A new canthinone glycoside isolated from the root barks of Ailanthus altissima with NO inhibitory activity.

One new canthinone glycoside (1), together with six known compounds (2-7) including three lignans (2-4), two coumarins (5-6) and one phenol (7) was isolated from the root barks of Ailanthus altissima. The structure of new compound 1 was established by the interpretation of UV, IR, MS and NMR data, while its absolute configuration was determined by acid hydrolysis and GIAO NMR calculations with DP4+ probability analysis. The inhibitory effects of all compounds on Nitric oxide (NO) production were investigated in lipopolysaccharide (LPS)-induced RAW 264.7 cells. Results showed that compounds 2 and 5 displayed NO production inhibitory activity with IC50 values of 30.1 and 15.3 µM, respectively.

Polyketides from the mangrove-derived fungus Penicillium robsamsonii HNNU0006.

Seven polyketides, including an undescribed depsidone (1) and six previously reported 3,6,8-trihydroxy-1-methylxanthone (2), 7-hydroxy-2-(2-hydroxypropyl)-5-methylchromone (3), methyl3-chloro-6-hydroxy-2-(4-hy-droxy-2-methoxy-6-methylphenoxy)-4- methoxybenzoate (4), xylarianin A (5), 4,5-dihydroxy-6-(6'-methylsalicyloxy)-2-hydro-xymethyl-2-cyclohexen-1-one (6) and alternariol (7), have been isolated from cultures of the mangrove-derived fungus Penicillium robsamsonii HNNU0006. The structure of compound 1 was elucidated by extensive spectroscopic analysis and X-ray crystallography. Furthermore, all the compounds were evaluated their cytotoxic activities, and compounds 1 and 7 showed weak cytotoxicity against two cell lines Vero and A549 with IC50 values ranging from 95.6 and 296.5 µM, relative to the positive control Etoposide phosphate with IC50 values of 24.5 and 18.7 µM, respectively.

Inhibitor of cardiolipin biosynthesis-related enzyme MoGep4 confers broad-spectrum anti-fungal activity.

Plant pathogens cause devastating diseases, leading to serious losses to agriculture. Mechanistic understanding of pathogenesis of plant pathogens lays the foundation for the development of fungicides for disease control. Mitophagy, a specific form of autophagy, is important for fungal virulence. The role of cardiolipin, mitochondrial signature phospholipid, in mitophagy and pathogenesis is largely unknown in plant pathogenic fungi. The functions of enzymes involved in cardiolipin biosynthesis and relevant inhibitors were assessed using a set of assays, including genetic deletion, plant infection, lipidomics, chemical-protein interaction, chemical inhibition, and field trials. Our results showed that the cardiolipin biosynthesis-related gene MoGEP4 of the rice blast fungus Magnaporthe oryzae regulates growth, conidiation, cardiolipin biosynthesis, and virulence. Mechanistically, MoGep4 regulated mitophagy and Mps1-MAPK phosphorylation, which are required for virulence. Chemical alexidine dihydrochloride (AXD) inhibited the enzyme activity of MoGep4, cardiolipin biosynthesis and mitophagy. Importantly, AXD efficiently inhibited the growth of 10 plant pathogens and controlled rice blast and Fusarium head blight in the field. Our study demonstrated that MoGep4 regulates mitophagy, Mps1 phosphorylation and pathogenesis in M. oryzae. In addition, we found that the MoGep4 inhibitor, AXD, displays broad-spectrum antifungal activity and is a promising candidate for fungicide development.

Yishen Tongbi decoction attenuates inflammation and bone destruction in rheumatoid arthritis by regulating JAK/STAT3/SOCS3 pathway.

