AccuStart II PCR SuperMix
Robust, user-friendly 1-tube PCR SuperMix reagents for routine, general purpose PCR
Features & Benefits
- User-friendly 2X concentrated master mix simplifies reaction setup with exceptional room-temperature stability (≥30 days at 22°C) and is impervious to repetitive freeze-thaw (≥ 20X)
- High-yielding, ultrapure modified Taq DNA polymerase delivers robust, reliable assay sensitivity
- Stringent, ultrapure antibody hotstart ensures sensitive and precise target amplification
AccuStart II PCR SuperMix is intended for molecular biology applications. This product is not intended for the diagnosis, prevention or treatment of a disease.
AccuStart II PCR SuperMix is a 2X concentrated, ready-to-use reaction cocktail for routine PCR amplification of DNA fragments up to 4 kb. It contains all components, except primers and template. AccuStart II PCR SuperMix simplifies reaction assembly, improves assay reproducibility, and reduces the risk of contamination. A key component is AccuStart II Taq DNA polymerase which contains monoclonal antibodies that bind to the polymerase and keep it inactive prior to the initial PCR denaturation step. Upon heat activation (1 minute at 94°C), the antibodies denature irreversibly, releasing fully active, unmodified Taq DNA polymerase. This enables specific and efficient primer extension with the convenience of room temperature reaction assembly.
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Exploring the diversity of the deep sea—four new species of the amphipod genus Oedicerina described using morphological and molecular methodsAbstract
Collections of the amphipod genus Oedicerina were obtained during six expeditions devoted to the study of deep-sea environments of the Pacific Ocean. The material revealed four species new to science. Two species (Oedicerina henrici sp. nov. and sp. nov.) were found at abyssal depths of the central eastern Pacific in the Clarion-Clipperton Zone; one species (sp. nov.) (Oedicerina claudei sp. nov.) was recovered in the Sea of Okhotsk (north-west Pacific), and one (Oedicerina lesci sp. nov.) in the abyss adjacent to the Kuril-Kamchatka Trench (KKT). The four new species differ from each other and known species by the shapes of the rostrum, coxae 1 and 4, basis of pereopod 7, armatures of pereonite 7, pleonites and urosomites. An identification key for all known species is provided. The study of the cytochrome c oxidase subunit I gene of the four new species and Oedicerina ingolfi collected in the North Atlantic confirmed their genetic distinction. However, small intraspecific variation within each of the studied species was observed. In the case of the new species occurring across the KKT, the same haplotype was found on both sides of the trench, providing evidence that the trench does not constitute an insurmountable barrier for population connectivity. None of the species have so far been found on both sides of the Pacific.
A DNA microarray for the authentication of giant tiger prawn (Penaeus monodon) and whiteleg shrimp (Penaeus (Litopenaeus) vannamei): a proof-of-principleAbstract
This proof-of-principle study describes the development of a rapid and easy-to-use DNA microarray assay for the authentication of giant tiger prawns and whiteleg shrimp. Following DNA extraction and conventional end-point PCR of a 16S rDNA segment, the PCR products are hybridised to species-specific oligonucleotide probes on DNA microarrays located at the bottom of centrifuge tubes (ArrayTubes) and the resulting signal patterns are compared to those of reference specimens. A total of 21 species-specific probes were designed and signal patterns were recorded for 47 crustacean specimens belonging to 16 species of seven families. A hierarchical clustering of the signal patterns demonstrated the specificity of the DNA microarray for the two target species. The DNA microarray can easily be expanded to other important crustaceans. As the complete assay can be performed within half a day and does not require taxonomic expertise, it represents a rapid and simple alternative to tedious DNA barcoding and could be used by crustacean trading companies as well as food control authorities for authentication of crustacean commodities.
