Ultrafast Picometer-Resolved Molecular Structure Imaging by Laser-Induced High-Order Harmonics.

Real-time visualization of molecular transformations is a captivating yet challenging frontier of ultrafast optical science and physical chemistry. While ultrafast x-ray and electron diffraction methods can achieve the needed subangstrom spatial resolution, their temporal resolution is still limited to hundreds of femtoseconds, much longer than the few femtoseconds required to probe real-time molecular dynamics. Here, we show that high-order harmonics generated by intense femtosecond lasers can be used to image molecules with few-ten-attosecond temporal resolution and few-picometer spatial resolution. This is achieved by exploiting the sensitive dependence of molecular recombination dipole moment to the geometry of the molecule at the time of harmonic emission. In a proof-of-principle experiment, we have applied this high-harmonic structure imaging (HHSI) method to monitor the structural rearrangement in NH_{3}, ND_{3}, and N_{2} from one to a few femtoseconds after the molecule is ionized by an intense laser. Our findings establish HHSI as an effective approach to resolve molecular dynamics with unprecedented spatiotemporal resolution, which can be extended to trace photochemical reactions in the future.

Association of immune-related adverse events with COVID-19 pneumonia in lung cancer patients receiving immune checkpoint inhibitors: a cross-sectional study in China.

BACKGROUND: Immune checkpoint inhibitors (ICIs) are commonly used to treat lung cancer patients, but their use can lead to immune-related adverse events (irAEs), which pose a challenge for treatment strategies. The impact of irAEs on the incidence of COVID-19 pneumonia in lung cancer patients during the ongoing COVID-19 pandemic is unclear. This study aims to investigate the association between irAEs and COVID-19 pneumonia in lung cancer patients receiving ICIs. METHODS: We conducted a cross-sectional study of lung cancer patients who received ICIs and were infected with COVID-19 due to the Omicron variant between December 2022 and February 2023 in China. We collected data on irAEs and COVID-19 outcomes. Logistic regression analyses were used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for the association between irAEs and the incidence of COVID-19 pneumonia. RESULTS: A total of 193 patients were enrolled, with 72 patients (37.30%) in the irAEs group and 121 patients (62.70%) in the non-irAEs group. Twenty-six patients (13.47%) developed COVID-19 pneumonia and 6 patients (3.11%) progressed to severe cases after COVID-19 infection. Multivariate logistic regression showed that the lung cancer patients who experienced irAEs was significantly associated with a higher incidence rate of COVID-19 pneumonia (OR = 9.56, 95%CI: 2.21-41.33; P = 0.0025). CONCLUSION: Our study suggests that lung cancer patients receiving ICIs and experiencing irAEs may have a higher risk of developing COVID-19 pneumonia due to the Omicron variant. Therefore, close monitoring of these patients during the COVID-19 pandemic is necessary to mitigate this risk.

Non-rhizosphere reinforces the contributions of Feammox and anammox to nitrogen loss than rhizosphere in riparian zones.

The emergence of anaerobic ammonium oxidation (anammox) coupled to iron reduction (named Feammox) refreshes the microbial pathways for nitrogen (N) loss. However, the ecological role of Feammox, compared with conventional denitrification and anammox, in microbial N attenuation in ecosystems remains unclear. Here, the specific contribution of Feammox to N loss and the underlying microbiome interactive characteristics in a riparian ecosystem were investigated through 15N isotope tracing and molecular analysis. Feammox was highlighted in the riparian interface soils and maximally contributed 14.2% of N loss. Denitrification remained the dominant contributor to N loss (68.0%-95.3%), followed by anammox (5.7%-19.1%) and Feammox (0-14.2%). The rates of Feammox and anammox significantly decreased in rhizosphere soils (0.15 ± 0.08 µg N g-1 d -1 for Feammox, 0.80 ± 0.39 µg N g-1 d -1 for anammox) compared with those in non-rhizosphere soils; however, the activities of denitrification remarkably increased in the rhizosphere (13.17 ± 3.71 µg N g-1 d -1). In rhizosphere soils, the competition between bioavailable organic matter (e.g., amino acids and carbohydrates) and ammonium for electron acceptor [i.e., Fe(III)] was the vital inducement for restricted Feammox, while the nitrite consumption boosted by heterotrophic denitrifiers was responsible for weakened anammox. The functional gene of autotrophic Acidimicrobiaceae bacterium A6, instead of heterotrophic Geobacteraceae spp., was significantly positively correlated with Feammox activity. Rare iron-reducing bacteria showed higher node degrees in the non-rhizosphere network than in the rhizosphere network. A syntrophic relationship was found between iron-reducing bacteria (e.g., Anaeromyxobacter, Geobacter) and iron-oxidizing bacteria (e.g., Sideroxydans) in the non-rhizosphere network and facilitated the Feammox pathway. This study provides an in-depth exploration of microbial driven N loss in a riparian ecosystem and introduces new insights into riparian management practices toward high-efficient N pollution alleviation.

