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Objective: Clinical practice guidelines (CPG) worldwide help steer the management of early-onset neonatal sepsis (EONS). These documents typically discourage the use of risk assessment tools. However, prior work has shown that the Kaiser Permanente calculator (Early-Onset Sepsis Calculator [EOScalc]) could be a useful tool in EONS risk assessment. This study aimed to determine the agreement between the recommendations of the Colombian EONS CPG and those of the EOSCalc tool in a cohort of newborns in Bogotá, Colombia., Study Design: Multicenter retrospective observational cohort study. We included newborns with a gestational age ≥ 34 weeks who were admitted to the neonatal care unit with a suspected diagnosis of EONS between 2017 and 2019. Agreement between the two tools was examined using Cohen's kappa under two scenarios (unequivocal and cautious)., Results: Of the 23.490 live births, 470 (1.71%) were admitted to the neonatal care unit with a presumptive diagnosis of EONS. This diagnosis was confirmed in seven patients by means of blood cultures, with group B streptococcus the most common organism (57%; 95% confidence interval [CI]: 18.4-90.1). A single death occurred among the patients with confirmed EONS (lethality: 14.3%). The overall incidence of EONS was 0.298 per 1,000 live births. After splitting the recommendations into two scenarios regarding antibiotic use, unequivocal and cautious, the agreement between EOSCalc and the CPG was below 15% (6 and 14%, respectively)., Conclusion: Recommendations from the Colombian EONS CPG show poor agreement with the EOSCalc, with the latter detecting all newborns with EONS. Although the use of EOSCalc is clinically and administratively advantageous, further prospective studies are warranted to determine the safety of its implementation., Key Points: · Colombian EONS CPGs recommend that an outsized number of newborns be given antibiotics.. · The KP EOSCalc risk assessment calculator shows poor agreement with CPG recommendations.. · The Colombian CPGs should be updated to include the use of risk assessment calculators.., Competing Interests: None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Evidence about the environmental impacts of shared mobility is fragmented and scattered. In this article a systematic literature review is presented. The review focuses on assessments that use Life-Cycle Assessment to quantify the environmental impacts of car sharing, carpooling, bikesharing, and scooter/moped sharing. The results of these assessments were analyzed, as well as the factors that influence these impacts. Business-to-consumer car sharing, peer-to-peer car sharing, carpooling, bikesharing, and scooter/moped sharing can all cause gains and losses in terms of changing the environmental impacts of passenger transportation. The findings presented here refute unconditional claims that shared mobility delivers environmental benefits. Factors that influence changes in environmental impacts from passenger transportation from shared mobility include travel behaviour, the design of shared mobility modes, and how such schemes are implemented, as well as the local context. Local governments and shared mobility organisations can benefit from the analysis presented here by deepening their understanding of these factors and considering the life-cycle phase where the greatest impacts are caused., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Foliar-applied insecticides are commonly used for adult western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), control in Nebraska but little efficacy data is available. Anecdotal reports of reduced efficacy in areas of northeast Nebraska led to the conduct of this study. Objectives were to (i) evaluate the efficacy of commercial applications of commonly used formulated insecticides (bifenthrin, lambda-cyhalothrin, chlorpyrifos, or tank mixes) for WCR control in 7 northeast Nebraska counties during 2019 and 2020 and (ii) conduct adult WCR concentration-response vial bioassays with bifenthrin, chlorpyrifos, and dimethoate active ingredients on a subset of field populations. Whole plant counts (WPC) were used to measure WCR densities in insecticide-treated and untreated maize fields before and after insecticide application. Field control was excellent with organophosphate/pyrethroid tank mixes as proportional change in mean WPC of treated fields was significantly reduced (>0.90) versus untreated fields where little change in WPC occurred. The exception was one treated Boone County field where proportional reduction in WPC was ≤0.78. Bioassays revealed LC50s and resistance ratios of most populations exposed to bifenthrin and dimethoate were not significantly different than the susceptible control. Most populations exhibited a low level of chlorpyrifos resistance when compared to the susceptible control. Field and lab data suggest the local onset of practical WCR field-evolved resistance to bifenthrin in Boone County and chlorpyrifos in Boone and Colfax counties. Results of this study will increase our understanding of WCR resistance evolution, serve as a comprehensive baseline for future research, and inform WCR management programs., (© The Author(s) 2023. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Exposure of lepidopteran pests to Bacillus thuringiensis ( Bt ) proteins has been shown to affect the behavior of larvae, including increased movement and avoidance of Bt -expressing plants or diet. Therefore, we hypothesized that the behavior of western bean cutworm, Striacosta albicosta (Smith) (Lepidoptera: Noctuidae), an important pest of maize, could be affected when exposed to Bt plants. To test this hypothesis, we conducted a series of artificial arena and on-plant experiments to determine S. albicosta neonate behavior when exposed to Bt and non- Bt plant tissue. Video tracking experiments presented neonate larvae with the choice of Bt or non- Bt pollen in a Petri dish for 15 min while being video recorded for analysis with EthoVision software. This study showed an increase in mean velocity and total time spent moving for larvae in the presence of Cry1F vs. non- Bt when compared with Vip3A vs. non- Bt or Cry1F vs. Vip3A. However, there was no difference in total distance moved or time spent in the food zone for all scenarios. Maize tissue choice experiments allowed neonatal larvae the choice of feeding on Bt or non- Bt tassel or leaves for 9 h in Petri dish arenas. This experiment showed that larvae preferred tassel tissue over leaves but did not indicate that larvae could distinguish between Bt and non- Bt tissue. In contrast, on-plant experiments (including a whole plant neonate dispersal study under controlled conditions and an in-field silking behavior experiment) indicated that the presence of Cry1F and Vip3A Bt toxins increased plant abandonment, suggesting that larvae are able to detect and avoid Bt toxins. The discrepancy of these results is likely due to the on-plant studies providing more field-realistic environmental conditions and a longer duration of exposure to Bt toxins for the behavioral experiments. Our results represent the first steps in understanding the complex behavior of S. albicosta when exposed to Bt plants. A better understanding of the response of larvae when exposed to Bt traits can aid in the management of this pest, particularly for the design of resistance management strategies and refuge design.