Characterization of Emerging Swine Viral Diseases through Oxford Nanopore Sequencing Using Senecavirus A as a ModelAbstract
Emerging viral infectious diseases present a major threat to the global swine industry. Since 2015, Senecavirus A (SVA) has been identified as a cause of vesicular disease in different countries and is considered an emerging disease. Despite the growing concern about SVA, there is a lack of preventive and diagnostic strategies, which is also a problem for all emerging infectious diseases. Using SVA as a model, we demonstrated that Oxford Nanopore MinION sequencing could be used as a robust tool for the investigation and surveillance of emerging viral diseases. Our results identified that MinION sequencing allowed for rapid, unbiased pathogen detection at the species and strain level for clinical cases. SVA whole genome sequences were generated using both direct RNA sequencing and PCR-cDNA sequencing methods, with an optimized consensus accuracy of 94% and 99%, respectively. The advantages of direct RNA sequencing lie in its shorter turnaround time, higher analytical sensitivity and its quantitative relationship between input RNA and output sequencing reads, while PCR-cDNA sequencing excelled at creating highly accurate sequences. This study developed whole genome sequencing methods to facilitate the control of SVA and provide a reference for the timely detection and prevention of other emerging infectious diseases.
Experimental infection reveals transmission of tilapia lake virus (TiLV) from tilapia broodstock to their reproductive organs and fertilized eggsAbstract
Early developmental stages of tilapia, including fertilized eggs were tested positive for TiLV in our previous study (Dong et al., 2017a). We, therefore, hypothesized that infected broodstock is able to pass the virus to their reproductive organs and then to the fertilized eggs. In order to prove this hypothesis, Nile tilapia (Oreochromis niloticus) broodstock were experimentally infected with TiLV by intramuscular injection and non-infected broodstock were used as control group. At day 6 post infection, eggs and semen from each breeding pair were aseptically collected for in vitro fertilization. Fertilized eggs at 3, 12 and 64 h post-fertilization were subjected to detection of TiLV by PCR, ISH, and cell culture. In parallel, blood, serum, liver and reproductive organs from each broodstock were subjected to TiLV analysis. The results revealed that all collected tissues (liver, blood, ovary and testis) from infected broodstock tested positive for TiLV by PCR, ISH, and cell culture. ISH revealed strong positive signals in hepatocytes surrounding blood vessels in the liver, connective tissue and membrane surrounding the oocytes in the ovary and the connective tissue close to blood vessels in the testis. These findings suggested that TiLV causes systemic infection in tilapia broodstock with the virus being able to spread into the reproductive organs, most likely through the blood circulatory system. Subsequently, the fertilized eggs produced by infected broodstock tested positive for TiLV by PCR and ISH revealed location of the virus inside the fertilized eggs. The results of this study suggested that TiLV can be transmitted vertically. We thus recommend for hatchery and multiplication center to use TiLV-tested negative broodstock for the production of TiLV-free tilapia seeds.
A novel 8.7-kb mitochondrial genome deletion accurately detects endometriosis in the plasma of symptomatic womenAbstract
Aim: To evaluate an 8.7-kb mitochondrial DNA (mtDNA) deletion as a potential biomarker of endometriosis. Materials & methods: We tested the diagnostic accuracy of the 8.7-kb deletion real-time PCR assay using 182 prospectively collected blood samples from females presenting with symptoms of endometriosis in a case–control format. Results: The assay differentiated between endometriosis and controls (area under curve: 0.74–0.89) with a statistically significant difference (p < 0.05) in 8.7-kb deletion levels measured for all disease subtypes and stages. No correlation was seen between 8.7-kb deletion levels and participant or specimen age, hormone status or menstrual phase. Conclusion: The diagnostic accuracy of the 8.7-kb deletion for endometriosis suggests potential utility in the clinic to improve patient management. Endometriosis occurs when endometrial tissue grows outside the uterus and affects approximately 5–10% of women of reproductive age, with infertility, a symptom of this condition, reported in 30–50% of patients [1–8]. Symptoms vary in severity, and include pelvic pain, painful menstrual cramps, discomfort during intercourse and chronic tiredness . This painful condition can significantly impact a patient’s quality of life, resulting in days off work and loss of productivity . Endometriosis is associated with substantial costs and has a comparable economic burden to that of other chronic diseases such as diabetes, Crohn’s disease and rheumatoid arthritis . Laparoscopic surgery followed by histopathological confirmation is needed to make a definitive diagnosis [5,12], but this is invasive and patients are, understandably, reluctant to go through with the procedure causing further delays in diagnosis and treatment . There is, however, an increasingly important role for a clinical or presumptive diagnosis of disease, which can support the initiation of treatment and management strategies to control symptoms . Ballard et al.  have reported the value of a diagnosis of endometriosis to the patient beyond the availability of treatment options including legitimizing access to social support, excusing absences from work and social events due to symptoms and providing a language with which to communicate about their disease. These can be considered a benefit of both presumptive and surgical diagnoses. Increasingly, molecular biomarkers are being used in many areas of medicine to detect and manage diseases [16–22]. However, to date, an endometriosis-specific biomarker has not been found that can be used successfully in clinical practice [23–25]. Nonstandard procedures for sample collection and data analysis have hampered researchers’ ability to find such a biomarker. However, recent efforts to harmonize sample collection and storage, analysis methods and the reporting of data, encouraged by the publication of the World Endometriosis Research Foundation EPHect Protocols , have contributed toward the development of disease-specific assays. In this paper, we describe the investigation of an 8.7-kb mitochondrial DNA (mtDNA) deletion as a potential biomarker for diagnosing endometriosis, including an initial assessment of diagnostic accuracy followed by an evaluation of disease specificity by comparing the biomarker’s frequency in plasma from women with: endometriosis and symptomatic controls, and endometrial cancer, ovarian cancer and breast cancer.
