Crucial roles of specialized chitinases in elytral and hindwing cuticles construction in Leptinotarsa decemlineata.

BACKGROUND: The Colorado potato beetle (CPB), Leptinotarsa decemlineata, is a major potato (Solanum tuberosum) pest, infesting over 16 million km2 and causing substantial economic losses. The insect cuticle forms an apical extracellular matrix (ECM) envelope covering exposed organs to direct morphogenesis and confer structural protection. While select chitinase (Cht) genes have proven essential for larval development, their potential activities directing ECM remodeling underlying adult wing maturation remain undefined. RESULTS: We investigated the expression patterns and performed an oral RNA interference (RNAi) screen targeting 19 LdChts in late-instar L. decemlineata larvae. Subsequently, we assessed their effects on adult eclosion and wing characteristics. Knockdown of LdCht5, LdCht7, LdCht10, LdIDGF2, and LdIDGF4, as well as others from group IV (LdCht15, LdCht12, LdCht17, and LdCht13) and group VII-X (LdCht2, LdCht11, LdCht1, and LdCht3), resulting in shrunken, misshapen elytra with reduced areal density, as well as transverse wrinkling and impaired wing-tip folding in hindwings. Scanning electron micrographs revealed eroded elytral ridges alongside thinned, ruptured hindwing veins, indicative of mechanical fragility post-LdCht suppression. Spectroscopic analysis uncovered biomolecular alterations underlying the elytral anomalies, including decreases in peaks representing chitin, proteins, and lipids. This loss of essential extracellular matrix components provides evidence for the fragility, wrinkling, and shrinkage observed in the RNAi groups. CONCLUSION: Our findings elucidate the crucial role of chitinases in the turnover of chitinous cuticles on beetle wings, offering insights into RNAi-based control strategies against this invasive pest. This article is protected by copyright. All rights reserved.

Key arginine residues in R2D2 dsRBD1 and dsRBD2 lead the siRNA recognition in Drosophila melanogaster RNAi pathway.

In Drosophila melanogaster, Dcr-2:R2D2 heterodimer binds to the 21 nucleotide siRNA duplex to form the R2D2/Dcr-2 Initiator (RDI) complex, which is critical for the initiation of siRNA-induced silencing complex (RISC) assembly. During RDI complex formation, R2D2, a protein that contains three dsRNA binding domains (dsRBD), senses two aspects of the siRNA: thermodynamically more stable end (asymmetry sensing) and the 5'-phosphate (5'-P) recognition. Despite several detailed studies to date, the molecular determinants arising from R2D2 for performing these two tasks remain elusive. In this study, we have performed structural, biophysical, and biochemical characterization of R2D2 dsRBDs. We found that the solution NMR-derived structure of R2D2 dsRBD1 yielded a canonical α1-ß1-ß2-ß3-α2 fold, wherein two arginine salt bridges provide additional stability to the R2D2 dsRBD1. Furthermore, we show that R2D2 dsRBD1 interacts with thermodynamically asymmetric siRNA duplex independent of its 5'-phosphorylation state, whereas R2D2 dsRBD2 prefers to interact with 5'-P siRNA duplex. The mutation of key arginine residues, R53 and R101, in concatenated dsRBDs of R2D2 results in a significant loss of siRNA duplex recognition. Our study deciphers the active roles of R2D2 dsRBDs by showing that dsRBD1 initiates siRNA recognition, whereas dsRBD2 senses 5'-phosphate as an authentic mark on functional siRNA.

The Expression and Function of Notch Involved in Ovarian Development and Fecundity in Basilepta melanopus.

Basilepta melanopus is a pest that severely affects oil tea plants, and the Notch signaling pathway plays a significant role in the early development of insect ovaries. In this study, we explored the function of the notch gene within the Notch signaling pathway in the reproductive system of B. melanopus. The functional domains and expression patterns of Bmnotch were analyzed. Bmnotch contains 45 epidermal growth factor-like (EGF-like) domains, one negative regulatory region, one NODP domain and one repeat-containing domain superfamily. The qPCR reveals heightened expression in early developmental stages and specific tissues like the head and ovaries. The RNA interference (RNAi)-based suppression of notch decreased its expression by 52.1%, exhibiting heightened sensitivity to dsNotch at lower concentrations. Phenotypic and mating experiments have demonstrated that dsNotch significantly impairs ovarian development, leading to reduced mating frequencies and egg production. This decline underscores the Notch pathway's crucial role in fecundity. The findings advocate for RNAi-based, Notch-targeted pest control as an effective and sustainable strategy for managing B. melanopus populations, signifying a significant advancement in forest pest control endeavors.

