ROPS performance during field upset and static testing.

Agriculture remains one of the most hazardous occupations in the U.S. By conservative estimates, tractor overturns alone claim 120 lives annually. A rollover protective structure (ROPS) and a seatbelt are a highly effective engineering safety control that can prevent many of these fatalities and reduce the severity of injuries associated with tractor overturn. SAE J2194 is a consensus performance standard established for agricultural ROPS. According to this standard, satisfactory ROPS performance can be demonstrated through static testing, field upset testing, or impact testing. A previous modeling study suggested that static testing may underpredict the strain induced in a ROPS during afield upset. In the current study, field upset testing and laboratory static testing results were compared. Field upset testing included six rear and six side upset tests performed according to SAE J2194 guidelines. Additionally, static testing was performed on a ROPS of the same model. The results support findings from the modeling study. Near the lowest sections of the ROPS, the plastic strain resulting from rear upset testing exceeded the plastic strain from static testing for 18 of 24 data points. Conversely, the ROPS plastic strain from side upset testing was typically less than plastic strain from laboratory static testing. However, data indicate that the side upset test may not be very repeatable. This study suggests that the longitudinal loading energy criterion for static testing might not be a conservative predictor of rear upset ROPS response.

Dynamic performance of the mechanism of an automatically deployable ROPS.

The mechanism for an automatically deployable ROPS (AutoROPS) has been designed and tested. This mechanism is part of an innovative project to provide passive protection against rollover fatality to operators of new tractors used in both low-clearance and unrestricted-clearance tasks. The device is a spring-action, telescoping structure that releases on signal to pyrotechnic squibs that actuate release pins. Upper post motion begins when the release pins clear an internal piston. The structure extends until the piston impacts an elastomeric ring and latches at the top position. In lab tests the two-post structure consistently deployed in less than 0.3 s and latched securely. Static load tests of the telescoping structure and field upset tests of the fully functional AutoROPS have been successfully completed.

Static load test performance of a telescoping structure for an automatically deployable ROPS.

The automatically deployable ROPS was developed as part of an innovative project to provide passive protection against overturn fatality to operators of new tractors used in both low-clearance and unrestricted-clearance tasks. The primary objective of this phase of the research was to build a telescoping structure that would prove that a ROPS can be built that will (1) reliably deploy on signal, (2) rise in a sufficiently short amount of time, (3) firmly latch in its deployed position, and (4) satisfy SAE J2194 testing requirements. The two-post structure had previously been found to meet deployment time criteria, and design analyses indicated that neither the slip-fit joint nor the latch pins would fail at test loading. Four directions of static loading were applied to the structure to satisfy SAE requirements. For the series of static loading tests, the raised structure was found to maintain a protective clearance zone after all loads were applied. The structure is overly stiff and should be redesigned to increase its ability to absorb ground-impact energy. Results of dynamic tests and field upset tests are reported in companion articles. The next phase of development is to optimize the structure so that it will plastically deform and absorb energy that would otherwise be transferred to the tractor chassis.

Preventing tractor rollover fatalities: performance of the NIOSH autoROPS.

Approximately 132 agricultural tractor overturn fatalities occur per year. The use of rollover protective structures (ROPS), along with seat belts, is the best known method for preventing these fatalities. One impediment to ROPS use, however, is low clearance situations, such as orchards and animal confinement buildings. To address the need for ROPS that are easily adapted to low clearance situations, the Division of Safety Research, National Institute for Occupational Safety and Health (NIOSH), developed an automatically deploying, telescoping ROPS (Auto-ROPS). The NIOSH AutoROPS consists of two subsystems. The first is a retractable ROPS that is normally latched in its lowered position for day-to-day use. The second subsystem is a sensor that monitors the operating angle of the tractor. Ifa rollover condition is detected by the sensor, the retracted ROPS will deploy and lock in the full upright position before ground contact. Static load testing and field upset tests of the NIOSH AutoROPS have been conducted in accordance with SAE standard J2194. Additionally, timed trials of the AutoROPS deployment mechanism were completed. The design of the retractable ROPS and sensor, as well as the results of the different testing phases are discussed.

Performance of an automatically deployable ROPS on ASAE tests.

