-specific development. The left ovaries exhibit cortex cells, zygotene germ cells, and transcriptional changes unique to the left side. Differential gene expression analysis further identifies specific markers and pathways active in these cell types, highlighting the asymmetry in ovarian development. A fine-scale analysis of the germ cell meiotic transcriptome reveals seven distinct clusters with gene expression patterns specific to various meiotic stages. The study also identifies signaling pathways and intercellular communications, particularly between pre-granulosa and germ cells. Spatial transcriptome analysis shows the asymmetry, demonstrating cortex cells exclusively in the left ovary, modulating neighboring cell types through putative secreted signaling molecules. Overall, this single-cell analysis provides insights into the molecular mechanisms of the asymmetric development of avian ovaries, particularly the significant role of cortex cells in the left ovary.

Apatinib and gamabufotalin co-loaded lipid/Prussian blue nanoparticles for synergistic therapy to gastric cancer with metastasis.

Due to the non-targeted release and low solubility of anti-gastric cancer agent, apatinib (Apa), a first-line drug with long-term usage in a high dosage often induces multi-drug resistance and causes serious side effects. In order to avoid these drawbacks, lipid-film-coated Prussian blue nanoparticles (PB NPs) with hyaluronan (HA) modification was used for Apa loading to improve its solubility and targeting ability. Furthermore, anti-tumor compound of gamabufotalin (CS-6) was selected as a partner of Apa with reducing dosage for combinational gastric therapy. Thus, HA-Apa-Lip@PB-CS-6 NPs were constructed to synchronously transport the two drugs into tumor tissue. In vitro assay indicated that HA-Apa-Lip@PB-CS-6 NPs can synergistically inhibit proliferation and invasion/metastasis of BGC-823 cells via downregulating vascular endothelial growth factor receptor (VEGFR) and matrix metalloproteinase-9 (MMP-9). In vivo assay demonstrated strongest anti-tumor growth and liver metastasis of HA-Apa-Lip@PB-CS-6 NPs administration in BGC-823 cells-bearing mice compared with other groups due to the excellent penetration in tumor tissues and outstanding synergistic effects. In summary, we have successfully developed a new nanocomplexes for synchronous Apa/CS-6 delivery and synergistic gastric cancer (GC) therapy.

Dietary ferulic acid improves growth performance of broilers via enhanced intestinal antioxidant capacity and barrier function.

Objective: In this study, the effects of dietary ferulic acid (FA) on the growth traits, antioxidant capacity, and intestinal barrier function of broilers were investigated. Methods: In total, 192 male Arbor Acres broilers were randomly allocated to one of three dietary groups (8 replicates of 8 birds each): control (CON) group (basal diet), FA100 group (basal diet + 100 mg/kg FA), or FA200 group (basal diet + 200 mg/kg FA). The duration of the feeding trial was 42 days. Results: higher average daily gain (ADG) and lower feed to gain (F/G) ratio during day 0 to day 21 were found in the FA100 and FA200 groups, while higher ADG and lower F/G during day 21 to day 42 were only found in FA200 group, compared to the CON group (p < 0.05). Serum levels of MDA and DAO on day 21 were lower in the FA100 and FA200 groups and those on day 42 were lower in the FA200 group, while GSH-Px level in the FA100 and FA200 groups on day 21 and that in the FA200 group on day 42 were increased (p < 0.05). On day 21, jejunal GSS expression was upregulated in the FA200 group (p < 0.05), while jejunal and ileal expression of NRF2 and Occludin as well as ileal expression of GPX1 and ZO1 were increased in the FA100 and FA200 groups compared to the CON group (p < 0.05). On day 42, mRNA expression of GSS, NRF2, SOD1, and GPX1 in the jejunum and ileum as well as Claudin2 in the jejunum and Occludin in the ileum were increased in the FA200 group (p < 0.05). Conclusion: Dietary FA addition could improve the growth performance, antioxidant capacity, and gut integrity of broilers. The current findings provided evidences that the adoption of FA can be as nutrition intervention measure to achieve high-efficient broiler production for poultry farmers.