British Red Squirrels Remain the Only Known Wild Rodent Host for Leprosy BacilliAbstract
Eurasian red squirrels (Sciurus vulgaris) in the British Isles are the most recently discovered animal reservoir for the leprosy bacteria Mycobacterium leprae and Mycobacterium lepromatosis. Initial data suggest that prevalence of leprosy infection is variable and often low in different squirrel populations. Nothing is known about the presence of leprosy bacilli in other wild squirrel species despite two others (Siberian chipmunk [Tamias sibiricus], and Thirteen-lined ground squirrel [Ictidomys tridecemlineatus]) having been reported to be susceptible to experimental infection with M. leprae. Rats, a food-source in some countries where human leprosy occurs, have been suggested as potential reservoirs for leprosy bacilli, but no evidence supporting this hypothesis is currently available. We screened 301 squirrel samples covering four species [96 Eurasian red squirrels, 67 Eastern gray squirrels (Sciurus carolinensis), 35 Siberian chipmunks, and 103 Pallas's squirrels (Callosciurus erythraeus)] from Europe and 72 Mexican white-throated woodrats (Neotoma albigula) for the presence of M. leprae and M. lepromatosis using validated PCR protocols. No DNA from leprosy bacilli was detected in any of the samples tested. Given our sample-size, the pathogen should have been detected if the prevalence and/or bacillary load in the populations investigated were similar to those found for British red squirrels.
Temporal escalation of Pyrethroid Resistance in the major malaria vector Anopheles coluzzii from Sahelo-Sudanian Region of northern NigeriaAbstract
Despite the highest global burden of malaria, information on bionomics and insecticide resistance status of malaria vectors is grossly lacking in the densely populated Sahelo-Sudanian region of Nigeria. To support evidence-based vector control we characterised transmission and resistance profiles of Anopheles coluzzii populations from three sites in northern Nigeria. High sporozoite infection (~19.51%) was found in the An. coluzzii populations. A high pyrethroid resistance was observed with only 1% mortality against deltamethrin, a high LD50 (96.57 µg/ml), and a high LT50 (170.27 min, resistance ratio of ~51 compared with the fully susceptible Ngoussou colony). Moderate carbamate resistance was observed. Synergist bioassays significantly recovered deltamethrin susceptibility implicating CYP450s (mortality = 85%, χ2 = 134.04, p < 0.0001) and esterases (mortality = 56%, χ2 = 47.31, p < 0.0001). Reduced bed net efficacy was also observed, with mortalities on exposure to the roof of PermaNet3.0 (PBO + deltamethrin) more than 22 times compared to the side panel (deltamethrin). TaqMan genotyping revealed a high frequency of 1014F kdr mutation (82%) with significant difference in genotype distribution associated with permethrin resistance [OR = 4.69 (CI:1.53–14.35, χ2 = 8.22 p = 0.004]. Sequencing of exons 18–21 of the VGSC led to detection of two additional nonsynonymous mutations, Ile10148Asn and Ser1156Gly. These findings highlight the threats posed by the highly resistant An. coluzzii to malaria control in Nigeria.
