Cataloging the phylogenetic diversity of human bladder bacterial isolates.

BACKGROUND: Although the human bladder is reported to harbor unique microbiota, our understanding of how these microbial communities interact with their human hosts is limited, mostly owing to the lack of isolates to test mechanistic hypotheses. Niche-specific bacterial collections and associated reference genome databases have been instrumental in expanding knowledge of the microbiota of other anatomical sites, such as the gut and oral cavity. RESULTS: To facilitate genomic, functional, and experimental analyses of the human bladder microbiota, we present a bladder-specific bacterial isolate reference collection comprising 1134 genomes, primarily from adult females. These genomes were culled from bacterial isolates obtained by a metaculturomic method from bladder urine collected by transurethral catheterization. This bladder-specific bacterial isolate reference collection includes 196 different species, including representatives of major aerobes and facultative anaerobes, as well as some anaerobes. It captures 72.2% of the genera found when re-examining previously published 16S rRNA gene sequencing of 392 adult female bladder urine samples. Comparative genomic analysis finds that the taxonomies and functions of the bladder microbiota share more similarities with the vaginal microbiota than the gut microbiota. Whole-genome phylogenetic and functional analyses of 186 bladder Escherichia coli isolates and 387 gut Escherichia coli isolates support the hypothesis that phylogroup distribution and functions of Escherichia coli strains differ dramatically between these two very different niches. CONCLUSIONS: This bladder-specific bacterial isolate reference collection is a unique resource that will enable bladder microbiota research and comparison to isolates from other anatomical sites.

Viral potential to modulate microbial methane metabolism varies by habitat.

Methane is a potent greenhouse gas contributing to global warming. Microorganisms largely drive the biogeochemical cycling of methane, yet little is known about viral contributions to methane metabolism (MM). We analyzed 982 publicly available metagenomes from host-associated and environmental habitats containing microbial MM genes, expanding the known MM auxiliary metabolic genes (AMGs) from three to 24, including seven genes exclusive to MM pathways. These AMGs are recovered on 911 viral contigs predicted to infect 14 prokaryotic phyla including Halobacteriota, Methanobacteriota, and Thermoproteota. Of those 24, most were encoded by viruses from rumen (16/24), with substantially fewer by viruses from environmental habitats (0-7/24). To search for additional MM AMGs from an environmental habitat, we generate metagenomes from methane-rich sediments in Vrana Lake, Croatia. Therein, we find diverse viral communities, with most viruses predicted to infect methanogens and methanotrophs and some encoding 13 AMGs that can modulate host metabolisms. However, none of these AMGs directly participate in MM pathways. Together these findings suggest that the extent to which viruses use AMGs to modulate host metabolic processes (e.g., MM) varies depending on the ecological properties of the habitat in which they dwell and is not always predictable by habitat biogeochemical properties.

Taxonomic considerations on Aerococcus urinae with proposal of subdivision into Aerococcus urinae, Aerococcus tenax sp. nov., Aerococcus mictus sp. nov., and Aerococcus loyolae sp. nov.

Average nucleotide identity analysis, based on whole genome sequences of 115 strains previously identified as Aerococcus urinae, an emerging uropathogen, discriminates at least six unique genomic taxa. The whole genome analysis affords clearer species boundaries over 16S rRNA gene sequencing and traditional phenotypic approaches for the identification and phylogenetic organization of Aerococcus species. The newly described species can be differentiated by matrix-assisted laser desorption ionization time-of-flight analysis of protein signatures. We propose the emendation of the description of A. urinae (type strain ATCC 51268T = CCUG 34223T=NCFB 2893) and the names of Aerococcus tenax sp. nov. (ATCC TSD-302T = DSM 115700T = CCUG 76531T=NR-58630T), Aerococcus mictus sp. nov. (ATCC TSD-301T = DSM 115699T = CCUG 76532T=NR-58629T), and Aerococcus loyolae sp. nov. (ATCC TSD-300T = DSM 115698T = CCUG 76533T=NR-58628T) for three of the newly identified genomic taxa.

Cataloging the Phylogenetic Diversity of Human Bladder Bacterial Isolates.