Insights into microbial interactive mechanism regulating dissimilatory nitrate reduction processes in riparian freshwater aquaculture sediments.

Aquaculture can substantially alter the accumulation and cycling of nutrients in sediments. However, the microbial mechanisms mediating sediment dissimilatory nitrate (NO3-) reduction in freshwater aquaculture ponds are still unclear, which rule the removal and retention of N element. In the present study, three microbial NO3- reduction processes in riparian aquaculture pond sediments (i.e., crab, shrimp and fish ponds) and natural freshwater sediments (i.e., lakes and rivers) were investigated via isotopic tracing and molecular analyses. The potential rates of denitrification, anaerobic ammonium oxidation (anammox) and dissimilatory nitrate reduction to ammonium (DNRA) significantly increased in the aquaculture ponds compared with the natural freshwaters. Denitrification contributed 90.40-94.22% to the total NO3- reduction (product as N2), followed by 2.49-5.82% of anammox (product as N2) and 2.09-5.18% of DRNA (product as NH4+). The availability of C and N substrates, rather than functional gene abundance, regulated the activities of NO3- reductions and microbiome composition. Microbial mechanism based on network analysis indicated that heterotrophic denitrifiers and DNRA bacteria (e.g., Bacillus, Micromonospora, Mycobacterium and Brachybacterium) determined the community structure and function for N conversions in aquaculture ponds, whereas the such microbial network in natural freshwater sediments was manipulated by autotrophic denitrifiers (e.g., Desulfuromonas, Polaromonas, Solitalea). Collectively, this study provides an in-depth exploration of microbial nitrogen removal in freshwater aquaculture areas and supports management strategies for N pollution caused by reclamation for aquaculture in riparian zones.

Rotational echo spectroscopy for accurate measurement of molecular alignment.

We measure the molecular alignment induced in gas using molecular rotational echo spectroscopy. Our results show that the echo intensity and the time interval between the local extremas of the echo responses depend sensitively on the pump intensities and the initial molecular rotational temperature, respectively. This allows us to accurately extract these experimental parameters from the echo signals and then further determine the molecular alignment in experiments. The accuracy of our method has been verified by comparing the simulation with the extracted parameters from the molecular alignment experiment performed with a femtosecond pump pulse.

Effect of EGFR amplification on the prognosis of EGFR-mutated advanced non-small-cell lung cancer patients: a prospective observational study.

BACKGROUND: Epidermal growth factor receptor (EGFR) amplification refers to the copy number increase of EGFR gene, and is often identified as a "bypass" way of Epidermal growth factor receptor Tyrosine kinase inhibitors (EGFR-TKI) resistance. We aimed to explore the effect of EGFR amplification on EGFR mutation treatment-naive advanced non-squamous non-small cell lung cancer (NSCLC) patients. METHODS: We conducted a prospective observational study in single center, enrolling advanced non-squamous NSCLC patients receiving Tyrosine kinase inhibitors (TKIs) between March 3, 2019, and February 1, 2022. Next-generation sequencing (NGS) was used to detect genetic alterations in tumor tissue samples. Progression-free survival (PFS) curves were performed using the Kaplan-Meier method. Univariate and multivariate analyses were used to evaluate factors affecting the efficacy of TKIs. RESULTS: A total of 117 treatment-naive advanced NSCLC patients were identified in this study. EGFR amplification was found in 22 of 117 (18.8%) patients with EGFR mutations. Of 22 patients with EGFR amplification, 10 patients harbored EGFR 19 del, 11 patients with 21-L858R. The median follow-up time was 22.47 months. The median PFS of the patients with or without EGFR amplification was 8.25 months and 10.67 months, respectively (log-rank test, P = 0.63). In multivariate analysis, EGFR amplification was not an independent prognosis factor for the patients receiving first-line TKIs [HR = 1.38, 95%CI (0.73-2.58), P = 0.321]. Subgroup analysis revealed that EGFR amplification is a risk factor for progression in the brain metastasis population. [HR = 2.28, 95%CI (1.01, 5.14), P = 0.047]. CONCLUSION: EGFR amplification is not an independent prognosis factor for PFS in advanced non-squamous NSCLC patients receiving first-line TKIs. However, it is an independent risk factor for PFS in the brain metastasis population.

