Improved Assessment of Soil Nonextractable Residues of the Pyrethroid Insecticide Cyphenothrin.

The metabolic fate of pyrethroid insecticide cyphenothrin (1) [(RS)-α-cyano-3-phenoxybenzyl (1RS)-cis-trans-2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopropanecarboxylate] in soils was investigated using 14C-labeled (1R)-cis/trans isomers at the cyclopropane ring. Both isomers degraded with half-lives of 19.0-47.4 days, and 48.9-56.0% and 27.5-38.7% of the applied radioactivity (AR) were mineralized to CO2 and incorporated into nonextractable residues (NER), respectively, after 120 days at 20 °C. NER analyses revealed 37.5-42.2% (cis-1) and 44.9-54.1% (trans-1) of each residue at 30/120 days were comprised of 14C-amino acids (AAs) as microbial products. Assuming that 50% of microbial biomass is AAs, it was estimated that 11.3-22.9%AR (cis-1, 75.0-84.4% of NER) and 13.9-30.4%AR (trans-1, 89.8-108.2% of NER) were nonhazardous biogenic NER (bio-NER), while type I/II xenobiotic NER (xeno-NER) characterized by silylation was insignificant at 0.9-1.0%/2.8-3.3%AR (cis-1). Detailed 14C-AA quantitation indicated a high relevance of the tricarboxylic acid cycle and pyruvate pathway during bio-NER formation, offering new insights into the microbial assimilation of the chrysanthemic moiety.

A modified high-resolution melting-based assay (HRM) to identify the SARS-CoV-2 N501Y variant.

High-resolution melting (HRM) analysis is a PCR-based method that can be used as a screening assay to identify SARS-CoV-2 variants. However, conventional HRM assays hardly detect slight melting temperature differences at the A-T to T-A transversion. As the N501Y substitution results from A-T to T-A transversion in A23063, few or no studies have shown that a conventional HRM assay can identify N501Y variants. This study successfully developed an HRM assay for identifying the N501Y mutation. Two HRM assays were used in the N501 site because the discrimination results were affected by the virus copy numbers. One is a conventional HRM assay (detectable at 103-106 copies/mL) and the other is a modified HRM assay by adding the wild-type fragment (detectable at 105-1010 copies/mL). Using viral RNAs from cultured variants (Alpha, Beta, and Gamma), a modified HRM assay correctly identified three N501Y variants because of high-copy-number RNAs in those viral samples. The sensitivity and specificity of the N501Y assay were 93.3% and 100%, respectively, based on 209 clinical samples (105 for N501; 104 for N501Y). These results suggest that our HRM-based assay is a powerful tool for rapidly identifying various SARS-CoV-2 variants.

Rapid Identification of SARS-CoV-2 Omicron BA.5 Spike Mutation F486V in Clinical Specimens Using a High-Resolution Melting-Based Assay.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariant BA.5 emerged as of February 2022 and replaced the earlier Omicron subvariants BA.1 and BA.2. COVID-19 genomic surveillance should be continued as new variants seem to subsequently appear, including post-BA.5 subvariants. A rapid assay is needed to differentiate between the currently dominant BA.5 variant and other variants. This study successfully developed a high-resolution melting (HRM)-based assay for BA.4/5-characteristic spike mutation F486V detection and demonstrated that our assay could discriminate between BA.1, BA.2, and BA.5 subvariants in clinical specimens. The mutational spectra at two regions (G446/L452 and F486) for the variant-selective HRM analysis was the focus of our assay. The mutational spectra used as the basis to identify each Omicron subvariant were as follows: BA.1 (G446S/L452/F486), BA.2 (G446/L452/F486), and BA.4/5 (G446/L452R/F486V). Upon mutation-coding RNA fragment analysis, the wild-type fragments melting curves were distinct from those of the mutant fragments. Based on the analysis of 120 clinical samples (40 each of subvariants BA.1, BA.2, and BA.5), this method's sensitivity and specificity were determined to be more than 95% and 100%, respectively. These results clearly demonstrate that this HRM-based assay is a simple screening method for monitoring Omicron subvariant evolution.

Current Vaccine Platforms in Enhancing T-Cell Response.