Although the human bladder is reported to harbor unique microbiota, our understanding of how these microbial communities interact with their human hosts is limited, mostly owing to the lack of isolates to test mechanistic hypotheses. Niche-specific bacterial collections and associated reference genome databases have been instrumental in expanding knowledge of the microbiota of other anatomical sites, e.g., the gut and oral cavity. To facilitate genomic, functional, and experimental analyses of the human bladder microbiota, here we present a bladder-specific bacterial reference collection comprised of 1134 genomes. These genomes were culled from bacterial isolates obtained by a metaculturomic method from bladder urine collected by transurethral catheterization. This bladder-specific bacterial reference collection includes 196 different species, including representatives of major aerobes and facultative anaerobes, as well as some anaerobes. It captures 72.2 % of the genera found when we reexamined previously published 16S rRNA gene sequencing of 392 adult female bladder urine samples. Comparative genomic analysis found that the taxonomies and functions of the bladder microbiota shared more similarities with the vaginal microbiota than the gut microbiota. Whole-genome phylogenetic and functional analyses of 186 bladder E. coli isolates and 387 gut E. coli isolates supports the hypothesis that phylogroup distribution and functions of E. coli strains differ dramatically between these two very different niches. This bladder-specific bacterial reference collection is a unique resource that will enable hypothesis-driven bladder microbiota research and comparison to isolates from other anatomical sites.

Comparative Genomic Study of Streptococcus anginosus Reveals Distinct Group of Urinary Strains.

Streptococcus anginosus is a prevalent member of the human flora. While it has been found in the microbiota of "healthy" asymptomatic individuals, it has also been associated with genitourinary tract infections and bacteremia. Based upon multilocus sequence analysis, two subspecies and two genomosubspecies have been characterized for the species. We previously conducted whole-genome sequencing of 85 S. anginosus isolates from the urinary tract. Here, we present genomic analysis of this species, including isolates from the urinary tract as well as gut and fecal, vaginal, oral, respiratory, and blood and heart samples. Average nucleotide identity and core genome analysis revealed that these strains form two distinct groups. Group 1 is comprised of the S. anginosus type strain and other previously identified S. anginosus subspecies and genomosubspecies, including isolates from throughout the human body. In contrast, group 2 consists of predominantly urinary streptococci (n = 77; 85.6%). Both of these S. anginosus groups are distinct from other members of the Streptococcus anginosus group (SAG) species S. intermedius and S. constellatus. Genes conserved among all strains of one group but not in any strains in the other group were next identified. Group 1 strains included genes found in S. intermedius and S. constellatus, suggesting that they were lost within the ancestor of the group 2 strains. In contrast, genes unique to the group 2 strains were homologous to more distant streptococci, indicative of acquisition via horizontal gene transfer. These genes are ideal candidates for use as marker genes to distinguish between the two groups in the human microbiota. IMPORTANCE Whole-genome analysis of S. anginosus strains provides greater insight into the diversity of this species than from marker genes alone. Our investigation of 166 publicly available S. anginosus genomes via average nucleotide identity and core genome analysis revealed two phylogenomically distinct groups of this species, with one group almost exclusively consisting of isolates from the urinary tract. In contrast, only 8 urinary strains were identified within the other group, which contained the S. anginosus type strain, as well as all identified subspecies and genomosubspecies. While genomic analysis suggested that this urinary group of S. anginosus is genomically different from the previously characterized S. anginosus subspecies, phenotypic characterization is still needed. Given prior reports of the prevalence of S. anginosus in the urinary tract of both continent and incontinent females, future studies are needed to investigate if the symptom state of the urinary tract is associated with these two different groups.

Nonmetallic N/C Nanozyme Performs Continuous Consumption of Glu for Inhibition of Colorectal Cancer Cells.

Colorectal cancer (CRC) is the third leading cause of cancer-related mortality. 5-Fluorouracil (5-FU) is the first choice for treatment of CRC, but it cannot avoid the negative effects from local high glucose (Glu) in tumor. Recently, 5-FU therapy has been combined with other treatment modalities for CRC synergistic therapy. Although these combination therapy strategies are more effective in cancer therapy, the toxicity side effects to the liver and cause metabolic acidosis still exist. Herein, we report an emerging amorphous honeycomb-like nitrogen-doped carbon (N/C) nanozyme with nicotinamide adenine dinucleotide (NADH) oxidase and catalase (CAT) activity and cascade it with natural glucose dehydrogenase (GDH) to realize NAD+ regeneration and further hyperglycemia management. In this case, by the coupling of N/C nanozyme with natural GDH to form a N/C-GDH system, the electron transfer route can switch from Glu to a common but limited electron receptor, i.e., NAD+ to ubiquitous large amounts of oxygen, achieving the purpose of sustainable consumption of Glu under NAD+ circulation and regeneration, and importantly escaping the generation of toxic H2O2. The combination of the N/C-GDH system and 5-FU on CRC cells was investigated to assess their synergistic bioeffects. Notably, our results showed that the N/C-GDH system and 5-FU in combination significantly suppress the proliferation of human colon cancer cells (HCT-116) by reducing the sugar level and induced apoptosis compared with either material or drug used alone. This work expands the nanozymes in blood Glu management as well as the promising cancer cell inhibition and provides the possibility of nonmetallic nanomaterials in the realization of effective treatment of cancer.

