Unlocking the potential of chemically modified peptide nucleic acids for RNA-based therapeutics.

RNA therapeutics have emerged as next-generation therapy for the treatment of many diseases. Unlike small molecules, RNA targeted drugs are not limited by the availability of binding pockets on the protein, but rather utilize Watson-Crick (WC) base-pairing rules to recognize the target RNA and modulate gene expression. Antisense oligonucleotides (ASOs) present a powerful therapeutic approach to treat disorders triggered by genetic alterations. ASOs recognize the cognate site on the target RNA to alter gene expression. Nine single-stranded ASOs have been approved for clinical use and several candidates are in late-stage clinical trials for both rare and common diseases. Several chemical modifications, including phosphorothioates, locked nucleic acid, phosphorodiamidate, morpholino, and peptide nucleic acids (PNAs), have been investigated for efficient RNA targeting. PNAs are synthetic DNA mimics where the deoxyribose phosphate backbone is replaced by N-(2-aminoethyl)-glycine units. The neutral pseudopeptide backbone of PNAs contributes to enhanced binding affinity and high biological stability. PNAs hybridize with the complementary site in the target RNA and act by a steric hindrance--based mechanism. In the last three decades, various PNA designs, chemical modifications, and delivery strategies have been explored to demonstrate their potential as an effective and safe RNA-targeting platform. This review covers the advances in PNA-mediated targeting of coding and noncoding RNAs for a myriad of therapeutic applications.

Recent advancements in bionanomaterial applications of peptide nucleic acid assemblies.

Peptide nucleic acid (PNA) is a unique combination of peptides and nucleic acids. PNA can exhibit hydrogen bonding interactions with complementary nucleobases like DNA/RNA. Also, its polyamide backbone allows easy incorporation of biomolecules like peptides and proteins to build hybrid molecular constructs. Because of chimeric structural properties, PNA has lots of potential to build diverse nanostructures. However, progress in the PNA material field is still immature compared with its massive applications in antisense oligonucleotide research. Examples of well-defined molecular assemblies have been reported with PNA amphiphiles, self-assembling guanine-PNA monomers/dimers, and PNA-decorated nucleic acids/ polymers/ peptides. All these works indicate the great potential of PNA to be used as bionanomaterials. The review summarizes the recent reports on PNA-based nanostructures and their versatile applications. Additionally, this review shares a perspective to promote a better understanding of controlling molecular assembly by the systematic structural modifications of PNA monomers.

K-ras mutation detected by peptide nucleic acid-clamping polymerase chain reaction, Ki-67, S100P, and SMAD4 expression can improve the diagnostic accuracy of inconclusive pancreatic EUS-FNB specimens.

OBJECTIVES: We aimed to assess the diagnostic utility of an immunohistochemical panel including calcium-binding protein P, p53, Ki-67, and SMAD family member 4 and K-ras mutation for diagnosing pancreatic solid lesion specimens obtained by endoscopic ultrasound-guided fine-needle biopsy and to confirm their usefulness in histologically inconclusive cases. METHODS: Immunohistochemistry and peptide nucleic acid-clamping polymerase chain reaction for K-ras mutation were performed on 96 endoscopic ultrasound-guided fine-needle biopsy specimens. The diagnostic efficacy of each marker and the combination of markers was calculated. The diagnostic performances of these markers were evaluated in 27 endoscopic ultrasound-guided fine-needle biopsy specimens with histologically inconclusive diagnoses. A classification tree was constructed. RESULTS: K-ras mutation showed the highest accuracy and consistency. Positivity in more than two or three of the five markers showed high diagnostic accuracy (94.6 % and 93.6 %, respectively), and positivity for more than three markers showed the highest accuracy for inconclusive cases (92.0 %). A classification tree using K-ras mutation, Ki-67, S100P, and SMAD4 showed high diagnostic performance, with only two misclassifications in inconclusive cases. CONCLUSIONS: K-ras mutation detection via peptide nucleic acid-clamping polymerase chain reaction is a stable and accurate method for distinguishing between pancreatic ductal adenocarcinoma and non-pancreatic ductal adenocarcinoma lesions. A classification tree using K-ras mutation, Ki-67, S100P, and SMAD4 helps increase the diagnostic accuracy of cases that are histologically difficult to diagnose.