Functions of RNAi Pathways in Ribosomal RNA Regulation.

Argonaute proteins, guided by small RNAs, play crucial roles in gene regulation and genome protection through RNA interference (RNAi)-related mechanisms. Ribosomal RNAs (rRNAs), encoded by repeated rDNA units, constitute the core of the ribosome being the most abundant cellular transcripts. rDNA clusters also serve as sources of small RNAs, which are loaded into Argonaute proteins and are able to regulate rDNA itself or affect other gene targets. In this review, we consider the impact of small RNA pathways, specifically siRNAs and piRNAs, on rRNA gene regulation. Data from diverse eukaryotic organisms suggest the potential involvement of small RNAs in various molecular processes related to the rDNA transcription and rRNA fate. Endogenous siRNAs are integral to the chromatin-based silencing of rDNA loci in plants and have been shown to repress rDNA transcription in animals. Small RNAs also play a role in maintaining the integrity of rDNA clusters and may function in the cellular response to rDNA damage. Studies on the impact of RNAi and small RNAs on rRNA provide vast opportunities for future exploration.

Advances in discovery of novel investigational agents for functional cure of chronic hepatitis B: A comprehensive review of phases II and III therapeutic agents.

Chronic hepatitis B virus (HBV) infection affects over 295 million people globally and an estimated 1.6 million people in the United States. It is associated with significant morbidity and mortality due to cirrhosis, liver failure, and liver cancer. Antiviral therapy with oral nucleos(t)ide analogues is associated with high rates of virologic suppression, which in turn has been associated with a decreased risk of liver complications. However, current antiviral regimens are limited by concerns with adverse effects, adherence, resistance, long-term treatment, and ongoing risk for liver events. Novel investigational agents are currently in development and are targeted at achieving functional cure with sustained hepatitis B surface antigen (HBsAg) loss and suppression of HBV DNA. Herein we review key evidence from phases II and III trials defining the efficacy and safety profiles for key investigational agents for functional cure of chronic hepatitis B, including core/capsid inhibitors, entry inhibitors, RNA interference (siRNA/ASO), HBsAg inhibitors, Toll-like receptor agonists, checkpoint inhibitors, and therapeutic vaccines.

Molecular identification and related functional characterization of the FKBP52 gene in immunity of Locusta migratoria manilensis (Orthoptera: Oedipodidae).

Metarhizium anisopliae is an important class of entomopathogenic fungi used for the biocontrol of insects, but its virulence is affected by insect immunity. We identified a novel FK506 binding protein gene that was differentially expressed between control and Metarhizium-treated Locusta migratoria manilensis. We hypothesized that this protein played an important role in Metarhizium infection of L. migratoria and could provide new insights for developing highly efficient entomopathogenic fungi. We, therefore, cloned the specific gene and obtained its purified protein. The gene was then named FKBP52, and its dsRNA (dsFKBP52) was synthesized and used for gene interference. Bioassay results showed that the mortality of L. migratoria treated with dsFKBP52 + Metarhizium was significantly lower than that of other treatments. Furthermore, immune-related genes (MyD88, Dorsal, Cactus, and Defensin) in L. migratoria treated with dsFKBP52 + Metarhizium showed significant upregulation compared to that treated with Metarhizium only. However, the activities of peroxidase (POD), superoxide dismutase (SOD), and calcineurin (CaN) showed fluctuations. These results suggest that the FKBP52 gene may play a crucial role in the innate immunity of L. migratoria. The effect of its silencing indicated that this immunity-related protein might be a potential target for insect biocontrol.

Molecular mechanisms of cytochrome P450-mediated detoxification of tetraniliprole, spinetoram, and emamectin benzoate in the fall armyworm, Spodoptera frugiperda (J.E. Smith).