Ascorbate oxidase-like nanozyme with high specificity for inhibition of cancer cell proliferation and online electrochemical DOPAC monitoring.

Despite the extensive investigation of the nanozymes exhibit their favorable performance compared to natural enzymes, nevertheless, the highly specific nanozyme still needs to be developed so that it can meet the requirements of exploring the mechanism as well as administration of related diseases and selective monitoring in biological system. In this study, self-assembled glutathione-Cu/Cu2O nanoparticles (GSH-Cu/Cu2O NPs) that exhibits specific ascorbic acid (AA) oxidase-like catalytic activity were constructed for AA-activated and H2O2-reinforced cancer cell proliferation inhibition and selective neurochemical monitoring. Cu/Cu2O NPs demonstrates effective AA oxidase-like activity and no common characteristics of other redox mimic enzymes often present in nanozyme. In particular, we found that the AA oxidase-like activity of GSH-Cu/Cu2O nanozyme was significantly improved by about 40% by improving the activation ability toward oxygen. The synthesized nanozyme can induce the generation of active oxygen by accelerating the oxidation of AA, which effectively suppresses the proliferation of cancer cells. We constructed an online electrochemical system (OECS) though loading nanozyme with enhanced ascorbate oxidase activity into a microreactor and setting it in the upstream of the detector. This GSH-Cu/Cu2O NPs-integrated microreactor can completely eliminate AA interference of the physical level toward 3,4-dihydroxy phenylacetic acid (DOPAC) electrochemical measurement, and the nanozyme-based OECS is able to continuously capture DOPAC alteration in rat brain acidosis model. Our findings may inspire rational design of nanozymes with high specificity as well as nanozyme-based selectivity solution for in vivo detection and show promising opportunities for their involvement in neurochemistry investigation.

The Bladder Microbiome, Metabolome, Cytokines, and Phenotypes in Patients with Systemic Lupus Erythematosus.

Emerging studies reveal unique bacterial communities in the human bladder, with alteration of composition associated to disease states. Systemic lupus erythematosus (SLE) is a complex autoimmune disease that is characterized by frequent impairment of the kidney. Here, we explored the bladder microbiome, metabolome, and cytokine profiles in SLE patients, as well as correlations between microbiome and metabolome, cytokines, and disease profiles. We recruited a group of 50 SLE patients and 50 individually matched asymptomatic controls. We used transurethral catheterization to collect urine samples, 16S rRNA gene sequencing to profile bladder microbiomes, and liquid chromatography-tandem mass spectrometry to perform untargeted metabolomic profiling. Compared to controls, SLE patients possessed unique bladder microbial communities and increased alpha diversity. These differences were accompanied by differences in urinary metabolomes, cytokines, and patients' disease profiles. The SLE-enriched genera, including Bacteroides, were positively correlated with several SLE-enriched metabolites, including olopatadine. The SLE-depleted genera, such as Pseudomonas, were negatively correlated to SLE-depleted cytokines, including interleukin-8. Alteration of the bladder microbiome was associated with disease profile. For example, the genera Megamonas and Phocaeicola were negatively correlated with serum complement component 3, and Streptococcus was positively correlated with IgG. Our present study reveals associations between the bladder microbiome and the urinary metabolome, cytokines, and disease phenotypes. Our results could help identify biomarkers for SLE. IMPORTANCE Contrary to dogma, the human urinary bladder possesses its own unique bacterial community with alteration of composition associated with disease states. Systemic lupus erythematosus (SLE) is a complex autoimmune disease often characterized by kidney impairment. Here, we explored the bladder microbiome, metabolome, and cytokine profiles in SLE patients, as well as correlations between the microbiome and metabolome, cytokines, and disease profiles. Compared to controls, SLE patients possessed a unique bladder microbial community and elevated alpha diversity. These differences were accompanied by differences in bladder metabolomes, cytokines, and patients' disease profiles. SLE-enriched genera were positively correlated with several SLE-enriched metabolites. SLE-depleted genera were negatively correlated to SLE-depleted cytokines. Alteration of the bladder microbiome was associated with disease profile. Thus, our study reveals associations between the bladder microbiome and the bladder metabolome, cytokines, and disease phenotypes. These results could help identify biomarkers for SLE.