Hybrid peptide-PNA monomers as building blocks for the fabrication of supramolecular aggregates.

Advantages like biocompatibility, biodegradability and tunability allowed the exploitation of peptides and peptidomimetics as versatile therapeutic or diagnostic agents. Because of their selectivity towards transmembrane receptors or cell membranes, peptides have also been identified as suitable molecules able to deliver in vivo macromolecules, proteins or nucleic acids. However, after the identification of the homodimer diphenylalanine (FF) as an aggregative motif inside the Aß1-42 polypeptide, short and ultrashort peptides have been studied as building blocks for the fabrication of supramolecular, ordered nanostructures for applications in biotechnological, biomedical and industrial fields. In this perspective, many hybrid molecules that combine FF with other chemical entities have been synthesized and characterized. Two novel hybrid derivatives (tFaF and cFgF), in which the FF homodimer is alternated with the peptide-nucleic acid (PNA) heterodimer "g-c" (guanine-cytosine) or "a-t" (adenine-thymine) and their dimeric forms (tFaF)2 and (cFgF)2 were synthesized. The structural characterization performed by circular dichroism (CD), Fourier transform infrared (FTIR) and fluorescence spectroscopies highlighted the capability of all the FF-PNA derivatives to self-assemble into ß-sheet structures. As a consequence of this supramolecular organization, the resulting aggregates also exhibit optoelectronic properties already reported for other similar nanostructures. This photoemissive behavior is promising for their potential applications in bioimaging.

Response mechanism of a highly efficient partial nitritation-anammox (PN/A) process under antibiotic stress: Extracellular polymers, microbial community, and functional genes.

The Partial nitritation-Anammox (PN/A) process can be restricted when treating high ammonia nitrogen wastewater containing antibiotics. This study aims to explore the response mechanism of the PN/A process under antibiotic stress. Results showed the PN/A process achieved a nitrogen removal rate higher than 1.01 ± 0.03 kg N/m3/d under long-term sulfamethazine stress. The increase of extracellular polymers from 22.52 to 43.96 mg/g VSS was conducive to resisting antibiotic inhibitory. The increase of Denitratisoma and SM1A02 abundance as well as functional genes nirS and nirK indicated denitrifiers should play an important role in the stability of the PN/A system under sulfamethazine stress. In addition, antibiotic-resistant genes (ARGs) sul1 and intI1 significantly increased by 8.78 and 5.12 times of the initial values to maintain the resistance of PN/A process to sulfamethazine stress. This study uncovers the response mechanism of the PN/A process under antibiotic stress, offering a scientific basis and guidance for further application in the future.

A New Peptide Nucleic Acid Fluorescence In Situ Hybridization Probe for the Specific Detection of Salmonella Species in Food Matrices.

Salmonella spp. is among the most central etiological agents in foodborne bacterial disorders. To identify Salmonella spp., numerous new molecular techniques have been developed conversely to the traditional culture-based methods. In this work, a new peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method was developed for the specific detection of Salmonella species, allowing a faster analysis compared with the traditional methods (ISO 6579-1: 2017). The method was optimized based on a novel PNA probe (SalPNA1692) combined with a blocker probe to detect Salmonella in food samples through an assessment of diverse-rich and selective enrichment broths. Our findings indicated that the best outcome was obtained using a 24-h pre-enrichment step in buffered peptone water, followed by RambaQuick broth selective enrichment for 16 h. For the enrichment step performance validation, fresh ground beef was artificially contaminated with two ranges of concentration of inoculum: a low level (0.2-2 colony-forming units [CFUs]/25 g) and a high level (2-10 CFUs/25 g). The new PNA-FISH method presented a specificity of 100% and a detection limit of 0.5 CFU/25 g of food sample, which confirms the great potential of applying PNA probes in food analysis.