In the U.S., approximately 132 agricultural tractor overturn fatalities occur per year. The use of rollover protective structures (ROPS), along with seat belts, is the best-known method for preventing these fatalities. However, one impediment to ROPS use is low-clearance situations, such as orchards and animal confinement buildings. To address the need for ROPS that are easily adapted to low-clearance situations, the Division of Safety Research, National Institute for Occupational Safety and Health, developed a prototype automatically deploying, telescoping ROPS (AutoROPS). The NIOSH AutoROPS consists of two subsystems. The first is a retractable ROPS that is normally latched in its lowered position for day-to-day use. The second subsystem is a sensor that monitors the operating angle of the tractor. If an overturn condition is detected by the sensor, the retracted ROPS will deploy and lock in the full upright position before ground contact. Static load testing and field upset tests of the NIOSH AutoROPS have been conducted in accordance with SAE standard J2194. Additionally, timed trials of the AutoROPS deployment mechanism were completed. The results of these tests show that the NIOSH AutoROPS has significant potential to overcome the limitations of current ROPS designs for use in low clearance as well as unrestricted clearance operations.

Method enabling fast partial sequencing of cDNA clones.

Pyrosequencing is a nonelectrophoretic single-tube DNA sequencing method that takes advantage of cooperativity between four enzymes to monitor DNA synthesis. To investigate the feasibility of the recently developed technique for tag sequencing, 64 colonies of a selected cDNA library from human were sequenced by both pyrosequencing and Sanger DNA sequencing. To determine the needed length for finding a unique DNA sequence, 100 sequence tags from human were retrieved from the database and different lengths from each sequence were randomly analyzed. An homology search based on 20 and 30 nucleotides produced 97 and 98% unique hits, respectively. An homology search based on 100 nucleotides could identify all searched genes. Pyrosequencing was employed to produce sequence data for 30 nucleotides. A similar search using BLAST revealed 16 different genes. Forty-six percent of the sequences shared homology with one gene at different positions. Two of the 64 clones had unique sequences. The search results from pyrosequencing were in 100% agreement with conventional DNA sequencing methods. The possibility of using a fully automated pyrosequencer machine for future high-throughput tag sequencing is discussed.

Pyrosequencing sheds light on DNA sequencing.

DNA sequencing is one of the most important platforms for the study of biological systems today. Sequence determination is most commonly performed using dideoxy chain termination technology. Recently, pyrosequencing has emerged as a new sequencing methodology. This technique is a widely applicable, alternative technology for the detailed characterization of nucleic acids. Pyrosequencing has the potential advantages of accuracy, flexibility, parallel processing, and can be easily automated. Furthermore, the technique dispenses with the need for labeled primers, labeled nucleotides, and gel-electrophoresis. This article considers key features regarding different aspects of pyrosequencing technology, including the general principles, enzyme properties, sequencing modes, instrumentation, and potential applications.

Quantification of HIV-1 using multiple quantitative polymerase chain reaction standards and bioluminometric detection.

A non-gel-based quantification assay based on competitive PCR and bioluminometric detection has been developed. Samples containing human immunodeficiency virus type 1 (HIV-1) DNA and three quantitative standards at discrete concentrations were coamplified by PCR with primers annealing in the polymerase gene region. The quantitative standards contained the same primer binding sequences and had the same amplicon length as the wild-type DNA, but differed in an internal homopolymeric stretch (A, C, or T) over three base pairs. The PCR products were captured onto a solid support and treated with NaOH to separate the strands. Discrimination between the wild-type DNA and the three quantitative standard amplicons was achieved on the solid support by four parallel extension reactions with 3'-end specific primers. Inorganic pyrophosphate (PPi) released as a result of successful extension was converted to ATP by ATP sulfurylase and the level of ATP was sensed by firefly luciferase, generating a proportional amount of visible light which was detected by a luminometer. Here, we show that the obtained calibration curves, using the signal intensities of the three quantitative standards, enabled determination of the amount of target HIV-1 DNA.

Improved performance of pyrosequencing using single-stranded DNA-binding protein.

In modern biology, there is a critical need to develop a high-throughput and inexpensive platform for DNA sequencing. Pyrosequencing is a nonelectrophoretic single-tube DNA sequencing method that takes advantage of cooperativity between four enzymes to monitor DNA synthesis. In these studies, single-stranded DNA-binding protein (SSB) was added to the primed DNA template prior to the Pyrosequencing reaction. The addition of SSB to a Pyrosequencing reaction system resulted in a read length of more than 30 nucleotides. Improvements were observed as: (i) increased efficiency of the enzymes, (ii) reduced mispriming, as measured by nonspecific signals, (iii) an increase in signal intensity during the reaction, (iv) higher accuracy in reading the number of identical adjacent nucleotides in difficult templates, and (v) longer reads. The usefulness of these results for future Pyrosequencing applications is discussed.

Mutation detection by pyrosequencing: sequencing of exons 5-8 of the p53 tumor suppressor gene.