Declines of the monarch butterfly population have prompted large-scale plantings of milkweed to restore the population. In North America, there are >73 species of milkweed to choose from for these nationwide plantings. However, it is unclear how different milkweed species affect monarch caterpillar physiology, particularly detoxification enzyme activity and gene expression, given the highly variable cardenolide composition across milkweed species. Here, we investigate the effects of a high cardenolide, tropical milkweed species and a low cardenolide, swamp milkweed species on pyrethroid sensitivity as well as detoxification enzyme activity and expression in monarch caterpillars. Caterpillars fed on each species through the fifth-instar stage and were topically treated with bifenthrin after reaching this final-instar stage. Esterase, glutathione S-transferase, and cytochrome P450 monooxygenase activities were quantified as well as the expression of selected esterase, glutathione S-transferase, ABC transporter, and cytochrome P450 monooxygenase transcripts. There were no significant differences in survival 24 h after treatment with bifenthrin. However, bifenthrin significantly increased glutathione S-transferase activity in caterpillars feeding on tropical milkweed and significantly decreased esterase activity in caterpillars feeding on tropical and swamp milkweed. Significant differential expression of ABC transporter, glutathione S-transferase, and esterase genes was observed for caterpillars feeding on tropical and swamp milkweed and not receiving bifenthrin treatment. Furthermore, significant differential expression of glutathione S-transferase and esterase genes was observed for bifenthrin-treated and -untreated caterpillars feeding on tropical milkweed relative to swamp milkweed. These results suggest that feeding on different milkweed species can affect detoxification and development mechanisms with which monarch caterpillars rely on to cope with their environment., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is considered one of the most economically important pests of maize ( Zea mays L.) in the United States (U.S.) Corn Belt with costs of management and yield losses exceeding USD ~1-2 billion annually. WCR management has proven challenging given the ability of this insect to evolve resistance to multiple management strategies including synthetic insecticides, cultural practices, and plant-incorporated protectants, generating a constant need to develop new management tools. One of the most recent developments is maize expressing double-stranded hairpin RNA structures targeting housekeeping genes, which triggers an RNA interference (RNAi) response and eventually leads to insect death. Following the first description of in planta RNAi in 2007, traits targeting multiple genes have been explored. In June 2017, the U.S. Environmental Protection Agency approved the first in planta RNAi product against insects for commercial use. This product expresses a dsRNA targeting the WCR snf7 gene in combination with Bt proteins (Cry3Bb1 and Cry34Ab1/Cry35Ab1) to improve trait durability and will be introduced for commercial use in 2022.

The monarch butterfly, Danaus plexippus L., has evolved to be insensitive to milkweed cardenolides via genetic modifications of Na+/K+-ATPase. There is concern for insecticide exposures near agriculture, with little information on monarch caterpillar toxicology. It is unclear how cardenolide insensitivity may affect the sensitivity of monarch caterpillars to pyrethroid insecticides. Additionally, potassium fertilizers may affect monarch caterpillar physiology and cardenolide sequestration. Here, we investigated the growth, survival, and development of caterpillars exposed to the cardenolide ouabain, bifenthrin, and potassium chloride (KCl) alone and in combination. Caterpillars were either exposed to 1) ouabain from third- to fifth-instar stage, 2) KCl at fifth-instar stage, 3) KCl and bifenthrin at fifth-instar stage, or 4) combinations of ouabain at third-instar stage + KCl + bifenthrin at fifth-instar stage. Caterpillar weight, diet consumption, frass, and survival were recorded for the duration of the experiments. It was observed that 1-3 mg ouabain/g diet increased body weight and diet consumption, whereas 50 mg KCl/g diet decreased body weight and diet consumption. Caterpillars feeding on KCl and treated with 0.2 µg/µl bifenthrin consumed significantly less diet compared to individuals provided untreated diet. However, there was no effect on survival or body weight. Combinations of KCl + ouabain did not significantly affect caterpillar survival or body weight following treatment with 0.1 µg/µl bifenthrin. At the concentrations tested, there were no effects observed for bifenthrin sensitivity with increasing cardenolide or KCl concentrations. Further studies are warranted to understand how milkweed-specific cardenolides, at increasing concentrations, and agrochemical inputs can affect monarch caterpillar physiology near agricultural landscapes., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Insecticide exposure has been identified as a contributing stressor to the decline in the North American monarch butterfly Danaus plexippus L. (Lepidoptera: Nymphalidae) population. Monarch toxicity data are currently limited and available data focuses on lethal endpoints. This study examined the 72-h toxicity of two pyrethroid insecticides, bifenthrin and β-cyfluthrin, and their effects on growth and diet consumption. The toxicity of bifenthrin to caterpillars was lower than β-cyfluthrin after 72 h. Survival was the most sensitive endpoint for bifenthrin, but diet consumption and caterpillar growth were significantly reduced at sublethal levels of β-cyfluthrin. Using AgDRIFT spray drift assessment, the aerial application of bifenthrin or β-cyfluthrin is predicted to pose the greatest risk to fifth-instar caterpillars, with lethal insecticide deposition up to 28 m for bifenthrin and up to 23 m for β-cyfluthrin from treated edges of fields. Low boom ground applications are predicted to reduce distances of lethal insecticide exposure to 2 m from the treated field edge for bifenthrin and β-cyfluthrin. Growth and survival of fifth-instar monarch caterpillars developing within the margins of a treated field may be significantly impacted following foliar applications of bifenthrin or β-cyfluthrin. These findings provide evidence that pyrethroid insecticides commonly used for soybean pest control are a potential risk to monarch caterpillars in agricultural landscapes., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America.)

The genome of Diabrotica undecimpunctata virus 2 (DuV2), a putative positive-sense, single-stranded RNA virus identified from the southern corn rootworm transcriptome, comprises 5,313 nucleotides, including a short poly(A) tail. The two open reading frames encode a nonstructural polyprotein (p156) and a putative capsid protein (p25)., (Copyright © 2020 Liu et al.)