Background: Yishen-Tongbi Decoction (YSTB), a traditional Chinese prescription, has been used to improve syndromes of rheumatoid arthritis (RA) for many years. Previous research has shown that YSTB has anti-inflammatory and analgesic properties. However, the underlying molecular mechanism of the anti-RA effects of YSTB remains unclear. Purpose and study design: The purpose of this research was to investigate how YSTB affected mice with collagen-induced arthritis (CIA) and RAW264.7 cells induced with lipopolysaccharide (LPS). Results: The findings show that YSTB could significantly improve the clinical arthritic symptoms of CIA mice (mitigate paw swelling, arthritis score, thymus and spleen indices, augment body weight), downregulated expression of pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß), IL-6 and IL-17, while upregulated the level of anti-inflammatory like IL-10 and transforming growth factor-ß (TGF-ß). Meanwhile, YSTB inhibits bone erosion and reduces inflammatory cell infiltration, synovial proliferation, and joint destruction in CIA mice. In addition, we found that YSTB was able to suppress the LPS-induced inflammation of RAW264.7 cells, which was ascribed to the suppression of nitric oxide (NO) production and reactive oxygen species formation (ROS). YSTB also inhibited the production of inducible nitric oxide synthase and reduced the releases of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6 in LPS-induced RAW264.7 cells. Furthermore, the phosphorylation expression of JAK2, JAK3, STAT3, p38, ERK and p65 protein could be suppressed by YSTB, while the expression of SOCS3 could be activated. Conclusion: Taken together, YSTB possesses anti-inflammatory and prevention bone destruction effects in RA disease by regulating the JAK/STAT3/SOCS3 signaling pathway.

Hernandonine-mediated autophagic cell death in hepatocellular carcinoma: Interplay of p53 and YAP signaling pathways.

Hepatocellular carcinoma (HCC), the primary form of liver cancer, is the third leading cause of cancer-related death globally. Hernandonine is a natural alkaloid derived from Hernandia nymphaeifolia that has been shown to exert various biological functions. In a previous study, hernandonine was shown to suppress the proliferation of several solid tumor cell lines without affecting normal human cell lines. However, little is known about the effect of hernandonine on HCC. Therefore, this study aimed to investigate the effect and mechanism of hernandonine on HCC in relation to autophagy. We found that hernandonine inhibited HCC cell growth in vitro and in vivo. In addition, hernandonine elicited autophagic cell death and DNA damage in HCC cells. RNA-seq analysis revealed that hernandonine upregulated p53 and Hippo signaling pathway-related genes in HCC cells. Small RNA interference of p53 resulted in hernandonine-induced autophagic cell death attenuation. However, inhibition of YAP sensitized HCC cells to hernandonine by increasing the autophagy induction. This is the first study to illustrate the complex involvement of p53 and YAP in the hernandonine-induced autophagic cell death in human HCC cells. Our findings provide novel evidence for the potential of hernandonine as a therapeutic agent for HCC treatment.

Research progress of ectopic thyroid cancer in thyroglossal duct cyst: A case report and literature review.

RATIONALE: Thyroglossal duct carcinoma, a rare clinical condition characterized by ectopic thyroid adenocarcinoma within thyroglossal duct cysts (TGDCs), typically confirmed through intraoperative rapid pathology, this condition generally has a favorable prognosis. Nevertheless, comprehensive treatment guidelines across all disease stages are lacking, the purpose of this study is to report 1 case of the disease and propose the treatment plan for each stage of the disease. PATIENT CONCERNS: A patient presented with thyroid swelling, classified as C-TIRADS 4A following a physical examination. Preoperative thyroid puncture identified papillary thyroid carcinoma, and genetic testing revealed a BRAF gene exon 15-point mutation. Ancillary tests showed a slightly decreased thyroid stimulating hormone (TSH) level (0.172) with no other significant abnormalities. DIAGNOSES: Preoperative fine-needle aspiration cytology (FNAC) confirmed right-side thyroid cancer. Intraoperative exploration uncovered a TGDC and intraoperative rapid pathology confirmed thyroglossal duct carcinoma. INTERVENTIONS: A Sistrunk operation and ipsilateral thyroidectomy were performed. OUTCOMES: Postoperative recovery was satisfactory. LESSONS: Thyroglossal duct carcinoma is a rare disease affecting the neck. Due to limited clinical cases and the favorable prognosis associated with this condition, there is currently no established set of diagnostic and treatment guidelines. According to tumor size, lymph node metastasis, thyroid status and other factors, the corresponding treatment methods were established for each stage of thyroglossal duct cancer, which laid the foundation for the subsequent treatment development of this disease.

Online monitoring lignocellulosic particles by focus beam reflectance measurement for efficient bioprocessing.