The induction of T cell-mediated immunity is crucial in vaccine development. The most effective vaccine is likely to employ both cellular and humoral immune responses. The efficacy of a vaccine depends on T cells activated by antigen-presenting cells. T cells also play a critical role in the duration and cross-reactivity of vaccines. Moreover, pre-existing T-cell immunity is associated with a decreased severity of infectious diseases. Many technical and delivery platforms have been designed to induce T cell-mediated vaccine immunity. The immunogenicity of vaccines is enhanced by controlling the kinetics and targeted delivery. Viral vectors are attractive tools that enable the intracellular expression of foreign antigens and induce robust immunity. However, it is necessary to select an appropriate viral vector considering the existing anti-vector immunity that impairs vaccine efficacy. mRNA vaccines have the advantage of rapid and low-cost manufacturing and have been approved for clinical use as COVID-19 vaccines for the first time. mRNA modification and nanomaterial encapsulation can help address mRNA instability and translation efficacy. This review summarizes the T cell responses of vaccines against various infectious diseases based on vaccine technologies and delivery platforms and discusses the future directions of these cutting-edge platforms.

Discrimination of SARS-CoV-2 Omicron Sublineages BA.1 and BA.2 Using a High-Resolution Melting-Based Assay: a Pilot Study.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. As of March 2022, Omicron variant BA.2 is rapidly replacing variant BA.1. As variant BA.2 may cause more severe disease than variant BA.1, variant BA.2 requires continuous monitoring. The current study aimed to develop a novel high-resolution melting (HRM) assay for variants BA.1 and BA.2 and to determine the sensitivity and specificity of our method using clinical samples. Here, we focused on the mutational spectra at three regions in the spike receptor-binding domain (RBD; R408, G446/L452, and S477/T478) for the variant-selective HRM analysis. Each variant was identified based on the mutational spectra as follows: no mutations (Alpha variant); L452R and T478K (Delta variant); G446S and S477N/T478K (Omicron variant BA.1); and R408S and S477N/T478K (Omicron variant BA.2). Upon analysis of mutation-coding RNA fragments, the melting curves of the wild-type fragments were distinct from those of the mutant fragments. The sensitivity and specificity of this method were determined as 100% and more than 97.5%, respectively, based on 128 clinical samples (40 Alpha, 40 Delta, 40 Omicron variant BA.1/BA.1.1, and 8 Omicron variant BA.2). These results suggest that this HRM-based assay is a promising screening method for monitoring the transmission of Omicron variants BA.1 and BA.2. IMPORTANCE This study seeks to apply a novel high-resolution melting (HRM) assay to identify and discriminate BA.1 and BA.2 sublineages of the SARS-CoV-2 Omicron variant. Variant BA.2 may cause more severe disease than variant BA.1, meaning that identifying this variant is an important step toward improving the care of patients suffering from COVID-19. However, screening for these variants remains difficult, as current methods mostly rely on next-generation sequencing, which is significantly costlier and more time-consuming than other methods. We believe that our study makes a significant contribution to the literature because we show that this method was 100% sensitive and over 97.5% specific in our confirmation of 128 clinical samples.

Biodistribution and immunity of adenovirus 5/35 and modified vaccinia Ankara vector vaccines against human immunodeficiency virus 1 clade C.

Previously, we developed a chimeric adenovirus type 5 with type 35 fiber (Ad5/35), which has high tropism to dendritic cells and low hepatoxicity. For further clinical use, we constructed two recombinant vectors expressing human immunodeficiency virus 1 (HIV-1) clade C gag (Ad5/35-Cgag and MVA-Cgag). The biodistribution of the two viral vectors in a mouse model and immunity in monkeys were assessed. The mice received a single intramuscular injection with the vectors alone. The gag gene in the tissues were periodically detected using a real-time quantitative polymerase chain reaction. The distribution of Ad5/35 was also detected using an in vivo imaging system, followed by luciferase-expressing Ad5/35 administration. We found that Ad5/35-Cgag DNA and luciferase activity were detectable until 8 weeks post-administration, whereas MVA-Cgag was undetectable 72 h post-administration. Furthermore, viral administration did not increase serum aspartate aminotransferase and alanine aminotransferase levels in either mouse or monkey models. Moreover, intramuscular administration of Ad5/35-Cgag induced the gag-specific antibody level and IFNγ-secreting PBMCs, the boost with MVA-Cgag further increased the responses and lasted more than 20 weeks from the initial administration. These data demonstrate that Ad5/35 and MVA vectors are safe for in vivo use, and prime-boost with Ad5/35-MVA vaccines is suitable for clinical use against HIV-1 clade C.