The genome sequence of a novel small RNA virus, tentatively named Diabrotica undecimpunctata virus 1 (DuV1), was discovered from the transcriptome of the southern corn rootworm, Diabrotica undecimpunctata howardi Barber. DuV1 has a positive-sense, single-stranded RNA genome that encodes a single polyprotein of 3,401 amino acids with limited similarity to other viruses., (Copyright © 2020 Liu et al.)

Historically, genetically engineered (GE) plants that have incorporated genes conferring insect protection have primarily used Cry proteins derived from Bacillus thuringiensis ( Bt ) to achieve their insecticidal phenotype. As a result, regulators have developed a level of familiarity and confidence in reviewing plants incorporating these insecticidal proteins. However, new technologies have been developed that produce GE plants that incorporate pest protection by triggering an RNA interference (RNAi) response or proteins other than Bt Cry proteins. These technologies have new modes of action. Although the overall assessment paradigm for GE plants is robust, there are ongoing discussions about the appropriate tests and measurement endpoints needed to inform non-target arthropod assessment for technologies that have a different mode of action than the Bt Cry proteins. As a result, increasing attention is being paid to the use of sublethal endpoints and their value for environmental risk assessment (ERA). This review focuses on the current status and history of sublethal endpoint use in insect-active GE crops, and evaluates the future use of sublethal endpoints for new and emerging technologies. It builds upon presentations made at the Workshop on Sublethal Endpoints for Non-target Organism Testing for Non- Bt GE Crops (Washington DC, USA, 4-5 March 2019), and the discussions of government, academic and industry scientists convened for the purpose of reviewing the progress and status of sublethal endpoint testing in non-target organisms., (Copyright © 2020 Roberts, Boeckman, Mühl, Romeis, Teem, Valicente, Brown, Edwards, Levine, Melnick, Rodrigues, Vélez, Zhou and Hellmich.)

Exploiting the RNA interference (RNAi) gene mechanism to silence essential genes in pest insects, leading to toxic effects, has surfaced as a promising new control strategy in the past decade. While the first commercial RNAi-based products are currently coming to market, the application against a wide range of insect species is still hindered by a number of challenges. In this review, we discuss the current status of these RNAi-based products and the different delivery strategies by which insects can be targeted by the RNAi-triggering double-stranded RNA (dsRNA) molecules. Furthermore, this review also addresses a number of physiological and cellular barriers, which can lead to decreased RNAi efficacy in insects. Finally, novel non-transgenic delivery technologies, such as polymer or liposomic nanoparticles, peptide-based delivery vehicles and viral-like particles, are also discussed, as these could overcome these barriers and lead to effective RNAi-based pest control., (Copyright © 2020 Christiaens, Whyard, Vélez and Smagghe.)

Background: RNA interference (RNAi) triggered by maize plants expressing RNA hairpins against specific western corn rootworm (WCR) transcripts have proven to be effective at controlling this pest. To provide robust crop protection, mRNA transcripts targeted by double-stranded RNA must be sensitive to knockdown and encode essential proteins., Results: Using WCR adult feeding assays, we identified Sec23 as a highly lethal RNAi target. Sec23 encodes a coatomer protein, a component of the coat protein (COPII) complex that mediates ER-Golgi transport. The lethality detected in WCR adults was also observed in early instar larvae, the life stage causing most of the crop damage, suggesting that WCR adults can serve as an alternative to larvae for dsRNA screening. Surprisingly, over 85% transcript inhibition resulted in less than 40% protein knockdown, suggesting that complete protein knockdown is not necessary for Sec23 RNAi-mediated mortality. The efficacy of Sec23 dsRNA for rootworm control was confirmed in planta; T 0 maize events carrying rootworm Sec23 hairpin transgenes showed high levels of root protection in greenhouse assays. A reduction in larval survival and weight were observed in the offspring of WCR females exposed to Sec23 dsRNA LC 25 in diet bioassays., Conclusion: We describe Sec23 as RNAi target for in planta rootworm control. High mortality in exposed adult and larvae and moderate sublethal effects in the offspring of females exposed to Sec23 dsRNA LC 25 , suggest the potential for field application of this RNAi trait and the need to factor in responses to sublethal exposure into insect resistance management programs. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

Striacosta albicosta (Smith) is a maize pest that has recently expanded its geographical range into the eastern United States and southeastern Canada. Aerial application of pyrethroids, such as bifenthrin, has been a major practice adopted to manage this pest. Reports of field failure of pyrethroids have increased since 2013. Striacosta albicosta populations were collected in 2016 and 2017 from maize fields in Nebraska, Kansas, and Canada and screened with bifenthrin active ingredient in larval contact dose-response bioassays. Resistance ratios estimated were generally low in 2016 (1.04- to 1.32-fold) with the highest LC50 in North Platte, NE (66.10 ng/cm2) and lowest in Scottsbluff, NE (50.10 ng/cm2). In 2017, O'Neill, NE showed the highest LC50 (100.66 ng/cm2) and Delhi, Canada exhibited the lowest (6.33 ng/cm2), resulting in a resistance ratio variation of 6.02- to 15.90-fold. Implications of bifenthrin resistance levels were further investigated by aerial application simulations. Experiments were conducted with a spray chamber where representative S. albicosta populations were exposed to labeled rates of a commercial bifenthrin formulation. Experiments resulted in 100% mortality for all populations, instars, insecticide rates, and carrier volumes, suggesting that levels of resistance estimated for bifenthrin active ingredient did not seem to impact the efficacy of the correspondent commercial product under controlled conditions. Results obtained from this research indicate that control failures reported in Nebraska could be associated with factors other than insecticide resistance, such as issues with the application technique, environmental conditions during and/or after application, or the insect's natural behavior. Data generated will assist future S. albicosta resistance management programs., (© The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America.)