The Application of Peptide Nucleic Acids (PNA) in the Inhibition of Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) Gene Expression in a Cell-Free Transcription/Translation System.

Proprotein convertase subtilisin/kexin 9 (PCSK9) is a protein that plays a key role in the metabolism of low-density lipoprotein (LDL) cholesterol. The gain-of-function mutations of the PCSK9 gene lead to a reduced number of surface LDL receptors by binding to them, eventually leading to endosomal degradation. This, in turn, is the culprit of hypercholesterolemia, resulting in accelerated atherogenesis. The modern treatment for hypercholesterolemia encompasses the use of biological drugs against PCSK9, like monoclonal antibodies and gene expression modulators such as inclisiran-a short, interfering RNA (siRNA). Peptide nucleic acid (PNA) is a synthetic analog of nucleic acid that possesses a synthetic peptide skeleton instead of a phosphate-sugar one. This different structure determines the unique properties of PNA (e.g., neutral charge, enzymatic resistance, and an enormously high affinity with complementary DNA and RNA). Therefore, it might be possible to use PNA against PCSK9 in the treatment of hypercholesterolemia. We sought to explore the impact of three selected PNA oligomers on PCSK9 gene expression. Using a cell-free transcription/translation system, we showed that one of the tested PNA strands was able to reduce the PCSK9 gene expression down to 74%, 64%, and 68%, as measured by RT-real-time PCR, Western blot, and HPLC, respectively. This preliminary study shows the high applicability of a cell-free enzymatic environment as an efficient tool in the initial evaluation of biologically active PNA molecules in the field of hypercholesterolemia research. This cell-free approach allows for the omission of the hurdles associated with transmembrane PNA transportation at the early stage of PNA selection.

Impact of Iron Supplementation on Hospital Length of Stay for Pneumonia or Skin and Skin Structure Infections: A Retrospective Cohort Study.

Objectives: Pathogenic organisms utilize iron to survive and replicate and have evolved many processes to extract iron from human hosts. The goal of this study was to elucidate the impact of iron supplementation given in the setting of acute infection. Methods: This was a retrospective cohort study of Veterans Affairs patients who received intravenous antibiotics for pneumonia or skin and skin structure infections. Five-thousand subjects were included in each of the 2 cohorts: iron-receiving and non-iron-receiving. Data was analyzed using Fischer's Exact test if categorical and independent t-tests if continuous. Primary and secondary objectives analyzed with Cox proportional hazard regression and outcome rates estimated utilizing Kaplan-Meier method. Results: Five-thousand patients were included in each cohort. The iron cohort was significantly older (Mean-years: Iron = 71.6, No-iron = 68.9; mean-difference = 2.7, P < .0001) with reduced renal function (Mean-eGFR[mL/min/1.73 m²]: Iron = 67.2, No-iron = 77.4; mean-difference = 10.2, P < .0001). For the primary outcome, the iron cohort had a significantly longer mean length of hospital stay (10.4 days) compared to the no-iron cohort (8.7 days) (mean difference 1.7 days, P < .0001). Secondary outcome analysis showed the iron cohort received intravenous antibiotics for longer (Iron = 8.2 days, No-iron = 7.1 days; mean-difference = 1.1 days, P < .0001) with a higher proportion of 30-day readmissions (Iron = 15.6%, No-iron = 12.8%; proportion difference = 2.8%, P < .0001). No significant difference was found between cohort proportions for 30-day mortality (Iron = 12.7%, No-iron = 11.3%, proportion difference = 1.4%, P = .052). Conclusions: Baseline characteristic differences between cohorts is representative of patients who would be expected to require iron replacement therapy. Given the magnitude of primary and secondary-outcomes, further studies controlling for these factors would be warranted.