A rapid screening assay for L452R and T478K spike mutations in SARS-CoV-2 Delta variant using high-resolution melting analysis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission has been reported worldwide and novel SARS-CoV-2 variants continue to emerge. A novel SARS-CoV-2 strain, the Delta variant (B.1.617.2), is spreading worldwide. The Delta variant has reportedly high infectivity and immune evasion potency. In June 2021, the World Health Organization categorized it as a variant of concern (VOC). Therefore, it is vital to develop tests that can exclusively identify the Delta variant. Here, we developed a rapid screening assay to detect characteristic mutations observed in the Delta variant using high-resolution melting (HRM) analysis. In this assay, we determined L452R and T478K, among which T478K is an identifier of the Delta variant since L452R is seen in other strains (Kappa and Epsilon variants). Additionally, nested PCR-based HRM analysis, which involved RT-PCR (1st PCR) and HRM analysis (2nd PCR), was developed to improve the specificity and sensitivity. Our method discriminated between the L452R mutant and wild-type L452. In addition, HRM analysis distinguished the T478K mutant from the wild-type T478. Seven clinical samples containing the Delta variant were successfully identified as L452R/T478K mutants. These results indicate that this HRM-based genotyping method can identify the Delta variant. This simple method should contribute to rapid identification of the Delta variant and the prevention of infection spread.

Vaccination inhibits the human adenoviral transduction in a mouse keratoconjunctivitis model.

Adenovirus infections are a major cause of epidemic keratoconjunctivitis (EKC), which can lead to corneal subepithelial infiltrates and multifocal corneal opacity. In the current study, we investigated the use of an E1/E3-deleted adenovirus serotype 5 (Ad5) vector as a vaccine administered intramuscularly (IM) or intranasally (IN) against subsequent challenges with a luciferase-expressing Ad5 (Ad5-Luci) vector via eyedrop. We evaluated the adaptive immune response to Ad5 vector vaccination and confirmed a robust polyfunctional CD8 T cell response in splenic cells. Neutralizing Ad5 antibodies were also measured in the sera of vaccinated mice as well as Ad5 antibody in the eye wash solutions. Upon challenge with Ad5-Luci vector 8 weeks post the primary immunization, transduction was significantly reduced by > 70% in the vaccinated mice, which was slightly better in IM- vs. that in IN-vaccinated animals. Resistance to subsequent challenge was observed 10 months post primary IM vaccination, with sustained reduction up to 60% in the Ad5-Luci vector transduction. Passive immunization of naive mice with antisera from IM to vaccinated mice subsequently challenged with the Ad5-Luci vector resulted in approximately 40% loss in transduction efficiency. Furthermore, the mice that received IM immunization with or without CD8 T cell depletion showed > 40% and 70% reductions, respectively, in Ad8 genomic copies after Ad8 topical challenge. We conclude that Ad-vector vaccination successfully induced an adaptive immune response that prevented subsequent Ad transduction in the cornea and conjunctiva-associated tissues in a mouse model of adenovirus keratoconjunctivitis, and that both cellular and humoral immunity play an important role in preventing Ad transduction.

Prophylactic and therapeutic vaccine against Pseudomonas aeruginosa keratitis using bacterial membrane vesicles.

PURPOSE: Pseudomonas aeruginosa (P. aeruginosa) infection is one of the major causes of keratitis. However, effective prophylactic and therapeutic vaccines against P. aeruginosa keratitis have yet to be developed. In this study, we explored the use of P. aeruginosa membrane vesicles (MVs) as a prophylactic vaccine as well as the use of immune sera derived from P. aeruginosa MV-immunized animals as a treatment for P. aeruginosa corneal infections in C57BL/6 mice. METHODS: C57BL/6 mice were intramuscularly immunized with P. aeruginosa MVs; the mouse corneas were then scarified and topically infected with several P. aeruginosa strains, followed by determination of corneal clinical score and corneal bacterial load. Next, immune sera derived from P. aeruginosa MV-immunized ICR mice were administered intraperitoneally to naïve C57BL/6 mice, followed by topical P. aeruginosa challenge. Finally, the immune sera were also used as a topical treatment in the mice with established P. aeruginosa corneal infections. RESULTS: P. aeruginosa-specific IgG and IgA antibodies induced by intramuscular immunization were detected not only in the sera but also in the eye-wash solution. Both active and passive immunization significantly inhibited P. aeruginosa corneal infection. Finally, topical treatment with immune sera in the mice with established P. aeruginosa corneal infections notably decreased the corneal clinical score and corneal bacterial load. CONCLUSIONS: P. aeruginosa keratitis can be attenuated by vaccination of P. aeruginosa MVs and topical application of P. aeruginosa MV-specific immune sera.