Multilevel quantum interference in the formation of high-order fractional molecular alignment echoes.

We theoretically investigate the formation of the high-order fractional alignment echo in OCS molecule and systematically study the dependence of echo intensity on the intensities and time delay of the two excitation pulses. Our simulations reveal an intricate dependence of the intensity of high-order fractional alignment echo on the laser conditions. Based on the analysis with rotational density matrix, this intricate dependence is further demonstrated to arise from the interference of multiple quantum pathways that involve multilevel rotational transitions. Our result provides a comprehensive multilevel picture of the quantum dynamics of high-order fractional alignment echo in molecular ensembles, which will facilitate the development of "rotational echo spectroscopy."

Lhermitte-Duclos disease: A case report and literature review. / 小脑发育不良性神经节细胞瘤1ä¾‹åŠæ–‡çŒ®å¤ä¹ .

Lhermitte-Duclos disease (LDD) is a type of rare brain tumor located in posterior fossa. A patient with LDD located in the left cerebellum and vermis was admitted by the Department of Neurosurgery, Xiangya Hospital, Central South University. MRI scan showed slightly heterogeneous enhancement at the region close to vermis. The patient underwent partial resection on August 11, 2016 without postoperative chemoradiotherapy. The progress free survival was 11 months and the overall survival was 17 months. What the case reveals is that the partial resection is not beneficial to these patients with LDD as the residual lesion probably recurs in a short term after operation. The pathogenesis, diagnosis and treatment of LDD are explored and summarized in combination with relevant literature.

Measuring the rotational temperature and pump intensity in molecular alignment experiments via high harmonic generation.

We demonstrate a method to simultaneously measure the rotational temperature and pump intensity in laser-induced molecular alignment by the time-resolved high harmonic spectroscopy (HHS). It relies on the sensitive dependence of the arising times of the local minima and maxima of the harmonic yields at the rotational revivals on the pump intensity and rotational temperature. By measuring the arising times of these local extrema from the time-resolved harmonic signals, the rotational temperature and pump intensity can be accurately measured. We have demonstrated our method using N2 molecules. The validity and robustness of our method are tested with different harmonic orders and by changing the gas pressures as well as the distance between the gas exit and the optical axis. Moreover, we have also demonstrated the versatility of our method by applying it to CO2 molecules.

Dual role of mitochondria in producing melatonin and driving GPCR signaling to block cytochrome c release.

G protein-coupled receptors (GPCRs) are classically characterized as cell-surface receptors transmitting extracellular signals into cells. Here we show that central components of a GPCR signaling system comprised of the melatonin type 1 receptor (MT1), its associated G protein, and ß-arrestins are on and within neuronal mitochondria. We discovered that the ligand melatonin is exclusively synthesized in the mitochondrial matrix and released by the organelle activating the mitochondrial MT1 signal-transduction pathway inhibiting stress-mediated cytochrome c release and caspase activation. These findings coupled with our observation that mitochondrial MT1 overexpression reduces ischemic brain injury in mice delineate a mitochondrial GPCR mechanism contributing to the neuroprotective action of melatonin. We propose a new term, "automitocrine," analogous to "autocrine" when a similar phenomenon occurs at the cellular level, to describe this unexpected intracellular organelle ligand-receptor pathway that opens a new research avenue investigating mitochondrial GPCR biology.

All-optical measurement of high-order fractional molecular echoes by high-order harmonic generation.

An all-optical measurement of high-order fractional molecular echoes is demonstrated by using high-order harmonic generation (HHG). Excited by a pair of time-delayed short laser pulses, the signatures of full and high order fractional (1/2 and 1/3) alignment echoes are observed in the HHG signals measured from CO 2 molecules at various time delays of the probe pulse. By increasing the time delay of the pump pulses, much higher order fractional (1/4) alignment echo is also observed in N 2O molecules. With an analytic model based on the impulsive approximation, the spatiotemporal dynamics of the echo process are retrieved from the experiment. Compared to the typical molecular alignment revivals, high-order fractional molecular echoes are demonstrated to dephase more rapidly, which will open a new route towards the ultrashort-time measurement. The proposed HHG method paves an efficient way for accessing the high-order fractional echoes in molecules.