Lignocellulose presents a promising alternative to fossil fuels. Monitoring the mass and size changes of lignocellulosic particles without disrupting the process can assist in adjusting pretreatment and enzymatic hydrolysis, where conventional sieving methods fall short. A method utilizing focused beam reflectance measurement (FBRM) was developed to establish mathematical correlations between FBRM chord information (chord length and count) and particle characteristics (weight and size) quantified through sieving. Results indicate particle size exhibits a linear correlation with the square weighted median chord length (Lsqr) with R2 at 0.93. Further, real-time bulk particle mass can be predicted using Lsqr and chord count (R2 0.98). These correlations are applicable in range 53 µm to 358.5 µm. Real-time monitoring of enzymatic hydrolysis of corn stalks has demonstrated the practical applicability of FBRM. This study introduces a novel approach for online characterization of lignocellulosic particles, thereby enhancing lignocellulosic biorefineries.

Modular Access to Functionalized Oxetanes as Benzoyl Bioisosteres.

Bioisosterism is a valuable principle exploited in drug discovery to fine-tune physicochemical properties of bioactive compounds. Functionalized 3-aryl oxetanes, as an important class of bioisosteres for benzoyl groups (highly prevalent structures in approved drugs), have been rarely utilized in agrochemicals and pharmaceuticals due to significant synthetic challenges. Here, we present a modular synthetic strategy based on the unexplored yet readily available reagents, oxetanyl trichloroacetimidates, inspired by Schmidt glycosylation, enabling easy access to a library of functionalized oxetanes. This operationally simple protocol leverages the vast existing libraries of aryl halides and various nucleophiles. The power and generality of this approach is demonstrated by late-stage functionalization of complex molecules, as well as the rapid synthesis of oxetane analogues of bioactive molecules and marketed drugs. Preliminary mechanistic study suggests that the oxygen atom in the oxetane ring plays a crucial role in stabilizing the carbocation intermediates.

Effect of family intervention on relapse rate of Chinese patients with alcohol-dependent.

Objective: This study explored the impact of a family intervention on the relapse rate of Chinese patients with alcohol dependence. Methods: A total of 151 male patients with alcohol dependence who were discharged from the Substance Dependence Department of the Wenzhou Seventh People's Hospital from January to December 2020 were selected. They were divided into the control (n = 73) and experimental (n = 78) groups. Patients in both groups received routine alcohol cessation treatment. Moreover, patients in the experimental group were followed up by a professional psychiatrist to carry out individual family intervention. The Family Function Rating Scale (FAD), a Self-made general information questionnaire, and the Chinese version of the Family Intimacy and Adaptability Scale (FACESI-CV) were performed. Re-drinking rate and readmission rate were assessed. Results: Family intervention could reduce relapse rate (31, 39.74%) and rehospitalization (27, 34.62%) compared with the control group. After family training, FAD factor scores were improved in the experiment group in comparison with the control group. Family training improved communication (18.2 ± 3.7), role (20.8 ± 2.5), emotional response (10.8 ± 1.8), emotional involvement (13.7 ± 1.2), behavioral control (19.8 ± 1.2), and overall functionality (23.5 ± 2.1) in the experiment group in comparison with the control group. After family training, intimacy (70.5 ± 8.7) and adaptability (64.1 ± 6.9) in the experiment group was higher than in the control group. After family intervention, Michigan Alcohol Dependence Scale (MAST) (9.21 ± 0.68) and Short-Form 36 (SF-36) (80.32 ± 4.47) in the experiment group were higher than the control group. Conclusion: Family intervention for families of patients with alcohol dependence can improve their family function, increase their family intimacy and adaptability, and reduce the rate of relapse.

Effect of thermal treatment of illite on the bioavailability of copper and zinc in the aerobic composting of pig manure with corn straw.