Catechol-Siderophore Mimics Convey Nucleic Acid Therapeutics into Bacteria.

Antibacterial resistance is a major threat for human health. There is a need for new antibacterials to stay ahead of constantly-evolving resistant bacteria. Nucleic acid therapeutics hold promise as powerful antibiotics, but issues with their delivery hamper their applicability. Here, we exploit the siderophore-mediated iron uptake pathway to efficiently transport antisense oligomers into bacteria. We appended a synthetic siderophore to antisense oligomers targeting the essential acpP gene in Escherichia coli. Siderophore-conjugated PNA and PMO antisense oligomers displayed potent antibacterial properties. Conjugates bearing a minimal siderophore consisting of a mono-catechol group showed equally effective. Targeting the lacZ transcript resulted in dose-dependent decreased ß-galactosidase production, demonstrating selective protein downregulation. Applying this concept to Acinetobacter baumannii also showed concentration-dependent growth inhibition. Whole-genome sequencing of resistant mutants and competition experiments with the endogenous siderophore verified selective uptake through the siderophore-mediated iron uptake pathway. Lastly, no toxicity towards mammalian cells was found. Collectively, we demonstrate for the first time that large nucleic acid therapeutics can be efficiently transported into bacteria using synthetic siderophore mimics.

Nucleobase and Linker Modification for Triple-Helical Recognition of Pyrimidines in RNA Using Peptide Nucleic Acids.

Triple-helical recognition of any sequence of double-stranded RNA requires high affinity Hoogsteen hydrogen binding to pyrimidine interruptions of polypurine tracts. Because pyrimidines have only one hydrogen bond donor/acceptor on Hoogsteen face, their triple-helical recognition is a formidable problem. The present study explored various five-membered heterocycles and linkers that connect nucleobases to backbone of peptide nucleic acid (PNA) to optimize formation of X•C-G and Y•U-A triplets. Molecular modeling and biophysical (UV melting and isothermal titration calorimetry) results revealed a complex interplay between the heterocyclic nucleobase and linker to PNA backbone. While the five-membered heterocycles did not improve pyrimidine recognition, increasing the linker length by four atoms provided promising gains in binding affinity and selectivity. The results suggest that further optimization of heterocyclic bases with extended linkers to PNA backbone may be a promising approach to triple-helical recognition of RNA.

Targeting a KRAS i-motif forming sequence by unmodified and gamma-modified peptide nucleic acid oligomers.

Growing interest in i-motif DNA as a transcriptional regulatory element motivates development of synthetic molecules capable of targeting these structures. In this study, we designed unmodified peptide nucleic acid (PNA) and gamma-modified PNA (γPNA) oligomers complementary to an i-motif forming sequence derived from the promoter of the KRAS oncogene. Biophysical techniques such as circular dichroism (CD) spectroscopy, CD melting, and fluorescence spectroscopy demonstrated the successful invasion of the i-motif by PNA and γPNA. Both PNA and γPNA showed very strong binding to the target sequence with high thermal stability of the resulting heteroduplexes. Interestingly fluorescence and CD experiments indicated formation of an intermolecular i-motif structure via the overhangs of target-probe heteroduplexes formed by PNA/γPNA invasion of the intramolecular i-motif. Targeting promoter i-motif forming sequences with high-affinity oligonucleotide mimics like γPNAs may represent a new approach for inhibiting KRAS transcription, thereby representing a potentially useful anti-cancer strategy.

Peptide nucleic acid conjugates and their antimicrobial applications-a mini-review.

Peptide nucleic acid (PNA) is a nucleic acid mimic with high specificity and binding affinity to natural DNA or RNA, as well as resistance to enzymatic degradation. PNA sequences can be designed to selectively silence gene expression, which makes PNA a promising tool for antimicrobial applications. However, the poor membrane permeability of PNA remains the main limiting factor for its applications in cells. To overcome this obstacle, PNA conjugates with different molecules have been developed. This mini-review focuses on covalently linked conjugates of PNA with cell-penetrating peptides, aminosugars, aminoglycoside antibiotics, and non-peptidic molecules that were tested, primarily as PNA carriers, in antibacterial and antiviral applications. The chemistries of the conjugation and the applied linkers are also discussed.