The fall armyworm (FAW) Spodoptera frugiperda (J.E. Smith) is a highly damaging invasive omnivorous pest that has developed varying degrees of resistance to commonly used insecticides. To investigate the molecular mechanisms of tolerance to tetraniliprole, spinetoram, and emamectin benzoate, the enzyme activity, synergistic effect, and RNA interference were implemented in S. frugiperda. The functions of cytochrome P450 monooxygenase (P450) in the tolerance to tetraniliprole, spinetoram, and emamectin benzoate in S. frugiperda was determined by analysing changes in detoxification metabolic enzyme activity and the effects of enzyme inhibitors on susceptibility to the three insecticides. 102 P450 genes were screened via transcriptome and genome, of which 67 P450 genes were differentially expressed in response to tetraniliprole, spinetoram, and emamectin benzoate and validated by quantitative real-time PCR. The expression patterns of CYP9A75, CYP340AA4, CYP340AX8v2, CYP340L16, CYP341B15v2, and CYP341B17v2 were analysed in different tissues and at different developmental stages in S. frugiperda. Silencing CYP340L16 significantly increased the susceptibility of S. frugiperda to tetraniliprole, spinetoram, and emamectin benzoate. Furthermore, knockdown of CYP340AX8v2, CYP9A75, and CYP341B17v2 significantly increased the sensitivity of S. frugiperda to tetraniliprole. Knockdown of CYP340AX8v2 and CYP340AA4 significantly increased mortality of S. frugiperda to spinetoram. Knockdown of CYP9A75 and CYP341B15v2 significantly increased the susceptibility of S. frugiperda to emamectin benzoate. These results may help to elucidate the mechanisms of tolerance to tetraniliprole, spinetoram and emamectin benzoate in S. frugiperda.

Identification of an active RNAi pathway in Candida albicans.

RNA interference (RNAi) is a fundamental regulatory pathway with a wide range of functions, including regulation of gene expression and maintenance of genome stability. Although RNAi is widespread in the fungal kingdom, well-known species, such as the model yeast Saccharomyces cerevisiae, have lost the RNAi pathway. Until now evidence has been lacking for a fully functional RNAi pathway in Candida albicans, a human fungal pathogen considered critically important by the World Health Organization. Here, we demonstrated that the widely used C. albicans reference strain (SC5314) contains an inactivating missense mutation in the gene encoding for the central RNAi component Argonaute. In contrast, most other C. albicans isolates contain a canonical Argonaute protein predicted to be functional and RNAi-active. Indeed, using high-throughput small and long RNA sequencing combined with seamless CRISPR/Cas9-based gene editing, we demonstrate that an active C. albicans RNAi machinery represses expression of subtelomeric gene families. Thus, an intact and functional RNAi pathway exists in C. albicans, highlighting the importance of using multiple reference strains when studying this dangerous pathogen.

Identification of Growth-Related Gene BAMBI and Analysis of Gene Structure and Function in the Pacific White Shrimp Litopenaeus vannamei.

As one of the most important aquaculture species in the world, the improvement of growth traits of the Pacific white shrimp (Litopenaeus vannamei), has always been a primary focus. In this study, we conducted SNP-specific locus analysis and identified a growth-related gene, BAMBI, in L. vannamei. We analyzed the structure and function of LvBAMBI using genomic, transcriptomic, metabolomic, and RNA interference (RNAi) assays. The LvBAMBI possessed highly conserved structural domains and widely expressed in various tissues. Knockdown of LvBAMBI significantly inhibited the gain of body length and weight of the shrimp, underscoring its role as a growth-promoting factor. Specifically, knockdown of LvBAMBI resulted in a significant downregulation of genes involved in lipid metabolism, protein synthesis, catabolism and transport, and immunity. Conversely, genes related to glucose metabolism exhibited significant upregulations. Analysis of differential metabolites (DMs) in metabolomics further revealed that LvBAMBI knockdown may primarily affect shrimp growth by regulating biological processes related to lipid and glucose metabolism. These results suggested that LvBAMBI plays a crucial role in regulating lipid metabolism, glucose metabolism, and protein transport in shrimp. This study provides valuable insights for future research and utilization of BAMBI genes in shrimp and crustaceans.

Functional Analysis of ß-Carotene Oxygenase 2 (BCO2) Gene in Yesso Scallop (Patinopecten yessoensis).

Carotenoids are essential nutrients for humans and animals, and carotenoid coloration represents an important meat quality parameter for many farmed animals. Increasingly, studies have demonstrated that vertebrate carotenoid cleavage oxygenases (CCOs) are essential enzymes in carotenoid metabolism and are therefore potential candidate genes for improving carotenoid deposition. However, our understanding of carotenoid bioavailability and CCOs functions in invertebrates, particularly marine species, is currently quite limited. We previously identified that a CCO homolog, PyBCO-like 1, was the causal gene for carotenoid coloration in the 'Haida golden scallop', a variety of Yesso scallop (Patinopecten yessoensis) characterized by carotenoid enrichment. Here, we found that another CCO-encoding gene named PyBCO2 (ß-carotene oxygenase 2) was widely expressed in P. yessoensis organs/tissues, with the highest expression in striated muscle. Inhibiting BCO2 expression in P. yessoensis through RNA interference led to increased carotenoid (pectenolone and pectenoxanthin) deposition in the striated muscle, and the color of the striated muscle changed from white to light orange. Our results indicate that PyBCO2 might be a candidate gene used for improving carotenoid content in normal Yesso scallops, and also in 'Haida golden scallops'.