Knowledge of dispersal and spatial dynamics of pest populations is fundamental for implementation of integrated pest management and integrated resistance management. This study evaluated 1) the effectiveness of egg white albumin protein to mark larvae and adults of two polyphagous and highly mobile pests, Spodoptera frugiperda (J.E. Smith) (fall armyworm) and Helicoverpa zea (Boddie) (corn earworm) (Lepidoptera: Noctuidae), and 2) the sensitivity of polyvinylidene difluoride membrane (dot blot) in detecting albumin on marked insects. Laboratory and field experiments tested egg albumin as a protein marker, which was detected using two enzyme-linked immunosorbent assay (ELISA), microplate, and dot blot. In the laboratory, 100% of the moths sprayed with 20% egg white solution acquired the albumin marker, which was detected through the last time point tested (5 d) after application. Egg albumin was not effective at long-term marking of larvae, detected only prior the molting to the next instar. Albumin application in field cages resulted in a high percentage of moths detected as marked at 24 h and 5 d for both species. Egg albumin applied in the open field resulted in 15% of the recaptured corn earworm moths marked with most of them collected 150 m from the application area, although some were captured as far as 1,600 m within approximately 6 d after adult emergence. The results indicated egg albumin is a suitable marker to study the dispersion of fall armyworm and corn earworm in the agroecosystem and dot blot was as effective to detect egg albumin as was indirect ELISA., (© The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America.)

Coffee production is a global industry valued at approximately 173 billion US dollars. One of the main challenges facing coffee production is the management of the coffee berry borer (CBB), Hypothenemus hampei, which is considered the primary arthropod pest of coffee worldwide. Current control strategies are inefficient for CBB management. Although biotechnological alternatives, including RNA interference (RNAi), have been proposed in recent years to control insect pests, characterizing the genetics of the target pest is essential for the successful application of these emerging technologies. In this study, we employed RNA-seq to obtain the transcriptome of three developmental stages of the CBB (larva, female and male) to increase our understanding of the CBB life cycle in relation to molecular features. The CBB transcriptome was sequenced using Illumina Hiseq and assembled de novo. Differential gene expression analysis was performed across the developmental stages. The final assembly produced 29,434 unigenes, of which 4,664 transcripts were differentially expressed. Genes linked to crucial physiological functions, such as digestion and detoxification, were determined to be tightly regulated between the reproductive and nonreproductive stages of CBB. The data obtained in this study help to elucidate the critical roles that several genes play as regulatory elements in CBB development.

Quantitative reverse transcription PCR (RT-qPCR) is one of the most efficient, reliable and widely used techniques to quantify gene expression. In this study, we evaluated the performance of six southern corn rootworm, Diabrotica undecimpunctata howardi (Barber), housekeeping genes (HKG), β-actin (Actin), β-tubulin (Tubulin), elongation factor 1 alpha (EF1α), glyceraldehyde-3 phosphate dehydrogenase (GAPDH), 40 S ribosomal protein S9 (RpS9) and ubiquitin-conjugating protein (Ubi), under different experimental conditions such as developmental stage, exposure of neonate and adults to dsRNA, exposure of adults to different temperatures, different 3 rd instar larva tissues, and neonate starvation. The HKGs were analyzed with four algorithms, including geNorm, NormFinder, BestKeeper, and delta-CT. Although the six HKGs showed a relatively stable expression pattern among different treatments, some variability was observed. Among the six genes, EF1α exhibited the lowest Ct values for all treatments while Ubi exhibited the highest. Among life stages and across treatments, Ubi exhibited the least stable expression pattern. GAPDH, Actin, and EF1α were among the most stable HKGs in the majority of the treatments. This research provides HKG for accurate normalization of RT-qPCR data in the southern corn rootworm. Furthermore, this information can contribute to future genomic and functional genomic research in Diabrotica species.

RNA interference (RNAi) is becoming a practical tool to control insect pests. Many mysteries of how double-stranded RNA (dsRNA) is transported into, within, and between cells to generate an efficient RNAi response in insects are still to be unraveled. This review provides an overview of the evidence that supports a key role of endocytosis in the uptake of dsRNA on both cellular and tissue levels. Additionally, other components of cellular membrane transport and their impact on the efficiency of RNAi in insects are explored. It is now evident that the membrane transport and potentially dsRNA release from the endosome may comprise some of the limiting factors in insects that are recalcitrant to dsRNA. This review concludes with the apparent connection between gene products that are necessary for cellular trafficking of dsRNA and highly lethal RNAi targets., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The cellular uptake of dsRNA after dietary exposure is critical for RNAi efficiency; however, the mechanism of its uptake in many insects remains to be understood. In this study, we evaluated the roles of the endocytic pathway genes Clathrin heavy chain (Chc), Clathrin adaptor protein AP50, ADP ribosylation factor-like 1 (Arf72A), Vacuolar H+ ATPase 16 kDa subunit (Vha16), and small GTPase Rab7 and putative sid-1-like genes (silA and silC) in RNAi response in western corn rootworm (WCR) using a two-stage dsRNA exposure bioassay. Silencing of Chc, Vha16, and AP50 led to a significant decrease in the effects of laccase2 dsRNA reporter, indicating that these genes are involved in RNAi response. However, the knockdown of either Arf72A or Rab7 did not suppress the response to laccase2 dsRNA. The silencing of the silC gene did not lead to a significant reduction in mortality or increase in the expression of V-ATPase A reporter. While the silencing of the silA gene significantly decreased insect mortality, significant changes in V-ATPase A expression were not detected. These results suggest that clathrin-dependent endocytosis is a biological mechanism that plays an important role during RNAi response in WCR adults. The fact that no definitive support for the roles of silA or silC in RNAi response was obtained support the idea that RNAi response varies greatly in different insect species, demanding additional studies focused on elucidating their involvement in this mechanism., Competing Interests: KEN is an employee of Corteva Agriscience™, Agriculture Division of DowDuPont™. EF is a former employee of Corteva Agriscience™, Agriculture Division of DowDuPont™. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials. There are no disputes pertaining to the ownership of the data presented in the paper and all contributions are attributed below. All authors state that they adhere to the PLoS One Conflict of Interest Guidelines.