Dimethyl phenyl piperazine iodide (DMPP) induces glioma regression by inhibiting angiogenesis.

1,1-Dimethyl-4-phenyl piperazine iodide (DMPP) is a synthetic nicotinic acetylcholine receptor (nAChR) agonist that could reduce airway inflammation. In this study, we demonstrated that DMPP could dramatically inhibit glioma size maintained on the chick embryonic chorioallantoic membrane (CAM). We first performed MTT and BrdU incorporation experiments on U87 glioma cells in vitro to understand the mechanism involved. We established that DMPP did not significantly affect U87 cell proliferation and survival. We speculated that DMPP directly caused the tumor to regress by affecting the vasculature in and around the implanted tumor on our chick CAM model. Hence, we conducted detailed analysis of DMPP's inhibitory effects on angiogenesis. Three vasculogenesis and angiogenesis in vivo models were used in the study which included (1) early chick blood islands formation, (2) chick yolk-sac membrane (YSW) and (3) CAM models. The results revealed that DMPP directly suppressed all developmental stages involved in vasculogenesis and angiogenesis - possibly by acting through Ang-1 and HIF-2α signaling. In sum, our results show that DMPP could induce glioma regression grown on CAM by inhibiting vasculogenesis and angiogenesis.

Association between serum C-reactive protein (CRP) and Omicron variant COVID-19 pneumonia in cancer patients: A multicenter cross-sectional study at the end of 2022 in China.

Cancer patients with COVID-19 have a higher infection rate and mortality rate than non-cancer patients. However, there are few studies on the correlation between the serum C-reactive protein (CRP) and cancer patients with COVID-19. This study aims to investigate the association between serum CRP and the incidence of COVID-19 pneumonia in cancer patients at the end of 2022 in China. This cross-sectional study with a retrospective cohort between December 2022 and February 2023 assessed cancer patients complicated with COVID-19 infection in 2 Chinese institutions. Logistic regression analyses were used to compute Odds ratio (OR) and 95%CIs for the association between serum CRP and the incidence of COVID-19 pneumonia in cancer patients. A total of 213 cancer patients with COVID-19 were enrolled. Eighty-six patients (40.4%) developed COVID-19 pneumonia, among which 23 patients (10.8%) progressed to severe cases. Univariate Logistic regression showed that high CRP levels were found to be an unfavorable predictor of COVID-19 outcomes (OR = 17.9, 95%CI: 7.3, 43.6; P < .001). In the multivariate analysis, high CRP levels were associated with a higher incidence rate of COVID-19 pneumonia (OR = 9.8, 95%CI: 2.2, 43.8; P = .003). In the multivariate logistic regression model and smooth curve fitting, we found a correlation between CRP and COVID-19 pneumonia. The serum CRP was associated with the incidence of Omicron variant COVID- 19 pneumonia in cancer patients. Hence, cancer patients with high CRP level maybe need for timely computer tomography examination and more aggressive treatment.

Unveiling different antibiotic degradation mechanisms on dual reaction center catalysts with nitrogen vacancies via peroxymonosulfate activation.

Metal-nitrogen-site catalysts are widely recognized as effective heterogeneous catalysts in peroxymonosulfate (PMS)-based advanced oxidation processes. However, the selective oxidation mechanism for organic pollutants is still contradictory. In this work, manganese-nitrogen active centers and tunable nitrogen vacancies were synchronously constructed on graphitic carbon nitride (LMCN) through l-cysteine-assisted thermal polymerization to reveal different antibiotic degradation mechanisms. Benefiting from the synergism of manganese-nitrogen bond and nitrogen vacancies, the LMCN catalyst exhibited excellent catalytic activity for the degradation of tetracycline (TC) and sulfamethoxazole (SMX) antibiotics with first-order kinetic rate constants of 0.136 min-1 and 0.047 min-1, which were higher than those of other catalysts. Electron transfer dominated TC degradation at low redox potentials, while electron transfer and high-valent manganese (Mn (V)) were responsible for SMX degradation at high redox potentials. Further experimental studies unveiled that the pivotal role of nitrogen vacancies is to promote electron transfer pathway and Mn(V) generation, while nitrogen-coordinated manganese as the primary catalytic active site determines Mn(V) generation. In addition, the antibiotic degradation pathways were proposed and the toxicity of byproducts was analyzed. This work provides an inspiring idea for the controlled generation of reactive oxygen species by targeted activation of PMS.