Na Cocations and Hydrothermal Aging Cooperatively Boost the Regeneration of Phosphorus-Poisoned Pd/SSZ-13 for Passive NOx Adsorption.

Pd/SSZ-13 has been proposed as a passive NOx adsorber (PNA) for low-temperature NOx adsorption. However, it remains challenging for Pd/SSZ-13 to work efficiently when suffering from phosphorus poisoning. Herein, we report a simple and efficient strategy to regenerate the phosphorus-poisoned Pd/SSZ-13 based on the cooperation between hydrothermal aging treatment and Na cocations. It was found that hydrothermal aging treatment enabled the redispersion of Pd and P-containing species in phosphorus-poisoned Pd/SSZ-13. Meanwhile, the presence of Na cocations significantly reduced the formation of AlPO4 and retained more paired Al sites for highly dispersed Pd2+ ions, which was of great importance for the recovery of adsorption performance. To our satisfaction, the restoration ratio of the adsorption capacity of poisoned Pd/SSZ-13 was >90% after regeneration. Strikingly, the NOx adsorption activities of phosphorus-poisoned Pd/SSZ-13 with phosphorus loadings of 0.2 and 0.4 mmol g-1 almost completely recovered upon regeneration. This study demonstrates the promoting effect of Na cocations on the regeneration of phosphorus-poisoned Pd/SSZ-13 by hydrothermal aging treatment, which provides useful guidance for the design of PNA materials with excellent durability for cold-start application.

Peptide Nucleic Acids: Recent Developments in the Synthesis and Backbone Modifications.

Nucleic acid represents the ideal drug candidate for protein targets that are hard to target or against which drug development is not easy. Peptide nucleic acids (PNAs) are synthesized by attaching modified peptide backbones generally derived from repetitive N-2-aminoethyl glycine units in place of the regular phosphodiester backbone and represent synthetic impersonator of nucleic acids that offers an exciting research field due to their fascinating spectrum of biotechnological, diagnostic and potential therapeutic applications. The semi-rigid peptide nucleic acid backbone serves as a nearly-perfect template for attaching complimentary base pairs on DNA or RNA in a sequence-dependent manner as described by Watson-Crick models. PNAs and their analogues are endowed with exceptionally high affinity and specificity for receptor sites, essentially due to their polyamide backbone's uncharged and flexible nature. The present review compiled various strategies to modify the polypeptide backbone for improving the target selectivity and stability of the PNAs in the body. The investigated biological activities carried out on PNAs have also been summarized in the present review.

Genital mycobacteriosis caused by Mycobacterium marinum detected in two captive sharks by peptide nucleic acid-fluorescence in situ hybridization.

Mycobacterium marinum is a prevalent nontuberculous mycobacterium (NTM)-infecting teleosts. Conversely, little is known about mycobacteriosis in elasmobranchs, and M. marinum infection has never been reported from the subclass. This study investigated the histopathological characteristics and localization of this mycobacterium through molecular analysis of two captive sharks, a scalloped hammerhead Sphyrna lewini and a Japanese bullhead shark Heterodontus japonicus, exhibited in the same aquarium tank. We detected genital mycobacteriosis caused by M. marinum infection using molecular analyses, including polymerase chain reaction (PCR) and DNA sequencing targeting the 60 kDa heat-shock protein gene (hsp65), and peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) targeting the 16S rRNA gene. Both sharks showed granulomas in connective tissues of the gonads without central necrosis or surrounding fibrous capsules, which is unlike the typical mycobacterial granulomas seen in teleosts. This study reveals that elasmobranchs can be aquatic hosts of M. marinum. Because M. marinum is a representative waterborne NTM and a potential zoonotic agent, cautious and intensive research is needed to overcome a lack of data on the relationship between NTM and the aquatic environment in association with this subclass of Chondrichthyes.