The large amount of various types of heavy metals in animal manure applied to agricultural field has caused severe threat to the ecosystems of soil environments. In this study, the effect of thermal treatment of illite on the bioavailability of copper (Cu) and zinc (Zn) in the aerobic composting of pig manure with corn straw biochar was investigated. The objectives of this study were to characterize the variations in the bioavailability of Cu and Zn in the aerobic composting of pig manure added with illite treated with high temperatures and to identify the relatively dominant microbes involved in the formation of humus and passivation of heavy metals in pig manure composting based on 16S rRNA high-throughput sequencing analysis. The results showed that in comparison with the raw materials of pig manure, the bioavailability of Zn and Cu in the control and three experimental composting groups, i.e., group I (with untreated illite), group I-2 (with illite treated under 200°C), and group I-5 (with illite treated under 500°C), was decreased by 27.66 and 71.54%, 47.05 and 79.80%, 51.56 and 81.93%, and 58.15 and 86.60%, respectively. The results of 16S rRNA sequencing analysis revealed that in the I-5 group, the highest relative abundance was detected in Fermentimonas, which was associated with the degradation of glucose and fructose, and the increased relative abundances were revealed in the microbes associated with the formation of humus, which chelated with Zn and Cu to ultimately reduce the bioavailability of heavy metals and their biotoxicity in the compost. This study provided strong experimental evidence to support the application of illite in pig manure composting and novel insights into the selection of appropriate additives (i.e., illite) to promote humification and passivation of different heavy metals in pig manure composting.

Cross-frequency modulation of postural fluctuations and scalp EEG in older adults: error amplification feedback for rapid balance adjustments.

Virtual error amplification (VEA) in visual feedback enhances attentive control over postural stability, although the neural mechanisms are still debated. This study investigated the distinct cortical control of unsteady stance in older adults using VEA through cross-frequency modulation of postural fluctuations and scalp EEG. Thirty-seven community-dwelling older adults (68.1 ± 3.6 years) maintained an upright stance on a stabilometer while receiving either VEA or real error feedback. Along with postural fluctuation dynamics, phase-amplitude coupling (PAC) and amplitude-amplitude coupling (AAC) were analyzed for postural fluctuations under 2 Hz and EEG sub-bands (theta, alpha, and beta). The results revealed a higher mean frequency of the postural fluctuation phase (p = .005) and a greater root mean square of the postural fluctuation amplitude (p = .003) with VEA compared to the control condition. VEA also reduced PAC between the postural fluctuation phase and beta-band EEG in the left frontal (p = .009), sensorimotor (p = .002), and occipital (p = .018) areas. Conversely, VEA increased the AAC of posture fluctuation amplitude and beta-band EEG in FP2 (p = .027). Neither theta nor alpha band PAC or AAC were affected by VEA. VEA optimizes postural strategies in older adults during stabilometer stance by enhancing visuospatial attentive control of postural responses and facilitating the transition of motor states against postural perturbations through a disinhibitory process. Incorporating VEA into virtual reality technology is advocated as a valuable strategy for optimizing therapeutic interventions in postural therapy, particularly to mitigate the risk of falls among older adults.

Embryo Microinjection for Transgenesis in Drosophila.

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the construction of transgenic flies. Microinjection requires some types of equipment, including a microinjector, a micromanipulator, an inverted microscope, and a stereo microscope. Plasmids isolated with a plasmid miniprep kit are qualified for microinjection. Embryos at the pre-blastoderm or syncytial blastoderm stage, where nuclei share a common cytoplasm, are subjected to microinjection. A cell strainer eases the process of dechorionating embryos. The optimal time for dechorionation and desiccation of embryos needs to be determined experimentally. To increase the efficiency of embryo microinjection, needles prepared by a puller need to be beveled by a needle grinder. In the process of grinding needles, we utilize a foot air pump with a pressure gauge to avoid the capillary effect of the needle tip. We routinely inject 120-140 embryos for each plasmid and obtain at least one transgenic line for around 85% of plasmids. This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and presents a detailed protocol for embryo microinjection for transgenesis in Drosophila.

The receptor kinase OsANX limits precocious flowering and inflorescence over-branching and maintains pollen tube integrity in rice.

