Modification of tomato breeding traits and plant hormone signaling by target-AID, the genome-editing system inducing efficient nucleotide substitution.

Target activation-induced cytidine deaminase (Target-AID), a novel CRISPR/Cas9-based genome-editing tool, confers the base-editing capability on the Cas9 genome-editing system. It involves the fusion of cytidine deaminase (CDA), which catalyzes cytidine (C) to uridine (U) substitutions, to the mutated nickase-type nCas9 or deactivated-type dCas9. To confirm and extend the applicability of the Target-AID genome-editing system in tomatoes (Solanum lycopersicum L.), we transformed the model tomato cultivar "Micro-Tom" and commercial tomato cultivars using this system by targeting SlDELLA, which encodes a negative regulator of the plant phytohormone gibberellic acid (GA) signaling pathway. We confirmed that the nucleotide substitutions were induced by the Target-AID system, and we isolated mutants showing high GA sensitivity in both "Micro-Tom" and the commercial cultivars. Moreover, by successfully applying this system to ETHYLENE RECEPTOR 1 (SlETR1) with single sgRNA targeting, double sgRNA targeting, as well as dual-targeting of both SlETR1 and SlETR2 with a single sgRNA, we demonstrated that the Target-AID genome-editing system is a promising tool for molecular breeding in tomato crops. This study highlights an important aspect of the scientific and agricultural potential of the combinatorial use of the Target-AID and other base-editing systems.

Challenges and Prospects of New Plant Breeding Techniques for GABA Improvement in Crops: Tomato as an Example.

Over the last seven decades, γ-aminobutyric acid (GABA) has attracted great attention from scientists for its ubiquity in plants, animals and microorganisms and for its physiological implications as a signaling molecule involved in multiple pathways and processes. Recently, the food and pharmaceutical industries have also shown significantly increased interest in GABA, because of its great potential benefits for human health and the consumer demand for health-promoting functional compounds, resulting in the release of a plethora of GABA-enriched products. Nevertheless, many crop species accumulate appreciable GABA levels in their edible parts and could help to meet the daily recommended intake of GABA for promoting positive health effects. Therefore, plant breeders are devoting much effort into breeding elite varieties with improved GABA contents. In this regard, tomato (Solanum lycopersicum), the most produced and consumed vegetable worldwide and a fruit-bearing model crop, has received much consideration for its accumulation of remarkable GABA levels. Although many different strategies have been implemented, from classical crossbreeding to induced mutagenesis, new plant breeding techniques (NPBTs) have achieved the best GABA accumulation results in red ripe tomato fruits along with shedding light on GABA metabolism and gene functions. In this review, we summarize, analyze and compare all the studies that have substantially contributed to tomato GABA breeding with further discussion and proposals regarding the most recent NPBTs that could bring this process to the next level of precision and efficiency. This document also provides guidelines with which researchers of other crops might take advantage of the progress achieved in tomato for more efficient GABA breeding programs.

Author Correction: Efficient increase of ɣ-aminobutyric acid (GABA) content in tomato fruits by targeted mutagenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Utilization of a Genome-Edited Tomato (Solanum lycopersicum) with High Gamma Aminobutyric Acid Content in Hybrid Breeding.

γ-Aminobutyric acid (GABA) is a nonproteogenic amino acid with health-promoting functions. Although tomato fruits have a relatively high GABA content compared with other crops, levels must be further increased to effectively confer the health-promoting functions. In this study, we evaluated the potential of the genome-edited tomato as a breeding material for producing high-GABA hybrid tomatoes. Hybrid lines were produced by crossing the genome-edited tomato with a pure line tomato cultivar, "Aichi First", and were evaluated for GABA accumulation and other fruit traits. The hybrid lines showed high GABA accumulation in the fruits, which was sufficiently high for expecting health-promoting functions and had minimal effects on other fruit traits, suggesting that the high GABA is a dominant trait and that the genome-edited tomato would be useful as a parental line of hybrid cultivars. These results also indicate that genome editing technology is useful for the rapid breeding of high-GABA hybrid tomato cultivars.

Efficient increase of ɣ-aminobutyric acid (GABA) content in tomato fruits by targeted mutagenesis.