New vaccine production platforms used in developing SARS-CoV-2 vaccine candidates.

The threat of the current coronavirus disease pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is accelerating the development of potential vaccines. Candidate vaccines have been generated using existing technologies that have been applied for developing vaccines against other infectious diseases. Two new types of platforms, mRNA- and viral vector-based vaccines, have been gaining attention owing to the rapid advancement in their methodologies. In clinical trials, setting appropriate immunological endpoints plays a key role in evaluating the efficacy and safety of candidate vaccines. Updated information about immunological features from individuals who have or have not been exposed to SARS-CoV-2 continues to guide effective vaccine development strategies. This review highlights key strategies for generating candidate SARS-CoV-2 vaccines and considerations for vaccine development and clinical trials.

The human papillomavirus E6 protein targets apoptosis-inducing factor (AIF) for degradation.

Oncoprotein E6 of high-risk human papillomavirus (HPV) plays a critical role in inducing cell immortalization and malignancy. E6 downregulates caspase-dependent pathway through the degradation of p53. However, the effect of HPV E6 on other pathways is still under investigation. In the present study, we found that HPV E6 directly binds to all three forms (precursor, mature, and apoptotic) of apoptosis-inducing factor (AIF) and co-localizes with apoptotic AIF. This binding induced MG132-sensitive reduction of AIF expression in the presence of E6 derived from HPV16 (16E6), a cancer-causing type of HPV. Conversely, E6 derived from a non-cancer-causing type of HPV, HPV6 (6E6), did not reduce the levels of AIF despite its interaction with AIF. Flow cytometric analysis revealed that 16E6, but not 6E6, suppressed apoptotic AIF-induced chromatin degradation (an indicator of caspase-independent apoptosis) and staurosporine (STS, a protein kinase inhibitor)-induced apoptosis. AIF knockdown reduced STS-induced apoptosis in both of 16E6-expressing and 6E6-expressing cells; however, the reduction in 16E6-expressing cells was lower than that in 6E6-expressing cells. These findings indicate that 16E6, but not 6E6, blocks AIF-mediated apoptosis, and that AIF may represent a novel therapeutic target for HPV-induced cervical cancer.

Characterization of the reaction of decoupling ubiquinone with bovine mitochondrial respiratory complex I.

We previously produced the unique ubiquinone QT ("decoupling" quinone), the catalytic reduction of which in NADH-quinone oxidoreduction with bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I) is completely decoupled from proton translocation across the membrane domain. This feature is markedly distinct from those of typical short-chain quinones such as ubiquinone-1. To further characterize the features of the QT reaction with complex I, we herein synthesized three QT analogs, QT2-QT4, and characterized their electron transfer reactions. We found that all aspects of electron transfer (e.g. electron-accepting activity and membrane potential formation) vary significantly among these analogs. The features of QT2 as decoupling quinone were slightly superior to those of original QT. Based on these results, we conclude that the bound positions of QTs within the quinone binding cavity susceptibly change depending on their side-chain structures, and the positions, in turn, govern the behavior of QTs as electron acceptors.

Reduction of Synthetic Ubiquinone QT Catalyzed by Bovine Mitochondrial Complex I Is Decoupled from Proton Translocation.