Pyrethroid exposure alters internal and cuticle surface bacterial communities in Anopheles albimanusAbstract
A deeper understanding of the mechanisms underlying insecticide resistance is needed to mitigate its threat to malaria vector control. Following previously identified associations between mosquito microbiota and insecticide resistance, we demonstrate for the first time, the effects of pyrethroid exposure on the microbiota of F1 progeny of field-collected Anopheles albimanus. Larval and adult mosquitoes were exposed to the pyrethroids alphacypermethrin (only adults), permethrin, and deltamethrin. While there were no significant differences in bacterial composition between insecticide-resistant and insecticide-susceptible mosquitoes, bacterial composition between insecticide-exposed and non-exposed mosquitoes was significantly different for alphacypermethrin and permethrin exposure. Along with other bacterial taxa not identified to species, Pantoea agglomerans (a known insecticide-degrading bacterial species) and Pseudomonas fragi were more abundant in insecticide-exposed compared to non-exposed adults, demonstrating that insecticide exposure can alter mosquito bacterial communities. We also show for the first time that the cuticle surfaces of both larval and adult An. albimanus harbor more diverse bacterial communities than their internal microbial niches. Together, these findings demonstrate how insecticide pressure could be selecting for certain bacteria within mosquitoes, especially insecticide-metabolizing bacteria, thus potentially contributing to insecticide resistance.
High Plasmodium infection and multiple insecticide resistance in a major malaria vector Anopheles coluzzii from Sahel of Niger RepublicAbstract
Background: Information on insecticide resistance and the mechanisms driving it in the major malaria vectors is grossly lacking in Niger Republic, thus hindering control eforts. To facilitate evidence-based malaria control, the role of Anopheles coluzzii population from southern Niger, in malaria transmission, its insecticides resistance profle and the molecular mechanisms driving the resistance were characterized. Methods: Blood fed female Anopheles gambiae sensu lato resting indoor were collected at Tessaoua, Niger. Source of blood was established using PCR and infection with Plasmodium determined using TaqMan assay. Resistance profle was established with the major public health insecticides, and resistance intensity determined with deltamethrin. Synergist assays were conducted with piperonyl butoxide and diethyl maleate. Presence of L1014F and L1014S knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) was investigated using TaqMan genotyping, and strength of selection pressure acting on the Anopheles populations determined by assessing the genetic diversity of a fragment spanning exon-20 of the VGSC from alive and dead females. Results: High human blood index (96%) and high Plasmodium falciparum infection (~13%) was observed in the An. coluzzii population. Also, a single mosquito was found infected with Plasmodium vivax. High pyrethroid and organochloride resistance was observed with mortalities of less than 20% for deltamethrin, permethrin, α-cypermethrin, and DDT. A high LD50 (156.65 min) was obtained for deltamethrin, with a resistance ratio of~47.18 compared to the susceptible Ngoussou colony. Moderate carbamate resistance was observed, and a full susceptibility to organophosphates recorded. Synergist bioassays with piperonyl butoxide and diethyl maleate signifcantly recovered deltamethrin and DDT susceptibility, respectively implicating CYP450 s (mortality=82%, χ2=84.51, p<0.0001) and glutathione S-transferases (mortality=58%, χ2=33.96, p<0.001) in resistance. A high frequency of 1014F kdr mutation (82%) was established, with signifcant diference in genotype distribution associated with permethrin resistance [odds ratio=7.71 (95% CI 2.43–14.53, χ2=13.67, p=0.001]. Sequencing of intron-1 of the voltage-gated sodium channel (VGSC) revealed a low genetic diversity. Conclusion: High pyrethroid resistance highlight the challenges to the efectiveness of the pyrethroids-based ITNs and indoor residual spraying (IRS) against An. coluzzii in Niger. The pyrethroids-synergists LLINs and organophosphatebased IRS maybe the alternatives for malaria control in southern Niger
Community ecology across bacteria, archaea and microbial eukaryotes in the sediment and seawater of coastal Puerto Nuevo, Baja CaliforniaAbstract