Tyrosine hydroxylase plays crucial roles in larval cuticle formation and larval-pupal tanning in the rice stem borer, Chilo suppressalis.

The striped stem borer, Chilo suppressalis (Walker), a notorious pest infesting rice, has evolved a high level of resistance to many commonly used insecticides. In this study, we investigate whether tyrosine hydroxylase (TH), which is required for larval development and cuticle tanning in many insects, could be a potential target for the control of C. suppressalis. We identified and characterized the full-length cDNA (CsTH) of C. suppressalis. The complete open reading frame of CsTH (MW690914) was 1683 bp in length, encoding a protein of 560 amino acids. Within the first to the sixth larval instars, CsTH was high in the first day just after molting, and lower in the ensuing days. From the wandering stage to the adult stage, levels of CSTH began to rise and reached a peak at the pupal stage. These patterns suggested a role for the gene in larval development and larval-pupal cuticle tanning. When we injected dsCsTH or 3-iodotyrosine (3-IT) as a TH inhibitor or fed a larva diet supplemented with 3-IT, there were significant impairments in larval development and larval-pupal cuticle tanning. Adult emergence was severely impaired, and most adults died. These results suggest that CsTH might play a critical role in larval development as well as larval-pupal tanning and immunity in C. suppressalis, and this gene could form a potential novel target for pest control.

Structural Characterization and Physiological Role of Bombyx mori Fibroinase in the Silk Gland Development.

Silkworm is a highly valuable insect that produces silk through secretion by a silk gland. Within this gland, a type of cathepsin L protease called Fibroinase was identified as an enzyme for hydrolyzing the primary components of silk, including fibroin and sericin. Here, we determined the crystal structure of Fibroinase fromBombyx mori at a resolution of 1.56 Å. Comparative structural analysis revealed that Fibroinase adopted a similar structural pattern with papain-type cathepsin, consisting of an N-terminal domain and a C-terminal domain. The interface between the domains forms a substrate-binding cleft, where the E64 inhibitor noncovalently binds in a novel manner. Additionally, computational simulations combined with biochemical analysis allowed us to define the binding mode and inhibition mechanism of physiological inhibitor Bombyx cysteine protease inhibitor (BCPI) with Fibroinase. Moreover, the expression profiles and RNA interference of Fibroinase indicated its critical role in removing silk proteins in the silk gland lumen and the destruction of silk gland tissue during the larval-pupal metamorphosis. These findings enhance our understanding of the structural and biochemical features of Fibroinase and its inhibitors, while also providing evidence for the physiological role of Fibroinase in silk gland development.

Exploring the mechanism of nonylphenol-induced ovarian developmental delay of manila clams, Ruditapes philippinarum: Applying RNAi to toxicological analysis.

Nonylphenol (NP) contamination in the coastal environment of China poses ecological risks to aquatic organisms. However, the endocrine disruptive impacts of NP on bivalves, particularly on ovarian development, remain poorly understood. In this study, Manila clams Ruditapes philippinarum at the developing stage of gonad were exposed to 1.0 µg/L NP for 21 days. Utilizing RNA interference (RNAi) to suppress ER gene expression, we observed a delay in ovarian development as evidenced by histological observations under both NP and NPRi (NP with ER-RNAi) treatment, with Vtg elevation exclusive to the NP group. Comprehensive analyses encompassing transcriptomics, real-time quantitative PCR, and steroid hormone measurement revealed significant alterations in aldosterone synthesis, estrogen signaling, and thyroid hormone synthesis. These pathways showed similar perturbations in both NP and NPRi groups compared to controls. Notably, the NPRi group exhibited distinct enrichment in PPAR and insulin signaling pathways, may implicating these in ER function suppression. Steroid hormone biosynthesis was notably reduced in both treatments, pointing to a profound impact on hormone synthesis. The contrast between in vivo and in vitro findings suggests that NP's detrimental effects on ovarian development may primarily involve neuroendocrine regulation of steroidogenesis. This investigation highlights the complex dynamics of NP-induced endocrine disruption in bivalves, emphasizing the pivotal role of ER and associated pathways.