Immune Checkpoint Inhibitors and Tuberculosis Infection in Lung Cancer: A Case Series and Systematic Review With Pooled Analysis.

The association between immune checkpoint inhibitors (ICIs) and tuberculosis (TB) infection in patients with lung cancer remains largely elusive. We performed a systematic review and conducted a retrospective analysis of TB infection in patients with lung cancer and ICI exposure to assess the clinical characteristics and outcomes using PubMed, EMBASE, and the Cochrane Library. The time interval from ICI administration to diagnosis of TB between patients with and without a history of TB was compared using Kaplan-Meier analysis. A multivariate Cox regression model was used to identify potential risk factors associated with the time interval of TB development. Twenty-four studies including 53 patients with lung cancer were included. The median age of the patients was 64 years. Eight patients had a history of TB. The median time interval from ICI administration to TB diagnosis was 3 months. In retrospective analysis, 5 (1.16%, 95%CI 0.38% to 2.68%) patients with lung cancer developed TB during ICI treatment. The median time interval was 10.4 months. In a pooled analysis, the median time interval in the without-TB and with-TB groups was 7.00 and 2.35 months, respectively (P = .034). Multivariate Cox regression analyses revealed a history of TB to be an independent factor affecting the time interval of TB activation in patients with lung cancer and ICI exposure (HR 3.59; 95%CI 1.17 to 11.02; P = .026). Therefore, TB infection should be considered in patients with lung cancer during or after ICI treatment. Moreover, we found TB history to be a positive risk factor for a shorter median time interval from ICI to TB diagnosis in patients with lung cancer receiving ICI.

Potential of crop straw incorporation for replacing chemical fertilizer and reducing nutrient loss in Sichuan Province, China.

Sichuan Province is rich in crop straw, yet little is known about its spatial distribution pattern, potential in replacing chemical fertilizer and mitigating nutrient loss. Based on the statistical data and literature review, the spatial distribution and potential of nutrient resources in crop straw for replacing chemical fertilizers was evaluated in this study. The nutrient loss with both crop incorporation and chemical fertilizer application were examined using a nutrient release coefficient method and compared. Results showed that Chengdu Plain, Northeast and South Sichuan produced more than 95% of the total straw nutrient resources during the period of 2016-2020. The potential of crop straw to substitute potassium (K), nitrogen (N) and phosphorus (P) fertilizer were K2O 33.08-285.95 kg hm-2, N 9.52-82.32 kg hm-2 and P2O5 4.91-28.71 kg hm-2, respectively. If chemical fertilizer was substituted by all the available straw nutrient resources, N and P loss can be decreased by 55.12% and 65.84% in average in Sichuan Province. 343.93 t of N loss and 20.05 t of P loss can be reduced in plain areas, 122.88 t of N loss and 46.29 t of P loss can be reduced in mountainous and hilly areas, and 5.65 t of t N loss and 3.54 t of P loss can be reduced in plateau areas. It can be concluded that there were rich crop straw nutrient resources in Sichuan Province with obvious spatial variability, solid consideration should be put on to the proper use of crop straw nutrient resources, with the aim of chemical fertilizer reduction, nutrient loss reduction and sustainable development.

Acetylation of Atp5f1c Mediates Cardiomyocyte Senescence via Metabolic Dysfunction in Radiation-Induced Heart Damage.

Objective: Ionizing radiation (IR) causes cardiac senescence, which eventually manifests as radiation-induced heart damage (RIHD). This study is aimed at exploring the mechanisms underlying IR-induced senescence using acetylation proteomics. Methods: Irradiated mouse hearts and H9C2 cells were harvested for senescence detection. Acetylation proteomics was used to investigate alterations in lysine acetylation. Atp5f1c acetylation after IR was verified using coimmunoprecipitation (Co-IP). Atp5f1c lysine 55 site acetylation (Atp5f1c K55-Ac) point mutation plasmids were used to evaluate the influence of Atp5f1c K55-Ac on energy metabolism and cellular senescence. Deacetylation inhibitors, plasmids, and siRNA transfection were used to determine the mechanism of Atp5f1c K55-Ac regulation. Results: The mice showed cardiomyocyte and cardiac aging phenotypes after IR. We identified 90 lysine acetylation sites from 70 protein alterations in the heart in response to IR. Hyperacetylated proteins are primarily involved in energy metabolism. Among them, Atp5f1c was hyperacetylated, as confirmed by Co-IP. Atp5f1c K55-Ac decreased ATP enzyme activity and synthesis. Atp5f1c K55 acetylation induced cardiomyocyte senescence, and Sirt4 and Sirt5 regulated Atp5f1c K55 deacetylation. Conclusion: Our findings reveal a mechanism of RIHD through which Atp5f1c K55-Ac leads to cardiac aging and Sirt4 or Sirt5 modulates Atp5f1c acetylation. Therefore, the regulation of Atp5f1c K55-Ac might be a potential target for the treatment of RIHD.