RNA interference is a powerful tool against corn rootworm. Adults and neonates of southern corn rootworm, Diabrotica undecimpunctata howardi Barber (Coleoptera: Chrysomelidae), were exposed to the LC50 of vATPase-A and Snf7 double-stranded RNAs (dsRNAs), and the effects on female fecundity, egg viability, male fitness as measured by sperm viability and mating capacity, larval recovery along with dry weight, and instar determination 10 d after exposure to dsRNA, were determined. Significant reductions were observed for a number of parameters in dsRNA-exposed rootworms relative to control treatments. Female fecundity and larval recovery were significantly reduced after exposure to both dsRNAs. In addition, larval dry weight and recovery of 2nd and 3rd instars along with dry weight for 3rd instars were significantly reduced after neonate exposure to vATPase-A dsRNA. Neither dsRNA affected male capacity to mate or sperm viability after exposure to the respective LC50s. After 10 d of feeding on untreated corn roots, neonates that survived exposure for 2 d to the vATPase-A dsRNA LC50 exhibited lower dry weight than the control. There was significant gene knockdown in adult males and females after exposure for 5 d to LC50 of vATPase-A and Snf7 dsRNAs. The parameters are discussed in terms of fitness and possible outcomes after deployment of corn hybrids expressing dsRNAs., (© The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) is one of the major pests of maize and is in the same feeding guild as the noctuid pests Helicoverpa zea (Boddie) and Helicoverpa armigera (Hübner), recently reported in South and North America. The intraguild interactions of these species were assessed in laboratory and field conditions by determining the survival of larvae in interaction scenarios with non-Bt maize silks and ears. Moreover, a video tracking system was utilized to evaluate behavioral parameters during larval interactions in scenarios with or without food., Results: In intraguild interactions, S. frugiperda had greater survival (55-100%) when competing with Helicoverpa spp. in scenarios where larvae were the same instar or when they were larger (fourth versus second) than their competitor. Frequency and time in food of S. frugiperda larvae were negatively influenced by interactions. Larvae of S. frugiperda moved shorter distances (less than 183.03 cm) compared with H. zea., Conclusion: Overall, S. frugiperda had a competitive advantage over Helicoverpa spp. This study provides significant information regarding noctuid behavior and larval survival during intraguild interactions, which may impact pest prevalence and population dynamics, thereby affecting integrated pest management and insect resistance management of these species in maize. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

Parental RNAi (pRNAi) is an RNA interference response where the gene knockdown phenotype is observed in the progeny of the treated organism. pRNAi has been demonstrated in female western corn rootworms (WCR) via diet applications and has been described as a potential approach for rootworm pest management. However, it is not clear if plant-expressed pRNAi can provide effective control of next generation WCR larvae in the field. In this study, we evaluated parameters required to generate a successful pRNAi response in WCR for the genes brahma and hunchback . The parameters tested included a concentration response, duration of the dsRNA exposure, timing of the dsRNA exposure with respect to the mating status in WCR females, and the effects of pRNAi on males. Results indicate that all of the above parameters affect the strength of pRNAi phenotype in females. Results are interpreted in terms of how this technology will perform in the field and the potential role for pRNAi in pest and resistance management strategies. More broadly, the described approaches enable examination of the dynamics of RNAi response in insects beyond pRNAi and crop pests., Competing Interests: E.F., N.P.S. and K.E.N. are current employees of Dow AgroSciences. All authors state that they adhere to Genes′ Publication Ethics Statement.

The western corn rootworm (WCR), Diabrotica virgifera virgifera, is the most important pest of corn in the US Corn Belt. Economic estimates indicate that costs of control and yield loss associated with WCR damage exceed $US 1 billion annually. Historically, corn rootworm management has been extremely difficult because of its ability to evolve resistance to both chemical insecticides and cultural control practices. Since 2003, the only novel commercialized developments in rootworm management have been transgenic plants expressing Bt insecticidal proteins. Four transgenic insecticidal proteins are currently registered for rootworm management, and field resistance to proteins from the Cry3 family highlights the importance of developing traits with new modes of action. One of the newest approaches for controlling rootworm pests involves RNA interference (RNAi). This review describes the current understanding of the RNAi mechanisms in WCR and the use of this technology for WCR management. Further, the review addresses ecological risk assessment of RNAi and insect resistance management of RNAi for corn rootworm. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is an important maize pest throughout most of the U.S. Corn Belt. Bacillus thuringiensis (Bt) insecticidal proteins including modified Cry3Aa and Cry34/35Ab1 have been expressed in transgenic maize to protect against WCR feeding damage. To date, there is limited information regarding the WCR midgut target sites for these proteins. In this study, we examined whether a cadherin-like gene from Diabrotica virgifera virgifera (DvvCad; GenBank accession # EF531715) associated with WCR larval midgut tissue is necessary for Cry3Aa or Cry34/35Ab1 toxicity. Experiments were designed to examine the sensitivity of WCR to trypsin activated Cry3Aa and Cry34/35Ab1 after oral feeding of the DvvCad dsRNA to knockdown gene expression. Quantitative real-time PCR confirmed that DvvCad mRNA transcript levels were reduced in larvae treated with cadherin dsRNA. Relative cadherin expression by immunoblot analysis and nano-liquid chromatography - mass spectrometry (nanoLC-MS) of WCR neonate brush border membrane vesicle (BBMV) preparations exposed to DvvCad dsRNA confirmed reduced cadherin expression when compared to BBMV from untreated larvae. However, the larval mortality and growth inhibition of WCR neonates exposed to cadherin dsRNA for two days followed by feeding exposure to either Cry3Aa or Cry34/35Ab1 for four days was not significantly different to that observed in insects exposed to either Cry3Aa or Cry34/35Ab1 alone. In combination, these results suggest that cadherin is unlikely to be involved in the toxicity of Cry3Aa or Cry34/35Ab1 to WCR., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

RNA interference (RNAi) is being developed as a potential tool for insect pest management. Increased understanding of the RNAi pathway in target insect pests will provide information to use this technology effectively and to inform decisions related to resistant management strategies for RNAi based traits. Dicer 2 (Dcr2), an endonuclease responsible for formation of small interfering RNA's and Argonaute 2 (Ago2), an essential catalytic component of the RNA-induced silencing complex (RISC) have both been associated with the RNAi pathway in a number of different insect species including the western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). We identified both genes from a transcriptome library generated from different tissues and developmental stages of the western corn rootworm, an important target pest for transgenic plants expressing dsRNA targeting essential genes. The expression of these genes was suppressed by more than 90% after injecting gene specific dsRNA into adult rootworms. The injected beetles were then fed vATPase A dsRNA which has previously been demonstrated to cause mortality in western corn rootworm adults. The suppression of both RNAi pathway genes resulted in reduced mortality after subsequent exposure to lethal concentrations of vATPase A dsRNA as well as increased vATPase A expression relative to control treatments. Injections with dsRNA for a non-lethal target sequence (Laccase 2) did not affect mortality or expression caused by vATPase A dsRNA indicating that the results observed with Argo and Dicer dsRNA were not caused by simple competition among different dsRNA's. These results confirm that both genes play an important role in the RNAi pathway for western corn rootworms and indicate that selection pressures that potentially affect the expression of these genes may provide a basis for future studies to understand potential mechanisms of resistance.