A Child's urine is not sterile: A pilot study evaluating the Pediatric Urinary Microbiome.

INTRODUCTION: A bladder microbiome (urobiome) exists in adults. Data supports the effects of the adult urobiome on urinary tract health with associations between dysbiotic urobiomes and lower urinary tract disorders. Understanding urobiome origin is important since other microbiomes establish around birth and microbiome alterations are linked to disease development. However, the pediatric urobiome has not been well studied. OBJECTIVES: We sought to determine the age when the urobiome develops, compare the pediatric urobiome to microbiomes of adjacent urogenital niches, and compare the urobiomes between boys and girls and across age groups. STUDY DESIGN: Seventy-four children less than 18 years of age without recent antibiotic exposure were recruited, including 48 males and 26 females, aged 2 weeks to 209 months of age. Transurethral catheterized urine samples and samples from the perineum, urethra, vagina, and foreskin were collected. Specimens were assessed using the expanded quantitative urine culture protocol and by 16S rRNA gene sequencing. Dada2 was used to profile microbial compositions, and BLCA was used to identify microbial taxa. RESULTS: Bacteria were detected in 90.5% of urine samples and identified in children as young as 2 weeks of age. Microbial communities and compositions of the female bladder and other urogenital niches (urethra, perineum, and vagina) differed significantly by age. Lactobacillus predominated the bladder, urethral, and vaginal microbiomes in post-pubertal girls. Compared to female urinary microbiomes, those of males differed less substantially. Only perineal microbiomes differed significantly by age, whereas male urethral and foreskin microbiomes did not differ significantly. DISCUSSION: We identified that a urinary microbiome is established as early as infancy. In addition, the female urobiome changes throughout childhood, until the post-pubertal bacterial taxa becomes consistent with that seen in adult females. Whereas in boys, the urinary microbiome changed very little over time. In addition, the surrounding urogenital microbiomes differed less in boys as compared to females. Microbiomes established at a young age may have long-term influences on immune, metabolic, and neurobehavioral traits. The same may be true for the urobiome. Our study provides a foundation for future research to determine the influence of the pediatric urobiome on the development of urinary and even non-urinary disorders. CONCLUSIONS: A pediatric urobiome exists, with differences between males and females and can be detected at a young age with changes occurring throughout childhood. Similarities and differences are also seen between the pediatric urobiome and adjacent niches.

A mouse model displays host and bacterial strain differences in Aerococcus urinae urinary tract infection.

In recent years, the clinical significance of Aerococcus urinae has been increasingly recognized. A. urinae has been implicated in cases of urinary tract infection (UTI; acute cystitis and pyelonephritis) in both male and female patients, ranging from children to older adults. Aerococcus urinae can also be invasive, causing urosepsis, endocarditis, and musculoskeletal infections. Mechanisms of pathogenesis in A. urinae infections are poorly understood, largely due to the lack of an animal model system. In response to this gap, we developed a model of A. urinae urinary tract infection in mice. We compared A. urinae UTI in female C3H/HeN and C57BL/6 mice and compared four clinical isolates of A. urinae isolated from patients with UTI, urgency urinary incontinence, and overactive bladder. Our data demonstrate that host genetic background modulates A. urinae UTI. Female C57BL/6 female mice rapidly cleared the infection. Female C3H/HeN mice, which have inherent vesicoureteral reflux that flushes urine from the bladder up into the kidneys, were susceptible to prolonged bacteriuria. This result is consistent with the fact that A. urinae infections most frequently occur in patients with underlying urinary tract abnormalities or disorders that make them susceptible to bacterial infection. Unlike uropathogens such as E. coli, which cause infection and inflammation both of the bladder and kidneys in C3H/HeN mice, A. urinae displayed tropism for the kidney, persisting in kidney tissue even after clearance of bacteria from the bladder. Aerococcus urinae strains from different genetic clades displayed varying propensities to cause persistent kidney infection. Aerococcus urinae infected kidneys displayed histological inflammation, neutrophil recruitment and increased pro-inflammatory cytokines. These results set the stage for future research that interrogates host-pathogen interactions between A. urinae and the urinary tract.