The ability to sequence a large number of DNA samples rapidly and accurately for detection of all possible mutations is a critical goal for the future application of DNA sequencing in routine medical diagnostics. Pyrosequencing() is a non-electrophoretic real-time DNA sequencing method that uses the luciferase-luciferin light release as the detection signal for nucleotide incorporation into target DNA. For pyrosequencing of the human p53 gene, a nested multiplex PCR method for amplification of exons 5-8 was prepared. In order to investigate the use of pyrosequencing in mutation detection, DNA samples from skin-cancer patients were used. Two forms of nucleotide dispensation strategy were used, cyclic and programmed. Bi-directional pyrosequencing was performed and the overlapping sequence data produced were assembled to determine the sequence of the gene. Reliable sequencing data were obtained with both dispensation strategies, but some advantages were obtained using the programmed nucleotide dispensation approach, such as longer and faster reads, and fewer out-of-phase problems. The accuracy of pyrosequencing for detection of p53 mutations and allele distribution was demonstrated.

Method enabling pyrosequencing on double-stranded DNA.

Pyrosequencing is a new nonelectrophoretic, single-tube DNA sequencing method that takes advantage of co-operativity between four enzymes to monitor DNA synthesis (M. Ronaghi, M. Uhlén, and P. Nyrén, Science 281, 363-365). Pyrosequencing has so far only been performed on single-stranded DNA. In this paper different enzymatic strategies for template preparation enabling pyrosequencing on double-stranded DNA were studied. High quality data were obtained with several different enzyme combinations: (i) shrimp alkaline phosphatase and exonuclease I, (ii) calf intestine alkaline phosphatase and exonuclease I, (iii) apyrase and inorganic pyrophosphatase together with exonuclease I, and (iv) apyrase and ATP sulfurylase together with exonuclease I. In many cases, when the polymerase chain reaction was efficient exonuclease I could be omitted. In certain cases, additives such as dimethyl sulfoxide, single-stranded DNA-binding protein, and Klenow DNA polymerase improved the sequence quality. Apyrase was the fastest and most efficient of the three different nucleotide degrading enzymes tested. The data quality obtained on double-stranded DNA was comparable with that on single-stranded DNA. Pyrosequencing data for more than 30 bases could be generated on both long and short templates, as well as on templates with high GC content.

Direct analysis of single-nucleotide polymorphism on double-stranded DNA by pyrosequencing.

Pyrosequencing, a new method for DNA sequencing, is gaining widespread use for many different types of DNA analysis. The method takes advantage of four coupled enzymes in a single tube assay to monitor DNA synthesis in real time using a luminometric detection system. Here, we demonstrate the use of pyrosequencing for direct analysis of single-nucleotide polymorphism on double-stranded PCR product. Pyrosequencing data on the human glutathione peroxidase gene (GPX1) from several individuals were analysed and three different allelic variants were determined and confirmed. The possibility of further simplifying the sequencing and template-preparation steps is discussed.

Polymorphism in the pertussis toxin promoter region affecting the DNA-based diagnosis of Bordetella infection.

The pertussis toxin (PT) promoter region is a frequently used target for DNA-based diagnosis of pertussis and parapertussis infections. The reported polymorphism in this region has also allowed discrimination of species in mixtures with several Bordetella species by their specific PCR amplicon restriction patterns. In the present study, we investigated the degree of polymorphism in order to confirm the reliability of the assay. Five different sequence types of the amplified 239- or 249-bp region were found among the 33 Bordetella pertussis, B. parapertussis, and B. bronchiseptica American Type Culture Collection reference strains and patient isolates analyzed. According to the sequences that were obtained and according to the PT promoter sequences already available in the databases, restriction enzyme analysis with TaqI, BglI, and HaeII, which gave four different patterns, can be performed to reliably identify B. pertussis, B. parapertussis, and B. bronchiseptica.

Analysis of the p53 tumor suppressor gene by pyrosequencing.

Tumor suppressor genes are implicated in cell cycle progression. Inactivation of these genes predominantly occurs through mutations and/or allelic loss that involves both alleles. With inactivation by multiple mutations in a single gene, cloning of the amplified gene is necessary to determine whether the mutations reside on one or both alleles. Using pyrosequencing, a recently developed approach based on sequencing-by-synthesis, we studied genetic variability in the p53 tumor suppressor gene and could quantify the ratio between the mutated and wild-type amplified fragments. Furthermore, this sequencing technique also allows allelic determination of adjacent mutations with no cloning of amplified fragments.