The Cry1Fa protein from the bacterium Bacillus thuringiensis (Bt) is known for its potential to control lepidopteran pests, especially through transgenic expression in maize and cotton. The maize event TC1507 expressing the cry1Fa toxin gene became commercially available in the United States in 2003 for the management of key lepidopteran pests including the European corn borer, Ostrinia nubilalis, and the fall armyworm, Spodoptera frugiperda. A high-dose/refuge strategy has been widely adopted to delay evolution of resistance to event TC1507 and other transgenic Bt crops. Efficacy of this strategy depends on the crops expressing a high dose of the Bt toxin to targeted pests and adjacent refuges of non-Bt host plants serving as a source of abundant susceptible insects. While this strategy has proved effective in delaying O. nubilalis resistance, field-evolved resistance to event TC1507 has been reported in S. frugiperda populations in Puerto Rico, Brazil, and the southeastern United States. This paper examines available information on resistance to Cry1Fa in O. nubilalis and S. frugiperda and discusses how this information identifies opportunities to refine resistance management recommendations for Bt maize., (Copyright © 2016 Elsevier Inc. All rights reserved.)

RNA interference (RNAi) is a gene silencing mechanism that is present in animals and plants and is triggered by double stranded RNA (dsRNA) or small interfering RNA (siRNA), depending on the organism. In the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), RNAi can be achieved by feeding rootworms dsRNA added to artificial diet or plant tissues transformed to express dsRNA. The effect of RNAi depends on the targeted gene function and can range from an absence of phenotypic response to readily apparent responses, including lethality. Furthermore, RNAi can directly affect individuals that consume dsRNA or the effect may be transferred to the next generation. Our previous work described the potential use of genes involved in embryonic development as a parental RNAi technology for the control of WCR. In this study, we describe the use of chromatin-remodeling ATPases as target genes to achieve parental gene silencing in two insect pests, a coleopteran, WCR, and a hemipteran, the Neotropical brown stink bug, Euschistus heros Fabricius (Hemiptera: Pentatomidae). Our results show that dsRNA targeting chromatin-remodeling ATPase transcripts, brahma, mi-2, and iswi strongly reduced the fecundity of the exposed females in both insect species. Additionally, knockdown of chd1 reduced the fecundity of E. heros., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Maize plants expressing dsRNA for the management of Diabrotica virgifera virgifera are likely to be commercially available by the end of this decade. Honey bees, Apis mellifera, can potentially be exposed to pollen from transformed maize expressing dsRNA. Consequently, evaluation of the biological impacts of RNAi in honey bees is a fundamental component for ecological risk assessment. The insecticidal activity of a known lethal dsRNA target for D. v. virgifera, the vATPase subunit A, was evaluated in larval and adult honey bees. Activity of both D. v. virgifera (Dvv)- and A. mellifera (Am)-specific dsRNA was tested by dietary exposure to dsRNA. Larval development, survival, adult eclosion, adult life span and relative gene expression were evaluated. The results of these tests indicated that Dvv vATPase-A dsRNA has limited effects on larval and adult honey bee survival. Importantly, no effects were observed upon exposure of Am vATPase-A dsRNA suggesting that the lack of response involves factors other than sequence specificity. The results from this study provide guidance for future RNAi risk analyses and for the development of a risk assessment framework that incorporates similar hazard assessments., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

RNA interference (RNAi) is being developed as a potential tool for insect pest management and one of the most likely target pest species for transgenic plants that express double stranded RNA (dsRNA) is the western corn rootworm. Thus far, most genes proposed as targets for RNAi in rootworm cause lethality in the larval stage. In this study, we describe RNAi-mediated knockdown of two developmental genes, hunchback (hb) and brahma (brm), in the western corn rootworm delivered via dsRNA fed to adult females. dsRNA feeding caused a significant decrease in hb and brm transcripts in the adult females. Although total oviposition was not significantly affected, there was almost complete absence of hatching in the eggs collected from females exposed to dsRNA for either gene. These results confirm that RNAi is systemic in nature for western corn rootworms. These results also indicate that hunchback and brahma play important roles in rootworm embryonic development and could provide useful RNAi targets in adult rootworms to prevent crop injury by impacting the population of larval progeny of exposed adults. The ability to deliver dsRNA in a trans-generational manner by feeding to adult rootworms may offer an additional approach to utilizing RNAi for rootworm pest management. The potential to develop parental RNAi technology targeting progeny of adult rootworms in combination with Bt proteins or dsRNA lethal to larvae may increase opportunities to develop sustainable approaches to rootworm management involving RNAi technologies for rootworm control., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Penicillin G acylase (PGA) is used industrially to catalyze the hydrolysis of penicillin G to obtain 6-aminopenicillanic acid. In Escherichia coli, the most-studied microorganism for PGA production, this enzyme accumulates in the periplasmic cell space, and temperature plays an important role in the correct synthesis of its subunits., Results: This work investigates the influence of medium composition, cultivation strategy, and temperature on PGA production by recombinant E. coli cells. Shake flask cultures carried out using induction temperatures ranging from 18 to 28°C revealed that the specific enzyme activity achieved at 20°C (3000 IU gDCW-1) was 6-fold higher than the value obtained at 28°C. Auto-induction and high cell density fed-batch bioreactor cultures were performed using the selected induction temperature, with both defined and complex media, and IPTG and lactose as inducers. Final biomass concentrations of 100 and 120 gDCW L-1, and maximum enzyme productivities of 7800 and 5556 IU L-1 h-1, were achieved for high cell density cultures using complex and defined media, respectively., Conclusions: To the best of our knowledge, the volumetric enzyme activity and productivity values achieved using the complex medium are the highest ever reported for PGA production using E. coli. Overall PGA recovery yields of 64 and 72% after purification were achieved for crude extracts obtained from cells cultivated in defined and complex media, respectively. The complex medium was the most cost-effective for PGA production, and could be used in both high cell density and straightforward auto-induction protocols.