PNA6, a Lactosyl Analogue of Angiotensin-(1-7), Reverses Pain Induced in Murine Models of Inflammation, Chemotherapy-Induced Peripheral Neuropathy, and Metastatic Bone Disease.

Pain is the most significant impairment and debilitating challenge for patients with bone metastasis. Therefore, the primary objective of current therapy is to mitigate and prevent the persistence of pain. Thus, cancer-induced bone pain is described as a multifaceted form of discomfort encompassing both inflammatory and neuropathic elements. We have developed a novel non-addictive pain therapeutic, PNA6, that is a derivative of the peptide Angiotensin-(1-7) and binds the Mas receptor to decrease inflammation-related cancer pain. In the present study, we provide evidence that PNA6 attenuates inflammatory, chemotherapy-induced peripheral neuropathy (CIPN) and cancer pain confined to the long bones, exhibiting longer-lasting efficacious therapeutic effects. PNA6, Asp-Arg-Val-Tyr-Ile-His-Ser-(O-ß-Lact)-amide, was successfully synthesized using solid phase peptide synthesis (SPPS). PNA6 significantly reversed inflammatory pain induced by 2% carrageenan in mice. A second murine model of platinum drug-induced painful peripheral neuropathy was established using oxaliplatin. Mice in the oxaliplatin-vehicle treatment groups demonstrated significant mechanical allodynia compared to the oxaliplatin-PNA6 treatment group mice. In a third study modeling a complex pain state, E0771 breast adenocarcinoma cells were implanted into the femur of female C57BL/6J wild-type mice to induce cancer-induced bone pain (CIBP). Both acute and chronic dosing of PNA6 significantly reduced the spontaneous pain behaviors associated with CIBP. These data suggest that PNA6 is a viable lead candidate for treating chronic inflammatory and complex neuropathic pain.

Advances in Nucleic Acid Research: Exploring the Potential of Oligonucleotides for Therapeutic Applications and Biological Studies.

In recent years, nucleic acids have emerged as powerful biomaterials, revolutionizing the field of biomedicine. This review explores the multifaceted applications of nucleic acids, focusing on their pivotal role in various biomedical applications. Nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), possess unique properties such as molecular recognition ability, programmability, and ease of synthesis, making them versatile tools in biosensing and for gene regulation, drug delivery, and targeted therapy. Their compatibility with chemical modifications enhances their binding affinity and resistance to degradation, elevating their effectiveness in targeted applications. Additionally, nucleic acids have found utility as self-assembling building blocks, leading to the creation of nanostructures whose high order underpins their enhanced biological stability and affects the cellular uptake efficiency. Furthermore, this review delves into the significant role of oligonucleotides (ODNs) as indispensable tools for biological studies and biomarker discovery. ODNs, short sequences of nucleic acids, have been instrumental in unraveling complex biological mechanisms. They serve as probes for studying gene expression, protein interactions, and cellular pathways, providing invaluable insights into fundamental biological processes. By examining the synergistic interplay between nucleic acids as powerful biomaterials and ODNs as indispensable tools for biological studies and biomarkers, this review highlights the transformative impact of these molecules on biomedical research. Their versatile applications not only deepen our understanding of biological systems but also are the driving force for innovation in diagnostics and therapeutics, ultimately advancing the field of biomedicine.

Linking morphological features to anammox communities in a partial nitritation and anammox (PN/A) biofilm reactor.