Spinal Cord Injury Changes the Structure and Functional Potential of Gut Bacterial and Viral Communities.

Emerging data indicate that gut dysbiosis contributes to many human diseases, including several comorbidities that develop after traumatic spinal cord injury (SCI). To date, all analyses of SCI-induced gut dysbiosis have used 16S rRNA amplicon sequencing. This technique has several limitations, including being susceptible to taxonomic "blind spots," primer bias, and an inability to profile microbiota functions or identify viruses. Here, SCI-induced gut dysbiosis was assessed by applying genome- and gene-resolved metagenomic analysis of murine stool samples collected 21 days after an experimental SCI at the 4th thoracic spine (T4) or 10th thoracic spine (T10) spinal level. These distinct injuries partially (T10) or completely (T4) abolish sympathetic tone in the gut. Among bacteria, 105 medium- to high-quality metagenome-assembled genomes (MAGs) were recovered, with most (n = 96) representing new bacterial species. Read mapping revealed that after SCI, the relative abundance of beneficial commensals (Lactobacillus johnsonii and CAG-1031 spp.) decreased, while potentially pathogenic bacteria (Weissella cibaria, Lactococcus lactis _A, Bacteroides thetaiotaomicron) increased. Functionally, microbial genes encoding proteins for tryptophan, vitamin B6, and folate biosynthesis, essential pathways for central nervous system function, were reduced after SCI. Among viruses, 1,028 mostly novel viral populations were recovered, expanding known murine gut viral species sequence space ∼3-fold compared to that of public databases. Phages of beneficial commensal hosts (CAG-1031, Lactobacillus, and Turicibacter) decreased, while phages of pathogenic hosts (Weissella, Lactococcus, and class Clostridia) increased after SCI. Although the microbiomes and viromes were changed in all SCI mice, some of these changes varied as a function of spinal injury level, implicating loss of sympathetic tone as a mechanism underlying gut dysbiosis.IMPORTANCE To our knowledge, this is the first article to apply metagenomics to characterize changes in gut microbial population dynamics caused by a clinically relevant model of central nervous system (CNS) trauma. It also utilizes the most current approaches in genome-resolved metagenomics and viromics to maximize the biological inferences that can be made from these data. Overall, this article highlights the importance of autonomic nervous system regulation of a distal organ (gut) and its microbiome inhabitants after traumatic spinal cord injury (SCI). By providing information on taxonomy, function, and viruses, metagenomic data may better predict how SCI-induced gut dysbiosis influences systemic and neurological outcomes after SCI.

microRNA-137 downregulates MCL1 in ovarian cancer cells and mediates cisplatin-induced apoptosis.

Aim: miR-137 is downregulated in various cancers; however, its function in ovarian cancer remains unclear. Methods: The roles of miR-137 in apoptosis were accessed through IC50 values and DAPI assay. The regulation of MCL1 by miR-137 was investigated through luciferase reporter assay and immunoblot. Results: miR-137 mimic could decrease the IC50 value of cisplatin and promote apoptosis in OVCAR3 ovarian cancer cells. Using luciferase assay, results on a panel of anti-apoptotic proteins, we identified MCL1 as a target for miR-137 and the results were confirmed using immunoblot. Finally, the underlying pathway in which miR-137 may be involved was investigated by transcriptome sequencing. Conclusion: These results suggest that miR-137 downregulates MCL1 in ovarian cancer cells and mediates cisplatin-induced apoptosis.

Double-stranded RNA deaminase ADAR1 promotes the Zika virus replication by inhibiting the activation of protein kinase PKR.