Microbial communities control numerous biogeochemical processes critical for ecosystem function and health. Most analyses of coastal microbial communities focus on the characterization of bacteria present in either sediment or seawater, with fewer studies characterizing both sediment and seawater together at a given site, and even fewer studies including information about non-bacterial microbial communities. As a result, knowledge about the ecological patterns of microbial biodiversity across domains and habitats in coastal communities is limited–despite the fact that archaea, bacteria, and microbial eukaryotes are present and known to interact in coastal habitats. To better understand microbial biodiversity patterns in coastal ecosystems, we characterized sediment and seawater microbial communities for three sites along the coastline of Puerto Nuevo, Baja California, Mexico using both 16S and 18S rRNA gene amplicon sequencing. We found that sediment hosted approximately 500-fold more operational taxonomic units (OTUs) for bacteria, archaea, and microbial eukaryotes than seawater (p < 0.001). Distinct phyla were found in sediment versus seawater samples. Of the top ten most abundant classes, Cytophagia (bacterial) and Chromadorea (eukaryal) were specific to the sediment environment, whereas Cyanobacteria and Bacteroidia (bacterial) and Chlorophyceae (eukaryal) were specific to the seawater environment. A total of 47 unique genera were observed to comprise the core taxa community across environment types and sites. No archaeal taxa were observed as part of either the abundant or core taxa. No significant differences were observed for sediment community composition across domains or between sites. For seawater, the bacterial and archaeal community composition was statistically different for the Major Outlet site (p < 0.05), the site closest to a residential area, and the eukaryal community composition was statistically different between all sites (p < 0.05). Our findings highlight the distinct patterns and spatial heterogeneity in microbial communities of a coastal region in Baja California, Mexico.
Downregulation of female doublesex expression by oral-mediated RNA interference reduces number and fitness of Anopheles gambiae adult femalesAbstract
Mosquito-borne diseases affect millions worldwide, with malaria alone killing over 400 thousand people per year and affecting hundreds of millions. To date, the best strategy to prevent the disease remains insecticide-based mosquito control. However, insecticide resistance as well as economic and social factors reduce the effectiveness of the current methodologies. Alternative control technologies are in development, including genetic control such as the sterile insect technique (SIT). The SIT is a pivotal tool in integrated agricultural pest management and could be used to improve malaria vector control. To apply the SIT and most other newer technologies against disease transmitting mosquitoes, it is essential that releases are composed of males with minimal female contamination. The removal of females is an essential requirement because released females can themselves contribute towards nuisance biting and disease transmission. Thus, females need to be eliminated from the cohorts prior to release. Manual separation of Anopheles gambiae pupae or adult mosquitoes based on morphology is time consuming, is not feasible on a large scale and has limited the implementation of the SIT technique. The doublesex (dsx) gene is one of the effector switches of sex determination in the process of sex differentiation in insects. Both males and females have specific splicing variants that are expressed across the different life stages. Using RNA interference (RNAi) to reduce expression of the female specific (dsxF) variant of this gene has proven to have detrimental effects to the females in other mosquito species, such as Aedes aegypti. We tested oral RNAi on dsx (AgdsxF) in An. gambiae. Methods We studied the expression pattern of the dsx gene in the An. gambiae G3 strain. We knocked down AgdsxF expression in larvae through oral delivery of double stranded RNA (dsRNA) produced by bacteria and observed its effects in adults. Results Our results show that feeding of AgdsxF dsRNA can effectively reduce (> 66%) the mRNA of female dsx transcript and that there is a concomitant reduction in the number of female larvae that achieve adulthood. Control groups produced 52% (± 3.9% SE) of adult males and 48% (± 4.0% SE) females, while AgdsxF dsRNA treated groups had 72.1% (± 4.0% SE) males vs 27.8% females (± 3.3% SE). In addition, the female adults produce fewer progeny, 37.1% (± 8.2% SE) less than the controls. The knockdown was sex-specific and had no impact on total numbers of viable male adults, in the male dsx transcripts or male fitness parameters such as longevity or body size. Conclusions These findings indicate that RNAi could be used to improve novel mosquito control strategies that require efficient sex separation and male-only release of An. gambiae by targeting sex determination genes such as AgdsxF. The advantages of using RNAi in a controlled setting for mosquito rearing are numerous, as the dose and time of exposure are controlled, and the possibility of off-target effects and the waste of female production would be significantly reduced.