Bioanalytical Approaches to Support the Development of Antibody-Oligonucleotide Conjugate (AOC) Therapeutic Proteins.

RNA interference (RNAi) is a biological process that evolved to protect eukaryotic organisms from foreign genes delivered by viruses. This process has been adapted as a powerful tool to treat numerous diseases through the delivery of small-interfering RNAs (siRNAs) to target cells to alter aberrant gene expression.Antibody-oligonucleotide conjugates (AOCs) are monoclonal antibodies with complexed siRNA or antisense oligonucleotides (ASOs) that have emerged to address some of the challenges faced by naked or chemically conjugated siRNA, which include rapid clearance from systemic circulation and lack of selective delivery of siRNA to target cells.It is essential to characterize the ADME properties of an AOC during development to optimize distribution to target tissues, to minimize the impact of biotransformation on exposure, and to characterize the PK/PD relationship to guide translation. However, owing to the complexity of AOC structure, this presents significant bioanalytical challenges, and multiple bioanalytical measurements are required to investigate the pharmacokinetics and biotransformation of the antibody, linker, and siRNA payload.In this paper, we describe an analytical workflow that details in vivo characterization of AOCs through measurement of their distinct molecular components to provide the basis for greater understanding of their ADME properties. Although the approaches herein can be applied to in vitro characterization of AOCs, this paper will focus on in vivo applications. This workflow relies on high-resolution mass spectrometry as the principal means of detection and leverages chromatographic, affinity-based, and enzymatic sample preparation steps.

Novel RNA molecular bioengineering technology efficiently produces functional miRNA agents.

Genome-derived microRNAs (miRNA or miR) govern posttranscriptional gene regulation and play important roles in various cellular processes and disease progression. While chemo-engineered miRNA mimics or biosimilars made in vitro are widely available and used, miRNA agents produced in vivo are emerging to closely recapitulate natural miRNA species for research. Our recent works have demonstrated the success of high-yield, in vivo production of recombinant miRNAs by using human tRNA (htRNA) fused precursor miRNA (pre-miR) carriers. In this study, we aim to compare the production of bioengineered RNA (BioRNA) molecules with glycyl versus leucyl htRNA fused hsa-pre-miR-34a carriers, namely BioRNAGly and BioRNALeu, respectively, and perform initial functional assessment. We designed, cloned, overexpressed, and purified a total of 48 new BioRNA/miRNAs, and overall expression levels, final yields, and purities were revealed to be comparable between BioRNAGly and BioRNALeu molecules. Meanwhile, the two versions of BioRNA/miRNAs showed similar activities to inhibit non-small cell lung cancer cell viability. Interestingly, functional analyses using model BioRNA/miR-7-5p demonstrated that BioRNAGly/miR-7-5p exhibited greater efficiency to regulate a known target gene expression (EGFR) than BioRNALeu/miR-7-5p, consistent with miR-7-5p levels released in cells. Moreover, BioRNAGly/miR-7-5p showed comparable or slightly greater activities to modulate MRP1 and VDAC1 expression, compared with miRCURY LNA miR-7-5p mimic. Computational modeling illustrated overall comparable 3D structures for exemplary BioRNA/miRNAs with noticeable differences in htRNA species and payload miRNAs. These findings support the utility of hybrid htRNA/hsa-pre-miR-34a as reliable carriers for RNA molecular bioengineering, and the resultant BioRNAs serve as functional biologic RNAs for research and development.

RNA Interference-based Therapies for the Reduction of Cardiovascular Risk.

Globally, there remains an unwavering increase in the incidence of cvd - from 271 million in 1990 to 523 million in 2019. Among the several modifiable and non-modifiable risk factors of heart disease, dyslipidemia is an important and prevalent risk factor mediated by both genetics and lifestyle factors. Hence, lowering lipid levels, specifically, ldl-c levels (low-density lipoprotein cholesterol), is a key strategy in decreasing the risk of cardiovascular disease. A reduction of 20 mg/dl in ldl-c levels has been found to prevent 2-3 cases of coronary artery disease (cad) for every 1000 individuals. Studies have also found reductions in ldl-c levels to be associated with a mortality benefit. However, ldl-c levels reduction may not eliminate the risk of significant cardiovascular events.

An update on the therapeutic role of RNAi in NAFLD/NASH.