Evidence for the occurrence of Feammox coupled with nitrate-dependent Fe(II) oxidation in natural enrichment cultures.

Feammox is a newly discovered process of anaerobic ammonium oxidation driven by Fe(III) reduction. Nitrate-dependent Fe(II) oxidation (NDFO) is the coupling of Fe(II) oxidation and nitrate reduction to produce N2 under anaerobic conditions. It has not been reported whether the coupling of the two reactions exists in natural enrichment. In this study, enrichment culture experiments were carrired out to prove the occurrence of Feammox with NDFO. The results indicated that the nitrogen and iron cycle were formed during natural enrichment cultures, including Fe(III) reduction and NH4+-N was oxidation to NO3--N, NO2--N and N2, Fe(III) and Fe(II) were cyclically formed, and Fe(II) was oxidized with NO3--N reduced to N2. The removal efficiencies of ammonium nitrogen and total nitrogen in the incubation were about 92.9% and 20% respectively. Organic carbon experiments indicate that sodium acetate can promote the initial NO3--N removal and a low concentration of organic carbon limited the NDFO process because iron-oxidizing bacteria are mixotrophic microorganisms. The added 9,10-anthraquinone-2,6-disulfonate (AQDS) in the later stage can promote NDFO to remove nitrate, thereby increasing the TN removal efficiency to 50%. 15N-isotope tracer incubations provided direct evidence for the occurrence of Feammox coupled to NDFO, with rates producing 30N2 of Feammox (0.024-0.0288 mg N·L-1·d-1) and NDFO (0.0465-0.0833 mg N·L-1·d-1) in three groups (Wetland/Wheat soil/Sediment). 16S rRNA sequencing further demonstrated that Pseudomonas, Rhodanobacter, Acinetobacter and Thermomonas were the dominant generas among the enrichment cultures, and these bacteria belonged to FeOB and FeRB, which may further promote Feammox coupled to NDFO in the cultivation system.

Filming movies of attosecond charge migration in single molecules with high harmonic spectroscopy.

Electron migration in molecules is the progenitor of chemical reactions and biological functions after light-matter interaction. Following this ultrafast dynamics, however, has been an enduring endeavor. Here we demonstrate that, by using machine learning algorithm to analyze high-order harmonics generated by two-color laser pulses, we are able to retrieve the complex amplitudes and phases of harmonics of single fixed-in-space molecules. These complex dipoles enable us to construct movies of laser-driven electron migration after tunnel ionization of N2 and CO2 molecules at time steps of 50 attoseconds. Moreover, the angular dependence of the migration dynamics is fully resolved. By examining the movies, we observe that electron holes do not just migrate along the laser polarization direction, but may swirl around the atom centers. Our result establishes a general scheme for studying ultrafast electron dynamics in molecules, paving a way for further advance in tracing and controlling photochemical reactions by femtosecond lasers.

Rapid cultivation of anammox bacteria by forming free cells in a membrane bioreactor.

The low growth rate of anammox bacteria is one of the main obstacles to its broad use in removing biological nitrogen in sewage treatment. Free cells show the characteristics of relatively rapid growth. To increase the growth rate, this study cultivated free-living anammox bacteria using a membrane bioreactor. The particle size distributions showed that more than 95.48% of biomass was less than 50 µm in size, and the growth rate of free-living anammox bacteria was shortened to 5.68 days after cultivation. Candidatus Brocadia was the most important anammox genus, with a relative abundance of 8.83%. The key functional genes, including hzs, hdh, and hao, were identified and expressed using metagenomic and metaproteomic analyses. After the rapid cultivation of the free-living anammox bacteria, a potential method shortening the start-up time was proposed to rapidly form anammox biofilm by attaching to carriers. PRACTITIONER POINTS: The free-living anammox bacteria are cultivated in a membrane bioreactor. More than 95.48% of biomass size is less than 50 µm. The doubling time of these free anammox bacteria was 5.68 days. The key functional genes of anammox bacteria were identified and expressed. A novel method is proposed to rapidly form anammox biofilm.