Partial nitritation/anammox (PN/A) has been recognized as a cost-efficient process for wastewater nitrogen removal. The addition of carriers could help achieve biomass retention and enhance the treatment efficiency by forming the dense biofilm. However, accurately determining the abundance of anammox bacteria (AnAOB) to evaluate the biofilm development still remains challenging in practice without access to specialized facilities and experimental skills. In this study, we explored the feasibility of utilizing the morphological features of anammox biofilm as an indication of the biofilm development progression, and its correlation with microbial communities was also revealed. The time-series biofilms from an integrated fixed-film activated sludge (IFAS) system with stable PN/A performance were sampled representing the different biofilm development stages. The biofilm morphological features including color and texture were respectively quantified by red (R) coordinate and Local binary pattern (LBP) descriptor via image processing. Hierarchy clustering analysis proved that the extracted morphological descriptors could well distinguish the different stages (colonization, succession, and maturation) of biofilm development. The microbial community dynamics of time-series anammox biofilms were investigated using the amplicon sequence variant (ASV) analysis. Candidatus Brocadia, as the typical AnAOB, dominated in the whole communities of 16.3%-20.0%, moreover, the biofilm development was found to be driven by distinct Brocadia species. Linear regression evidenced that the Brocadia abundance could be directly correlated to the value of R and LBP, and the total variation of microbial communities could be significantly explained by the morphological features via redundancy analysis. This study demonstrates a new way to monitor the biofilm development by extracting the visible features of anammox aggregates, which can help facilitate the automated control of anammox-based bioprocess.

Large-scale (500 kg N/day) two-stage partial nitritation/anammox (PN/A) process for liquid-ammonia mercerization wastewater treatment: Rapid start-up and long-term operational performance.

The nitrogen pollution control of liquid-ammonia mercerization wastewater (LMWW) is one of the typical obstacle restricting the sustainability of textile industry. In this study, a 500 kg N/day two-stage partial nitritation/anammox (PN/A) process containing PN reactor filled with zeolite and biofilm anammox reactors was successfully started up in 45 days and operated stably with high shock resistance over one year for LMWW treatment. The large-scale process achieved an average ammonium removal efficiency (94.3 ± 2.3%), total nitrogen removal efficiency (89.4 ± 2.7%) and nitrogen removal rate (1.003 ± 0.386 kg N/m3/day) during one year engineering operation. Simultaneous denitrification was revealed by the contribution of 5.2% total nitrogen removed. High-throughput sequencing results showed that Nitrosomonas was the most dominant genus in PN reactor, and Ca. Anammoxoglobus and Ca. Kuenenia were the functional bacteria for nitrogen removal in anammox reactors. Compared to traditional nitrification-denitrification process, the large-scale process reduced a total operational cost of 46.03 CNY/kg N for LMWW. This study revealed the proposed process was quite reliable with fast start-up and high impact resistance to overcome the obstacle of nitrogen pollution control for LMWW economically and conducive to the sustainable development for textile industry.

Anammox granule destruction and reconstruction in a partial nitrification/anammox system under hydroxylamine stress.

Nitrite oxidizing bacteria (NOB) outcompeting anammox bacteria (AnAOB) poses a challenge to the practical implementation of the partial nitrification/anammox (PN/A) process for municipal wastewater. A granules-based PN/A bioreactor was operated for 260 d with hydroxylamine (NH2OH) added halfway through. qPCR results detected the different amounts of NOB among granules and flocs and the dynamic succession during operation. CLSM images revealed a unique layered structure of granules that NOB located inside led to the inhibition effect of NH2OH delayed. Besides, the physical and morphological characteristics revealed that anammox granules experienced destruction. AnAOB took the broken granules as an initial biofilm aggregate to reconstruct new granules. RT-qPCR and high throughput sequencing results suggested that functional gene expression and community structure were regulated for the AnAOB metabolism process. Correspondingly, the rapid proliferation (0.52 â†’ 1.99%) of AnAOB was realized, and the nitrogen removal rate achieved a nearly quadruple improvement (0.21 â†’ 0.83 kg-N/m3·d). This study revealed that anammox granules can self-reconstruct in the PN/A system when granules are disintegrated under NH2OH stress, broadening the feasibility of applying PN/A process.