CrRLK1L subfamily members are involved in diverse growth- and development-related processes in Arabidopsis. However, the functions of their counterparts in rice are unknown. Here, OsANX expression was detected in developing inflorescences, mature pollen grains, and growing pollen tubes, and it was localized to the plasma membrane in pollen grains and tobacco epidermal cells. Homozygous osanx progeny could not be segregated from the CRISPR/Cas9-edited mutants osanx-c1+/- and osanx-c2+/-, and such progeny were segregated only occasionally from osanx-c3+/-. Further, all three alleles showed osanx male but not female gamete transmission defects, in line with premature pollen tube rupture in osanx-c3. Additionally, osanx-c3 exhibited precocious flowering, excessively branched inflorescences, and an extremely low seed setting rate of 1.4 %, while osanx-c2+/- and osanx-c3+/- had no obvious defects in inflorescence development or the seed setting rate compared to wild-type Nipponbare (Nip). Consistent with this, the complemented line pPS1:OsANX-GFP/osanx-c2 (PSC), in which the lack of OsANX expression was inflorescence-specific, showed slightly earlier flowering and overly-branched panicles. Multiple inflorescence meristem transition-related and inflorescence architecture-related genes were expressed at higher levels in osanx-c3 than in Nip; thus, they may partially account for the aforementioned mutant phenotypes. Our findings broaden our understanding of the biological functions of OsANX in rice.

Engineering carbon source division of labor for efficient α-carotene production in Corynebacterium glutamicum.

Effective utilization of glucose, xylose, and acetate, common carbon sources in lignocellulose hydrolysate, can boost biomanufacturing economics. However, carbon leaks into biomass biosynthesis pathways instead of the intended target product remain to be optimized. This study aimed to enhance α-carotene production by optimizing glucose, xylose, and acetate utilization in a high-efficiency Corynebacterium glutamicum cell factory. Heterologous xylose pathway expression in C. glutamicum resulted in strain m4, exhibiting a two-fold increase in α-carotene production from xylose compared to glucose. Xylose utilization was found to boost the biosynthesis of pyruvate and acetyl-CoA, essential precursors for carotenoid biosynthesis. Additionally, metabolic engineering including pck, pyc, ppc, and aceE deletion, completely disrupted the metabolic connection between glycolysis and the TCA cycle, further enhancing α-carotene production. This strategic intervention directed glucose and xylose primarily towards target chemical production, while acetate supplied essential metabolites for cell growth recovery. The engineered strain C. glutamicum m8 achieved 30 mg/g α-carotene, 67% higher than strain m4. In fed-batch fermentation, strain m8 produced 1802 mg/L of α-carotene, marking the highest titer reported to date in microbial fermentation. Moreover, it exhibited excellent performance in authentic lignocellulosic hydrolysate, producing 216 mg/L α-carotene, 1.45 times higher than the initial strain (m4). These labor-division strategies significantly contribute to the development of clean processes for producing various valuable chemicals from lignocellulosic resources.

Assessment of chlorine and hydrogen peroxide on airborne bacteria: Disinfection efficiency and induction of antibiotic resistance.

Airborne pathogens severely threaten public health worldwide. Air disinfection is essential to ensure public health. However, excessive use of disinfectants may endanger environmental and ecological security due to the residual disinfectants and their by-products. This study systematically evaluated disinfection efficiency, induction of multidrug resistance, and the underlying mechanisms of disinfectants (NaClO and H2O2) on airborne bacteria. The results showed that airborne bacteria were effectively inactivated by atomized NaClO (>160 µg/L) and H2O2 (>320 µg/L) after 15 min. However, some bacteria still survived after disinfection by atomized NaClO (0-80 µg/L) and H2O2 (0-160 µg/L), and they exhibited significant increases in antibiotic resistance. The whole-genome sequencing of the resistant bacteria revealed distinct mutations that were responsible for both antibiotic resistance and virulence. This study also provided evidences and insights into possible mechanisms underlying the induction of antibiotic resistance by air disinfection, which involved intracellular reactive oxygen species formation, oxidative stress responses, alterations in bacterial membranes, activation of efflux pumps, and the thickening of biofilms. The present results also shed light on the role of air disinfection in inducing antibiotic resistance, which could be a crucial factor contributing to the global spread of antibiotic resistance through the air.

CRISPR-Based Modular Assembly for High-Throughput Construction of a UAS-cDNA/ORF Plasmid Library.