The non-motor adaptor HMMR dampens Eg5-mediated forces to preserve the kinetics and integrity of chromosome segregationAbstract
Mitotic spindle assembly and organization require forces generated by motor proteins. The activity of these motors is regulated by non-motor adaptor proteins. However, there are limited studies reporting the functional importance of adaptors on the balance of motor forces and the promotion of faithful and timely cell division. Here, we show that genomic deletion or siRNA silencing of the non-motor adaptor Hmmr/HMMR disturbs spindle microtubule organization and bipolar chromosome-kinetochore attachments with a consequent elevated occurrence of aneuploidy. Rescue experiments show a conserved motif in HMMR is required to generate inter-kinetochore tension and promote anaphase entry. This motif bears high homology with the kinesin Kif15 and is known to interact with TPX2, a spindle assembly factor. We find that HMMR is required to dampen kinesin Eg5-mediated forces through localizing TPX2 and promoting the formation of inhibitory TPX2-Eg5 complexes. In HMMR-silenced cells, K-fiber stability is reduced while the frequency of unattached chromosomes and the time needed for chromosome segregation are both increased. These defects can be alleviated in HMMR-silenced cells with chemical inhibition of Eg5, but not through the silencing of Kif15. Together, our findings indicate that HMMR balances Eg5-mediated forces to preserve the kinetics and integrity of chromosome segregation.
Distribution and incidence of atoxigenic Aspergillus flavus VCG in tree crop orchards in California: A strategy for identifying potential antagonists, the example of almondsAbstract
To identify predominant isolates for potential use as biocontrol agents, Aspergillus flavus isolates collected soils of almond, pistachio and fig orchard in the Central Valley of California were tested for their membership to 16 atoxigenic vegetative compatibility groups (VCGs), including YV36, the VCG to which AF36, an atoxigenic isolate commercialized in the United States as biopesticide, belongs. A surprisingly large proportion of isolates belonged to YV36 (13.3%, 7.2% and 6.6% of the total almond, pistachio and fig populations, respectively), while the percentage of isolates belonging to the other 15 VCGs ranged from 0% to 2.3%. In order to gain a better insight into the structure and diversity of atoxigenic A. flavus populations and to further identify predominant isolates, seventeen SSR markers were then used to genetically characterize AF36, the 15 type-isolates of the VCGs and 342 atoxigenic isolates of the almond population. There was considerable genetic diversity among isolates with a lack of differentiation among micro-geographical regions or years. Since isolates sharing identical SSR profiles from distinct orchards were rare, we separated them into groups of at least 3 closely-related isolates from distinct orchards that shared identical alleles for at least 15 out of the 17 loci. This led to the identification of 15 groups comprising up to 24 closely-related isolates. The group which contained the largest number of isolates were members of YV36 while five groups were also found to be members of our studied atoxigenic VCGs. These results suggest that these 15 groups, and AF36 in particular, are well adapted to various environmental conditions in California and to tree crops and, as such, are good candidates for use as biocontrol agents.
Radiation of the polymorphic Little Devil poison frog (Oophaga sylvatica) in EcuadorAbstract
Some South American poison frogs (Dendrobatidae) are chemically defended and use bright aposematic colors to warn potential predators of their unpalatability. Aposematic signals are often frequency-dependent where individuals deviating from a local model are at a higher risk of predation. However, extreme diversity in the aposematic signal has been documented in poison frogs, especially in Oophaga. Here, we explore the phylogeographic pattern among color-divergent populations of the Little Devil poison frog Oophaga sylvatica by analyzing population structure and genetic differentiation to evaluate which processes could account for color diversity within and among populations. With a combination of PCR amplicons (three mitochondrial and three nuclear markers) and genome-wide markers from a double-digested RAD (ddRAD) approach, we characterized the phylogenetic and genetic structure of 199 individuals from 13 populations (12 monomorphic and 1 polymorphic) across the O. sylvatica distribution. Individuals segregated into two main lineages by their northern or southern latitudinal distribution. A high level of genetic and phenotypic polymorphism within the northern lineage suggests ongoing gene flow. In contrast, low levels of genetic differentiation were detected among the southern lineage populations and support recent range expansions from populations in the northern lineage. We propose that a combination of climatic gradients and structured landscapes might be promoting gene flow and phylogenetic diversification. Alternatively, we cannot rule out that the observed phenotypic and genomic variations are the result of genetic drift on near or neutral alleles in a small number of genes.
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