Unhealthy lifestyles have given rise to a growing epidemic of metabolic liver diseases, including nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). NAFLD often occurs as a consequence of obesity, and currently, there is no FDA-approved drug for its treatment. However, therapeutic oligonucleotides, such as RNA interference (RNAi), represent a promising class of pharmacotherapy that can target previously untreatable conditions. The potential significance of RNAi in maintaining physiological homeostasis, understanding pathogenesis, and improving metabolic liver diseases, including NAFLD, is discussed in this article. We explore why NAFLD/NASH is an ideal target for therapeutic oligonucleotides and provide insights into the delivery platforms of RNAi and its therapeutic role in addressing NAFLD/NASH.

The small heat shock protein Hsp20.8 imparts tolerance to high temperatures in the leafminer fly, Liriomyza trifolii (Diptera: Agtomyzidae).

As an environmental factor, temperature impacts the distribution of species and influences interspecific competition. The molecular chaperones encoded by small heat shock proteins (sHsps) are essential for rapid, appropriate responses to environmental stress. This study focuses on Hsp20.8, which encodes a temperature-responsive sHsp in Liriomyza trifolii, an insect pest that infests both agricultural and ornamental crops. Hsp20.8 expression was highest at 39℃ in L. trifolii pupae and adults, and expression levels were greater in pupae than in adults. Recombinant Hsp20.8 was expressed in Escherichia coli and conferred a higher survival rate than the empty vector to bacterial cells exposed to heat stress. RNA interference experiments were conducted using L. trifolii adults and prepupae and the knockdown of Hsp20.8 expression increased mortality in L. trifolii during heat stress. The results expand our understanding of sHsp function in Liriomyza spp. and the ongoing adaptation of this pest to climate change. In addition, this study is also important for predicting the distribution of invasive species and proposing new prevention and control strategies based on temperature adaptation.

Using RNA interference targeting a nicotinic acetylcholine receptor subunit to counteract insecticide accommodation mechanisms: example of the ß1 subunit in the imidacloprid-accommodated American cockroach, Periplaneta americana.

Insecticide accommodation and resistance are limiting factors to the much-needed increase in agricultural production. Various physiological and cellular modifications, such as the changes of insecticide molecular targets, have been linked to these events. Thus, a previous study demonstrated that the imidacloprid accommodation set up by the cockroach Periplaneta americana after an exposure to a sublethal dose of this insecticide involves functional alterations of two nicotinic acetylcholine receptor (nAChR) subtypes. As RNA interference (RNAi) is one of the most promising strategies for controlling pest insects, we evaluated, in this study, the use of RNAi that targets the ß1 nAChR subunit to counteract the imidacloprid accommodation phenomenon in cockroaches. Interestingly, we showed that ingestion of dsRNA-ß1 increased the sensitivity to imidacloprid of accommodated cockroaches. Thus, we have demonstrated for the first time that RNAi that targets an nAChR subunit can counteract the accommodation mechanism to insecticide targeting nAChRs set up by an insect.

Neutrophils and NADPH Oxidases Are Major Contributors to Mild but Not Severe Ischemic Acute Kidney Injury in Mice.

Upregulation of free radical-generating NADPH oxidases (NOX), xanthine oxidoreductase (XOR), and neutrophil infiltration-induced, NOX2-mediated respiratory burst contribute to renal ischemia-reperfusion injury (IRI), but their roles may depend on the severity of IRI. We investigated the role of NOX, XOR, and neutrophils in developing IRI of various severities. C57BL/6 and Mcl-1ΔMyelo neutrophil-deficient mice were used. Oxidases were silenced by RNA interference (RNAi) or pharmacologically inhibited. Kidney function, morphology, immunohistochemistry and mRNA expression were assessed. After reperfusion, the expression of NOX enzymes and XOR increased until 6 h and from 15 h, respectively, while neutrophil infiltration was prominent from 3 h. NOX4 and XOR silencing or pharmacological XOR inhibition did not protect the kidney from IRI. Attenuation of NOX enzyme-induced oxidative stress by apocynin and neutrophil deficiency improved kidney function and ameliorated morphological damage after mild but not moderate/severe IRI. The IR-induced postischemic renal functional impairment (BUN, Lcn-2), tubular necrosis score, inflammation (TNF-α, F4/80), and decreases in the antioxidant enzyme (GPx3) mRNA expression were attenuated by both apocynin and neutrophil deficiency. Inhibition of NOX enzyme-induced oxidative stress or the lack of infiltration by NOX2-expressing neutrophils can attenuate reperfusion injury after mild but not moderate/severe renal IR.