Functional genomics screening offers a powerful approach to probe gene function and relies on the construction of genome-wide plasmid libraries. Conventional approaches for plasmid library construction are time-consuming and laborious. Therefore, we recently developed a simple and efficient method, CRISPR-based modular assembly (CRISPRmass), for high-throughput construction of a genome-wide upstream activating sequence-complementary DNA/open reading frame (UAS-cDNA/ORF) plasmid library. Here, we present a protocol for CRISPRmass, taking as an example the construction of a GAL4/UAS-based UAS-cDNA/ORF plasmid library. The protocol includes massively parallel two-step test tube reactions followed by bacterial transformation. The first step is to linearize the existing complementary DNA (cDNA) or open reading frame (ORF) cDNA or ORF library plasmids by cutting the shared upstream vector sequences adjacent to the 5' end of cDNAs or ORFs using CRISPR/Cas9 together with single guide RNA (sgRNA), and the second step is to insert a UAS module into the linearized cDNA or ORF plasmids using a single step reaction. CRISPRmass allows the simple, fast, efficient, and cost-effective construction of various plasmid libraries. The UAS-cDNA/ORF plasmid library can be utilized for gain-of-function screening in cultured cells and for constructing a genome-wide transgenic UAS-cDNA/ORF library in Drosophila.

Anti-neuroinflammatory andrastin-type meroterpenoids from the marine-derived fungus Penicillium chrysogenum HNNU w0032.

A new andrastin-type meroterpenoid penimerodione A (1), and three known analogues (2-4), were isolated from the culture of a marine-derived fungus Penicillium chrysogenum HNNU w0032 by the guidance of MS/MS-based molecular networking. The planar structure of 1 was established by extensive NMR spectroscopic and HRESIMS analyses, and the absolute configuration was elucidated by a single-crystal X-ray diffraction. Compound 1 showed significant inhibitory effect on NO production in LPS-stimulated BV-2 macrophages with an IC50 value of 5.9 ± 0.3 µM. The Western blot result revealed that compound 1 exerted an anti-neuroinflammatory effect via the MAPK signalling pathway.

[Dynamic Response of Aerobic Denitrification Bacteria to Water Quality in Baiyangdian Lake Under Different Hydrological Scenarios].

In order to explore the evolution law and driving mechanism of aerobic denitrification bacteria in Baiyangdian Lake under different hydrological scenarios, based on water quality survey and high-throughput sequencing technology, this study conducted a water quality factor analysis and aerobic denitrification bacteria α-diversity analysis, species composition, and network analysis. The results showed that the water body of Baiyangdian Lake was weakly alkaline, with the highest T and the lowest DO in the rainy season and the lowest T and the highest DO in the freezing season. There were significant differences between NH4+-N, NO2--N, NO3--N, TN, permanganate index, Fe, and Mn in Baiyangdian water under different hydrological scenarios (P < 0.01), and there was no significant difference in TP under different hydrological scenarios (P > 0.05). The largest category in water bodies under different hydrological scenarios was Proteobacteria, and the genera with a higher relative abundance were Magnetospirillum, Aeromonas, Pseudomonas, Azospirillum, and Bradyrhizobium. In addition, within the aerobic denitrifying bacteria community, there were significant differences in α-diversity (P < 0.001), with the highest abundance of microbial communities occurring during the freezing period, and the highest diversity and evenness of microbial communities during the dry and freezing periods. According to the RDA and Mantel analyses, the water quality driving factors of flora were different under different hydrological scenarios. The water quality driving factors of flora in the dry season were pH, NO3--N, NO2--N, and permanganate index; the driving factors of flora in the rainy season were pH, T, DO, NO2--N, and TP; the driving factors of flora in the normal season were NO2--N, Fe, and permanganate index; and the driving factors of flora in the freezing season were NO3--N and NONO2--N. Network analysis showed that there were temporal differences in species related to water quality driving factors. The genera related to water quality driving factors during the dry season were Magnetospirillum, Aeromonas, and Azoarcus, whereas the genera related to the rainy season were Magnetospirillum, Pseudomonas, and Aeromonas. The genera related to the normal season were Magnetospirillum, Pseudomonas, and Limnohabitans, and the genera related to the freezing period were Magnetospirillum, Azoarcus, and Pseudomonas. The relationship between key water quality factors (mainly T, DO, NO3--N, and permanganate index) and aerobic denitrification flora in different hydrological scenarios was gradually changing with time. In conclusion, the study on the evolution characteristics of aerobic denitrification bacteria in Baiyangdian Lake under different hydrological scenarios and the driving mechanism of environmental factors could provide a basis for understanding the evolution mechanism of aerobic denitrification bacteria in the natural environment.