Production, purification, and luminometric analysis of recombinant Saccharomyces cerevisiae MET3 adenosine triphosphate sulfurylase expressed in Escherichia coli.

ATP sulfurylase cDNA from MET3 on chromosome X of Saccharomyces cerevisiae was amplified and cloned, and recombinant ATP sulfurylase was expressed in Escherichia coli. The synthesis of ATP sulfurylase was directed by an expression system that employs the regulatory genes of the luminous bacterium Vibrio fischeri. A soluble, biologically active form was purified to electrophoretic homogeneity from lysates of recombinant E. coli by ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration. The specific activity of the purified enzyme was estimated to 140 U/mg. The apparent molecular mass of the recombinant enzyme was determined by gel filtration to be 470 kDa, which indicates that the active enzyme is an octamer of identical subunits (the molecular mass of a single subunit is 59.3 kDa). The ATP sulfurylase activity was monitored in real time by a very sensitive bioluminometric method.

Analyses of secondary structures in DNA by pyrosequencing.

A common problem in conventional DNA sequencing is the occurrence of DNA sequence compressions during gel electrophoresis, leading to misreading of the sequence. These compressions are usually due to secondary structures in the DNA fragment. In this study, we present a non-gel-based DNA sequencing technique that facilitates analysis of such DNA regions. A part of the polymorphic pertussis toxin promoter region in five different Bordetella species was successfully resolved by the new technique. The obtained sequence data revealed four related palindromic sequences. The ability of different DNA polymerases to read through such secondary structures is also described.

PCR-introduced loop structure as primer in DNA sequencing.

The need for a primer hybridization step before sequencing has been eliminated using a stem-loop structure generated by PCR. The loop structure is obtained by careful design of the PCR primer or by cloning the target DNA into a dedicated vector (pRIT 28HP). After solid-phase capture of the PCR product, the loop is formed by elution of the non-bound strand. Here, we show that both the immobilized and the eluted strand can be analyzed using conventional Sanger DNA sequencing and the novel pyrosequencing method as described previously. By using a stem-loop structure as a primer for DNA sequencing, the risk for mispriming is minimized.

Phylogeny of the family Moraxellaceae by 16S rDNA sequence analysis, with special emphasis on differentiation of Moraxella species.

Thirty-three strains previously classified into 11 species in the bacterial family Moraxellaceae were subjected to phylogenetic analysis based on 16S rRNA sequences. The family Moraxellaceae formed a distinct clade consisting of four phylogenetic groups as judged from branch lengths, bootstrap values and signature nucleotides. Group I contained the classical moraxellae and strains of the coccal moraxellae, previously known as Branhamella, with 16S rRNA similarity of > or = 95%. A further division of group I into five tentative clusters is discussed. Group II consisted of two strains representing Moraxella atlantae and Moraxella osloensis. These strains were only distantly related to each other (93.4%) and also to the other members of the Moraxellaceae (< or = 93%). Therefore, reasons for reclassification of these species into separate and new genera are discussed. Group III harboured strains of the genus Psychrobacter and strain 752/52 of [Moraxella] phenylpyruvica. This strain of [M.] phenylpyruvica formed an early branch from the group III line of descent. Interestingly, a distant relationship was found between Psychrobacter phenylpyruvicus strain ATCC 23333T (formerly classified as [M.] phenylpyruvica) and [M.] phenylpyruvica strain 752/52, exhibiting less than 96% nucleotide similarity between their 16S rRNA sequences. The establishment of a new genus for [M.] phenylpyruvica strain 752/52 is therefore suggested. Group IV contained only two strains of the genus Acinetobacter. Strategies for the development of diagnostic probes and distinctive sequences for 16S rRNA-based species-specific assays within group I are suggested. Although these findings add to the classificatory placements within the Moraxellaceae, analysis of a more comprehensive selection of strains is still needed to obtain a complete classification system within this family.

Bioluminometric method for real-time detection of reverse transcriptase activity.

A simple and sensitive technique for detection of reverse transcriptase (RT) activity in real time has been developed. The technique is based on continuous detection of the inorganic pyrophosphate formed in the RT-catalyzed reaction by a luminometric method. The technique has been used for continuous monitoring of RT-catalyzed DNA synthesis on both homo- and heteropolymeric templates. The assay is sensitive and yields linear responses between 1.5-960 mU of avian myeloblastosis virus RT (AMV-RT). The assay was used for detection of the inhibitory effect of dideoxythymidine (ddTTP) on the AMV-RT activity and also for real-time detection of single-base incorporation events catalyzed by AMV-RT. The possibility of using the new technique for other applications is discussed.