Zika virus (ZIKV) is a mosquito-borne flavivirus that has emerged as a threat to global health. The family of adenosine deaminases acting on dsRNA (ADARs) are human host factors important for the genetic diversity and evolution of ZIKV. Here, we further investigated the role of ADAR1 in ZIKV replication by utilizing CRISPR/Cas9-based gene editing and RNAi-based gene knockdown techniques. Both ADAR1 knockout and knockdown significantly reduced ZIKV RNA synthesis, protein levels, and viral titers in several human cell lines. Trans-complementation with the full-length ADAR1 form p150 or the shorter form p110 lacking the Zα domain restored viral replication levels suppressed by the ADAR1 knockout. Moreover, we observed that the nuclear p110 form was redistributed to the cytoplasm in response to ZIKV infection. ADAR1 was not involved in viral entry but promoted viral protein translation by impairing ZIKV-induced activation of protein kinase regulated by dsRNA (PKR). Of note, the RNA-editing activity of ADAR1 was not required to promote ZIKV replication. We also found that the proviral role of ADAR1 was partially mediated through its ability to suppress IFN production and PKR activation. Our work identifies ADAR1 as a proviral factor involved in ZIKV replication, suggesting that ADAR1 could be a potential antiviral target.

Establishment of an miR-137-knockout cell model using CRISPR/Cas9 genome editing.

MicroRNA-137 (miR-137) has been reported to be abnormally expressed in a variety of types of cancer, including ovarian cancer. However, the roles served by miR-137 in cancer are not fully understood. In the present study, 3 single guide RNAs (sgRNAs) were designed, synthesized and inserted into pXPR001 plasmids. The pXPR001-sgRNA plasmids were verified using sequencing and integrated into the genome of the ovarian cancer cell line, A2780, through lentiviral transduction, puromycin selection and single-cell culture. PCR products amplified from single-cell cultures using primers covering miR-137 targeting sites were sequenced to identify clones with miR-137 gene disruption. Genome editing was detected in 72% of the clones derived from sgRNA2, 4% from sgRNA3 and 0% from sgRNA1. Of the clones from sgRNA2, 32% contained 1 edited miR-137 allele and 40% contained 2 edited miR-137 alleles. The expression of miR-137 in clones #2 and #3 could not be detected by reverse transcription-quantitative polymerase chain reaction. In addition, an MTT assay demonstrated that clones #2 and #3 exhibited enhanced proliferation. In conclusion, an miR-137-knockout cell model was successfully established in A2780 cells using CRISPR/Cas9 technology.

miR-509-3p promotes cisplatin-induced apoptosis in ovarian cancer cells through the regulation of anti-apoptotic genes.

AIM: Previous observations have implicated miR-509-3p's ability in regulating cisplatin-triggered apoptosis in ovarian cancer. However, the underlying mechanisms were not fully understood. MATERIALS & METHODS: The roles of miR-509-3p in cellular apoptosis were assessed through MTT and DAPI assays. The confirmation of the regulation of BCL2 family members by miR-509-3p was investigated by luciferase reporter assay, western blot, quantitative real-time PCR and rescue experiments. RESULTS: MiR-509-3p can decrease the IC50 values of cisplatin and promote apoptosis in ovarian cancer cells. Furthermore, on a panel of anti-apoptotic proteins, we identified that miR-509-3p could regulate BCL2, BCL2L2 and MCL1 via their 3'UTRs. CONCLUSION: Our study demonstrates that miR-509-3p could sensitize ovarian cancer cells to cisplatin treatment by targeting multiple anti-apoptosis genes including BCL2.

Polyaniline/Fe3O4 nanoparticle composite: synthesis and reaction mechanism.

Polyaniline/Fe(3)O(4) nanoparticle composite was prepared by polymerizing aniline in the presence of Fe(3)O(4) nanoparticles upon the use of H(2)O(2) as oxidant. The polymerization was monitored by ultraviolet-visible absorption spectroscopy. The microstructure of the resultant composite was characterized by transmission electron microscopy. The molecular structure of the resultant polyaniline in the composite was investigated by both Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, while the magnetic property of the composite was characterized by vibrating sample magnetometer. Furthermore, the microwave absorption property of the resultant composite was measured in a frequency range of 2-18 GHz. Systematic investigations revealed that carboxylic acid in the buffer presented a determined role in the polymerization of aniline. To discover the role of carboxylic acid in the polymerization of aniline, more control experiments were designed and carried out by theoretical calculation in combination with electron spin resonance measurements. It was for the first time found out that carboxylic acid such as acetic acid and succinic acid can not only catalyze the polymerization of aniline but also facilitate the generation of hydroxyl radical via the decomposition of H(2)O(2).