Transgenic maize, Zea maize L., expressing the Cry1F protein from Bacillus thuringiensis has been registered for Spodoptera frugiperda (J. E. Smith) control since 2003. Unexpected damage to Cry1F maize was reported in 2006 in Puerto Rico and Cry1F resistance in S. frugiperda was documented. The inheritance of Cry1F resistance was characterized in a S. frugiperda resistant strain originating from Puerto Rico, which displayed >289-fold resistance to purified Cry1F. Concentration-response bioassays of reciprocal crosses of resistant and susceptible parental populations indicated that resistance is recessive and autosomal. Bioassays of the backcross of the F1 generation crossed with the resistant parental strain suggest that a single locus is responsible for resistance. In addition, cross-resistance to Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba, Cry2Aa and Vip3Aa was assessed in the Cry1F-resistant strain. There was no significant cross-resistance to Cry1Aa, Cry1Ba and Cry2Aa, although only limited effects were observed in the susceptible strain. Vip3Aa was highly effective against susceptible and resistant insects indicating no cross-resistance with Cry1F. In contrast, low levels of cross-resistance were observed for both Cry1Ab and Cry1Ac. Because the resistance is recessive and conferred by a single locus, an F1 screening assay was used to measure the frequency of Cry1F-resistant alleles from populations of Florida and Texas in 2010 and 2011. A total frequency of resistant alleles of 0.13 and 0.02 was found for Florida and Texas populations, respectively, indicating resistant alleles could be found in US populations, although there have been no reports of reduced efficacy of Cry1F-expressing plants.

Thermostable microbial lipases are potential candidates for industrial applications such as specialty organic syntheses as well as hydrolysis of fats and oils. In this work, basic biochemical engineering tools were applied to enhance the production of BTL2 lipase cloned in Escherichia coli BL321 under control of the strong temperature-inducible λP(L) promoter. Initially, surface response analysis was used to assess the influence of growth and induction temperatures on enzyme production, in flask experiments. The results showed that temperatures of 30 and 45°C were the most suitable for growth and induction, respectively, and led to an enzyme specific activity of 706,000 U/gDCW. The most promising induction conditions previously identified were validated in fed-batch cultivation, carried out in a 2L bioreactor. Specific enzyme activity reached 770,000 U/gDCW, corresponding to 13,000 U/L of culture medium and a lipase protein concentration of 10.8 g/L. This superior performance on enzyme production was a consequence of the improved response of λP(L) promoter triggered by the high induction temperature applied (45°C). These results point out to the importance of taking into account protein structure and stability to adequately design the recombinant protein production strategy for thermally induced promoters., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Five microbial lipase preparations from several sources were immobilized by hydrophobic adsorption on small or large poly-hydroxybutyrate (PHB) beads and the effect of the support particle size on the biocatalyst activity was assessed in the hydrolysis of olive oil, esterification of butyric acid with butanol and transesterification of babassu oil (Orbignya sp.) with ethanol. The catalytic activity of the immobilized lipases in both olive oil hydrolysis and biodiesel synthesis was influenced by the particle size of PHB and lipase source. In the esterification reaction such influence was not observed. Geobacillus thermocatenulatus lipase (BTL2) was considered to be inadequate to catalyze biodiesel synthesis, but displayed high esterification activity. Butyl butyrate synthesis catalyzed by BTL2 immobilized on small PHB beads gave the highest yield (≈90 mmol L(-1)). In biodiesel synthesis, the catalytic activity of the immobilized lipases was significantly increased in comparison to the free lipases. Full conversion of babassu oil into ethyl esters was achieved at 72 h in the presence of Pseudozyma antarctica type B (CALB), Thermomyces lanuginosus lipase (Lipex(®) 100 L) immobilized on either small or large PHB beads and Pseudomonas fluorescens (PFL) immobilized on large PHB beads. The latter preparation presented the highest productivity (40.9 mg of ethyl esters mg(-1) immobilized protein h(-1))., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Endemic pemphigus foliaceus (EPF) is an autoimmune disease, classically occurring in a restricted geographic area. Foci of EPF have been described in several Central and South American countries, often affecting young people and Amerindians, with some female predilection. Although most American EPF cases have been documented in Brazil, cases have been reported in Peru, Paraguay, El Salvador and Venezuela. An additional variant of EPF has been described in El Bagre, Colombia, (El Bagre-EPF) affecting older men and a few post-menopausal females. Finally, one additional type of EPF has been described in nomadic tribes affecting females of child bearing age in Tunisia, Africa., Aims: The main aim of this review is to summarize current knowledge about autoantigens, and immunologic and genetic studies in EPF., Material and Methods: We utilized a retrospective review of the literature, aiming to compile and compare the multiple geographic foci of EPF., Results: The primary autoantigens in EPF are still considered to be desmogleins in the case of the Tunisian and all American cases, in contradistinction to plakins and desmogleins in El Bagre-EPF. Although several autoantigens are been suggested, their biochemical nature needs further elucidation. Current knowledge still supports the concept that an antibody mediated immune response represents the principal pathophysiology in all variants of EPF., Conclusion: A strong genetic susceptibility appears to contribute to disease development in several people affected by these diseases; however, no specific genes have been confirmed at present. We conclude that further investigation is necessary to define these disorders immunologically and genetically.