We previously succeeded in site-specific chemical modifications of the inner part of the quinone binding pocket of bovine mitochondrial complex I through ligand-directed tosylate (LDT) chemistry using specific inhibitors as high-affinity ligands for the enzyme [Masuya, T., et al. (2014) Biochemistry 53, 2304-2317, 7816-7823]. To investigate whether a short-chain ubiquinone, in place of these specific inhibitors, serves as a ligand for LDT chemistry, we herein synthesized a LDT reagent QT possessing ubiquinone scaffold and performed LDT chemistry with bovine heart submitochondrial particles (SMP). Detailed proteomic analyses revealed that QT properly guides the tosylate group into the quinone binding pocket and transfers a terminal alkyne to nucleophilic amino acids His150 and Asp160 in the 49 kDa subunit. This result clearly indicates that QT occupies the inner part of the quinone binding pocket. Nevertheless, we noted that QT is a unique electron acceptor from complex I distinct from typical short-chain ubiquinones such as ubiquinone-1 (Q1) for several reasons; for example, QT reduction in NADH-QT oxidoreduction was almost completely insensitive to quinone-site inhibitors (such as bullatacin and piericidin A), and this reaction did not produce a membrane potential. On the basis of detailed comparisons of the electron transfer features between QT and typical short-chain quinones, we conclude that QT may accept electrons from an N2 cluster at a position different from that of typical short-chain quinones because of its unique side-chain structure; accordingly, QT reduction is unable to induce putative structural changes inside the quinone binding pocket, which are critical for driving proton translocation. Thus, QT is the first ubiquinone analogue, to the best of our knowledge, the catalytic reduction of which is decoupled from proton translocation through the membrane domain. Implications for mechanistic studies on QT are also discussed.

Enhanced vaccination effect against influenza by prebiotics in elderly patients receiving enteral nutrition.

AIM: We investigated the effect of prebiotics on the immunological response after influenza vaccination in enterally fed elderly individuals. The intervention group was given an enteral formula containing lactic acid bacteria-fermented milk products. In addition, two different types of other prebiotics, galacto-oligosaccharide and bifidogenic growth stimulator, were also given. The two prebiotics improved intestinal microbiota differently. In a control group, a standard formula without prebiotics was given. METHODS: An enteral formula with (intervention group [F]) or without (control group [C]) prebiotics was given through percutaneous endoscopic gastrostomy to elderly participants for 10 weeks. Influenza vaccine was inoculated at week 4. Nutritional and biochemical indices, intestinal micro bacteria and immunological indices were analyzed. RESULTS: The Bifidobacterium count in groups F and C at week 0 was 6.4 ± 1.9 and 6.6 ± 3.0 (log10 [count/g feces]), respectively. Although the count in group C decreased at week 10, the count in group F increased. The Bacteroides count in group F increased from 10.7 ± 0.9 to 11.4 ± 0.5, but decreased in group C from 11.2 ± 0.2 to 10.7 ± 0.4. Although the enhanced titers of H1N1, H3N2 and B antigens against the vaccine decreased thereafter in group C, these enhanced titers in group F could be maintained. CONCLUSION: Our findings suggest that prebiotics affect the intestinal microbiota and might maintain the antibody titers in elderly individuals.

Quantitative analysis of motifs contributing to the interaction between PLS-subfamily members and their target RNA sequences in plastid RNA editing.

In plant organelles, RNA editing alters specific cytidine residues to uridine in transcripts. Target cytidines are specifically recognized by pentatricopeptide repeat (PPR) proteins of the PLS subfamily, which have additional C-terminal E or E-DYW motifs. Recent in silico analysis proposed a model for site recognition by PLS-subfamily PPR proteins, with a correspondence of one PPR motif to one nucleotide, and with the C-terminal last S motif aligning with the nucleotide at position -4 with respect to the editing site. Here, we present quantitative biochemical data on site recognition by four PLS-subfamily proteins: CRR28 and OTP85 are DYW-class members, whereas CRR21 and OTP80 are E-class members. The minimal RNA segments required for high-affinity binding by these PPR proteins were experimentally determined. The results were generally consistent with the in silico-based model; however, we clarified that several PPR motifs, including the C-terminal L2 and S motifs of CRR21 and OTP80, are dispensable for the RNA binding, suggesting distinct contributions of each PPR motif to site recognition. We also demonstrate that the DYW motif interacts with the target C and its 5' proximal region (from -3 to 0), whereas the E motif is not involved in binding.

Apoptosis of antigen-specific CTLs contributes to low immune response in gut-associated lymphoid tissue post vaccination.