A Tumor-Activatable Liposomal Nanoprobe for Selective Visualization of Metastatic Lymph Nodes.

The precise identification of sentinel lymph nodes (SLNs) during surgery and assessment of their benign status is crucial for accurate tumor staging and optimal treatment strategizing. Currently, a deficiency exists in non-invasive in vivo diagnostic techniques that can accurately pinpoint SLNs during surgery while simultaneously evaluating their benign status. Here, a tumor-activatable liposomal nanoprobe (nTAL) is developed, remotely loaded with clinically approved photosensitizer, methyl aminolevulinate (MAL), to noninvasively visualize the tumor metastasis lymph nodes (LNs) with precision. Benefited from the highly efficient LNs draining of nanosized liposome and tumor cell-specific transformation of the non-fluorescent MAL to fluorescent protoporphyrin IX (PPIX), nTAL succeeded in targeting the SLNs and differentiated the metastatic from the benign ones with a positive correlation between PPIX generation and tumor cell infiltration in LNs. Moreover, the nTAL technology is capable of probing the early metastatic stage with a primary tumor size of 50 mm3. This study provides a new strategy for intraoperative visualization of real-time sentinel node dissection.

Intraoperative intravenous versus periarticular injection of glucocorticoids in improving clinical outcomes after total knee arthroplasty: A prospective, randomized and controlled study.

BACKGROUND: Glucocorticoids have been widely used in perioperative period for postoperative pain relief after total knee arthroplasty (TKA). However, the optimal administration protocols of glucocorticoids remain controversial. This study aims to compare the efficacy of glucocorticoids between intravenous and periarticular injection on clinical outcomes. METHODS: A total of 114 patients were randomly assigned to intravenous (IV) group (n = 57) and periarticular injection (PI) group (n = 57). The IV group received 10 mg dexamethasone intravenously and the PI group received periarticular injection of 10 mg dexamethasone during the procedure. The clinical outcomes were assessed using visual analogue scale (VAS), knee society score (KSS), range of motion (ROM), knee swelling, inflammation markers and complications after TKA. RESULTS: The VAS score during walking at 2nd day postoperatively was lower in the PI group compared with the IV group (2.08 ± 1.45 vs 2.73 ± 1.69, p = .039), and there was no significant difference at the other time points of VAS score in two groups. The inflammation markers, knee swelling, knee ROM and KSS score were not statistically different. Vomiting and other complications occurrence were not significantly different between the two groups. CONCLUSIONS: Intraoperative periarticular injection of glucocorticoids has similar analgesic effect compared to intravenous in the postoperative period following TKA and may be even more effective on the second postoperative day. In addition, periarticular injection of glucocorticoids does not impose an excess risk or complication on patients.

Effects of low-intensity pulsed ultrasound on the microorganisms of expressed prostatic secretion in patients with IIIB prostatitis.

To detect and analyze the changes of microorganisms in expressed prostatic secretion (EPS) of patients with IIIB prostatitis before and after low-intensity pulsed ultrasound (LIPUS) treatment, and to explore the mechanism of LIPUS in the treatment of chronic prostatitis (CP). 25 patients (study power was estimated using a Dirichlet-multinomial approach and reached 96.5% at α = 0.05 using a sample size of 25) with IIIB prostatitis who were effective in LIPUS treatment were divided into two groups before and after LIPUS treatment. High throughput second-generation sequencing technique was used to detect and analyze the relative abundance of bacterial 16 s ribosomal variable regions in EPS before and after treatment. The data were analyzed by bioinformatics software and database, and differences with P < 0.05 were considered statistically significant. Beta diversity analysis showed that there was a significant difference between groups (P = 0.046). LEfSe detected four kinds of characteristic microorganisms in the EPS of patients with IIIB prostatitis before and after LIPUS treatment. After multiple comparisons among groups by DESeq2 method, six different microorganisms were found. LIPUS may improve patients' clinical symptoms by changing the flora structure of EPS, stabilizing and affecting resident bacteria or opportunistic pathogens.