Background: Endemic pemphigus foliaceus (EPF) is the only known autoimmune disease presenting in circumscribed geographic areas., Aim: We aim to provide information concerning the natural course of EPF, including systemic compromise in the presteroid era, which has been largely unavailable in the current medical literature. MATERIAL #ENTITYSTARTX00026;, Methods: By a retrospective review of the literature we aim to compile and compare the focus of EPF and the current knowledge about them. The main aim of this review is to summarize our current knowledge of EPF, including data described almost one century ago; and, to include several unindexed reports, which may have not been available to many current scientists and health care personnel., Results: Foci of EPF have been described in several Central American and South American countries, affecting predominately young people and Amerindians, with an additional female predilection. Although most cases have occurred in Brazil, some cases have been reported in Peru, Paraguay, El Salvador, and Venezuela. Another variant of EPF has been described in El Bagre, Colombia, affecting older men and a few post-menopausal females. Finally, another type of EPF was described in nomadic tribes affecting females of child bearing age in Tunisia, Africa., Conclusion: Our understanding of EPF has been hampered by a lack of government attention to these diseases, especially in some South and Central American countries. Other factors that have made past studies of EPF difficult include 1) that the disease foci are often located in rural areas bordering the rain forest of underdeveloped countries; and 2) military conflicts in some of these areas.

We recently described a new variant of endemic pemphigus foliaceus (EPF) in El Bagre, Colombia, that resembles Senear-Usher syndrome and identified autoantibodies to desmoglein 1 (Dsg1), as well as to multiple known and unknown antigens including plectins, in the serum of these patients. Here, we developed a cost-effective ELISA assay capable of detecting the heterogeneous antibody population observed in these EPF patients, and useful for serum epidemiological studies. A protein extract obtained from trypsin-digested fresh bovine skin and further purified on a concanavalin A matrix was used as antigen. This extract contains an important conformational epitope (a 45 kDa tryptic fragment of the Dsg1 ectodomain), which is recognized by antibodies in serum from patients with all varieties of pemphigus foliaceus (PF), and from half of those with pemphigus vulgaris with active clinical disease. The cut-off and threshold values were normalized using human serum obtained from both endemic and non-endemic areas for PF. The efficiency of this ELISA was tested using 600 serum samples from controls and patients diagnosed with EPF, non-endemic PF and other bullous diseases. The overall sensitivity and specificity of the assay were determined to be 95% and 72%, respectively, with reproducibilities of 98% (intraassay) and 95% (interassay). Comparing the ELISA with other tests to detect EPF autoantibodies, this ELISA was the most sensitive, followed by direct immunofluorescence (DIF), indirect immunofluorescence using anti-IgG4 monoclonal antibodies and immunoprecipitation (IP), respectively. The most specific assay was IP, followed by DIF. Immunoblotting to Dsg1 exhibited both poor sensitivity and poor specificity, although plectins were well visualized. We conclude that this ELISA is an excellent tool for field serological studies, allowing testing of multiple serum samples simultaneously and for detecting, with appropriate restriction and sensitivity, the heterogeneous antibody population seen in patients with this variant of EPF. Finally, autoantibody serum levels obtained with this ELISA correlated well with the clinical activity and extent of disease in patients with El Bagre EPF.

A novel variant of endemic pemphigus foliaceus (EPF) was described among individuals in an area surrounding El Bagre, Colombia, South America. The population in this rural mining community is exposed to high environmental levels of mercury, used for gold extraction, as well as other minerals, metalloids, and trace elements (e.g., quartz, rutile, granite, magnetite, and almenite) and ultraviolet radiation. Fifty control subjects and fifty EPF patients in the endemic area were examined for the presence of mercury in skin biopsies and hair, using autometallographic and mass spectroscopic analyses, respectively. Simultaneously, serum levels of IgE were measured, and cutaneous tests for hypersensitivity reactions were performed. Using autometallography, mercuric sulfides/selenides were detected in 14 of 51 skin biopsies distributed similarly in the control and patient groups. However, significantly higher serum IgE levels and mercury concentrations in hair, urine, and nails were found in patients compared with controls. Microscopic analysis revealed mercuric sulfides/selenides concentrated within and around the sweat gland epithelium, as well as in dendritic cells. Five skin biopsies from EPF patients and five from controls that tested positive for the presence of mercuric sulfides/selenides by autometallography were randomly selected for electron microscopic analysis. This analysis revealed a mixed electron-dense and electron-light material closely associated with desmosomes in patients. However, there were intracellular vesicles containing an amalgam of electron-dense and electron-light materials only in the EPF patients. Thus, EPF-affected individuals are exposed to high levels of environmental mercuric sulfides/selenides and other elements. This is the first study reporting mercuric sulfides/selenides in skin biopsies from people living in a focus of EPF, and these compounds may play a role in the pathogenesis of autoimmunity.

Multiple antigens are recognized by sera from patients with pemphigus foliaceus (PF). Several have been identified including keratin 59, desmocollins, envoplakin, periplakin, and desmogleins 1 and 3 (Dsg1 and Dsg3). In addition, an 80 kDa antigen was identified as the N-terminal fragment of Dsg1 using as antigen source an insoluble epidermal cell envelope preparation. However, still unsolved was the identity of the most important antigenic moiety, a 45 kDa tryptic fragment which is recognized by all sera from patients with fogo selvagem, pemphigus foliaceus, by half of pemphigus vulgaris sera and by a new variant of endemic pemphigus in E1 Bagre, Colombia that resembles Senear-Usher syndrome. Here, we report the identification of the 45 kDa conformational epitope of a soluble tryptic cleavage product from viable bovine epidermis. To elucidate the nature of this peptide, viable bovine epidermis was trypsin-digested, and glycosylated peptides were partially purified on a concanavalin A (Con-A) affinity column. This column fraction was then used as an antigen source for further immunoaffinity purification. A PF patient's serum covalently coupled to a Staphylococcus aureus protein A column was incubated with the Con-A eluted products and the immuno-isolated antigen was separated by SDS-PAGE, transferred to a membrane, and visualized with Coomassie blue, silver and amido black stains. The 45 kD band was subjected to amino acid sequence analysis revealing the sequence, EXIKFAAAXREGED, which matched the mature form of the extracellular domain of bovine Dsg1. This study confirms the biological importance of the ectodomain of Dsg1 as well as the relevance of conformational epitopes in various types of pemphigus.