The gut-associated lymphoid tissue (GALT) represents a major reservoir of HIV in infected individuals. Vaccines can induce strong systemic immune responses but these have less impact on CD4 T cells activity and numbers in GALT. In this study, we vaccinated mice with an adenovirus vector that expressed the envelope gene from HIV and observed immune responses in the peripheral blood, spleen, liver, mesenteric lymph nodes, and Peyer's patches. We found that (1) the number of HIV-specific CD8 T cells was dramatically lower in GALT than in other tissues; (2) the programmed cell death protein-1 (PD-1) was expressed at high levels in HIV-specific CD8 T cells including memory T cells in GALT; and (3) high levels of HIV-specific CD8 T cell apoptosis were occurring in GALT. These results suggest that contributing to GALT becoming an HIV reservoir during infection is a combination of exhaustion and/or dysfunction of HIV-specific CTLs at that site. These results emphasize the importance of developing of an effective mucosal vaccine against HIV.

Recent Developments in Preclinical DNA Vaccination.

The advantages of genetic immunization of the new vaccine using plasmid DNAs are multifold. For example, it is easy to generate plasmid DNAs, increase their dose during the manufacturing process, and sterilize them. Furthermore, they can be stored for a long period of time upon stabilization, and their protein encoding sequences can be easily modified by employing various DNA-manipulation techniques. Although DNA vaccinations strongly increase Th1-mediated immune responses in animals, several problems persist. One is about their weak immunogenicity in humans. To overcome this problem, various genetic adjuvants, electroporation, and prime-boost methods have been developed preclinically, which are reviewed here.

Developments in Viral Vector-Based Vaccines.

Viral vectors are promising tools for gene therapy and vaccines. Viral vector-based vaccines can enhance immunogenicity without an adjuvant and induce a robust cytotoxic T lymphocyte (CTL) response to eliminate virus-infected cells. During the last several decades, many types of viruses have been developed as vaccine vectors. Each has unique features and parental virus-related risks. In addition, genetically altered vectors have been developed to improve efficacy and safety, reduce administration dose, and enable large-scale manufacturing. To date, both successful and unsuccessful results have been reported in clinical trials. These trials provide important information on factors such as toxicity, administration dose tolerated, and optimized vaccination strategy. This review highlights major viral vectors that are the best candidates for clinical use.

Prophylaxis and treatment of Alzheimer's disease by delivery of an adeno-associated virus encoding a monoclonal antibody targeting the amyloid Beta protein.

We previously reported on a monoclonal antibody (mAb) that targeted amyloid beta (Aß) protein. Repeated injection of that mAb reduced the accumulation of Aß protein in the brain of human Aß transgenic mice (Tg2576). In the present study, cDNA encoding the heavy and light chains of this mAb were subcloned into an adeno-associated virus type 1 (AAV) vector with a 2A/furin adapter. A single intramuscular injection of 3.0×10(10) viral genome of these AAV vectors into C57BL/6 mice generated serum anti-Aß Ab levels up to 0.3 mg/ml. Anti-Aß Ab levels in excess of 0.1 mg/ml were maintained for up to 64 weeks. The effect of AAV administration on Aß levels in vivo was examined. A significant decrease in Aß levels in the brain of Tg2576 mice treated at 5 months (prophylactic) or 10 months (therapeutic) of age was observed. These results support the use of AAV vector encoding anti-Aß Ab for the prevention and treatment of Alzheimer's disease.

The E domains of pentatricopeptide repeat proteins from different organelles are not functionally equivalent for RNA editing.

RNA editing in plants is an essential post-transcriptional process that modifies the genetic information encoded in organelle genomes. Forward and reverse genetics approaches have revealed the prevalent role of pentatricopeptide repeat (PPR) proteins in editing in both plastids and mitochondria, confirming the shared origin of this process in both organelles. The E domain at or near the C terminus of these proteins has been shown to be essential for editing, and is presumed to recruit the enzyme that deaminates the target cytidine residue. Here, we describe two mutants, otp71 and otp72, disrupted in genes encoding mitochondrial E-type PPR proteins with single editing defects in ccmFN 2 and rpl16 transcripts, respectively. Comparisons between the E domains of these proteins and previously reported editing factors from chloroplasts suggested that there are characteristic differences in the proteins between the two organelles. To test this, we swapped E domains between two mitochondrial and two chloroplast editing factors. In all cases investigated, E domains from the same organelle (chloroplast or mitochondria) were found to be exchangeable; however, swapping the E domain between organelles led to non-functional editing factors. We conclude that the E domains of mitochondrial and plastid PPR proteins are not functionally equivalent, and therefore that an important component of the putative editing complexes in the two organelles may be different.