γ-Aminobutyric acid (GABA) is a non-proteinogenic amino acid that has hypotensive effects. Tomato (Solanum lycopersicum L.) is among the most widely cultivated and consumed vegetables in the world and contains higher levels of GABA than other major crops. Increasing these levels can further enhance the blood pressure-lowering function of tomato fruit. Glutamate decarboxylase (GAD) is a key enzyme in GABA biosynthesis; it has a C-terminal autoinhibitory domain that regulates enzymatic function, and deleting this domain increases GAD activity. The tomato genome has five GAD genes (SlGAD1-5), of which two (SlGAD2 and SlGAD3) are expressed during tomato fruit development. To increase GABA content in tomato, we deleted the autoinhibitory domain of SlGAD2 and SlGAD3 using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)9 technology. Introducing a stop codon immediately before the autoinhibitory domain increased GABA accumulation by 7 to 15 fold while having variable effects on plant and fruit size and yield. This is the first study describing the application of the CRISPR/Cas9 system to increase GABA content in tomato fruits. Our findings provide a basis for the improvement of other types of crop by CRISPR/Cas9-based genetic modification.

Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion.

We applied a fusion of CRISPR-Cas9 and activation-induced cytidine deaminase (Target-AID) for point mutagenesis at genomic regions specified by single guide RNAs (sgRNAs) in two crop plants. In rice, we induced multiple herbicide-resistance point mutations by multiplexed editing using herbicide selection, while in tomato we generated marker-free plants with homozygous heritable DNA substitutions, demonstrating the feasibility of base editing for crop improvement.

An Agrobacterium tumefaciens Strain with Gamma-Aminobutyric Acid Transaminase Activity Shows an Enhanced Genetic Transformation Ability in Plants.

Agrobacterium tumefaciens has the unique ability to mediate inter-kingdom DNA transfer, and for this reason, it has been utilized for plant genetic engineering. To increase the transformation frequency in plant genetic engineering, we focused on gamma-aminobutyric acid (GABA), which is a negative factor in the Agrobacterium-plant interaction. Recent studies have shown contradictory results regarding the effects of GABA on vir gene expression, leading to the speculation that GABA inhibits T-DNA transfer. In this study, we examined the effect of GABA on T-DNA transfer using a tomato line with a low GABA content. Compared with the control, the T-DNA transfer frequency was increased in the low-GABA tomato line, indicating that GABA inhibits T-DNA transfer. Therefore, we bred a new A. tumefaciens strain with GABA transaminase activity and the ability to degrade GABA. The A. tumefaciens strain exhibited increased T-DNA transfer in two tomato cultivars and Erianthus arundinacues and an increased frequency of stable transformation in tomato.

Activating glutamate decarboxylase activity by removing the autoinhibitory domain leads to hyper γ-aminobutyric acid (GABA) accumulation in tomato fruit.

KEY MESSAGE: The C-terminal extension region of SlGAD3 is likely involved in autoinhibition, and removing this domain increases GABA levels in tomato fruits. γ-Aminobutyric acid (GABA) is a ubiquitous non-protein amino acid with several health-promoting benefits. In many plants including tomato, GABA is synthesized via decarboxylation of glutamate in a reaction catalyzed by glutamate decarboxylase (GAD), which generally contains a C-terminal autoinhibitory domain. We previously generated transgenic tomato plants in which tomato GAD3 (SlGAD3) was expressed using the 35S promoter/NOS terminator expression cassette (35S-SlGAD3-NOS), yielding a four- to fivefold increase in GABA levels in red-ripe fruits compared to the control. In this study, to further increase GABA accumulation in tomato fruits, we expressed SlGAD3 with (SlGAD3 OX ) or without (SlGAD3ΔC OX ) a putative autoinhibitory domain in tomato using the fruit ripening-specific E8 promoter and the Arabidopsis heat shock protein 18.2 (HSP) terminator. Although the GABA levels in SlGAD3 OX fruits were equivalent to those in 35S-SlGAD3-NOS fruits, GABA levels in SlGAD3ΔC OX fruits increased by 11- to 18-fold compared to control plants, indicating that removing the autoinhibitory domain increases GABA biosynthesis activity. Furthermore, the increased GABA levels were accompanied by a drastic reduction in glutamate and aspartate levels, indicating that enhanced GABA biosynthesis affects amino acid metabolism in ripe-fruits. Moreover, SlGAD3ΔC OX fruits exhibited an orange-ripe phenotype, which was associated with reduced levels of both carotenoid and mRNA transcripts of ethylene-responsive carotenogenic genes, suggesting that over activation of GAD influences ethylene sensitivity. Our strategy utilizing the E8 promoter and HSP terminator expression cassette, together with SlGAD3 C-terminal deletion, would facilitate the production of tomato fruits with increased GABA levels.

How and why does tomato accumulate a large amount of GABA in the fruit?

Gamma-aminobutyric acid (GABA) has received much attention as a health-promoting functional compound, and several GABA-enriched foods have been commercialized. In higher plants, GABA is primarily metabolized via a short pathway called the GABA shunt. The GABA shunt bypasses two steps (the oxidation of α-ketoglutarate to succinate) of the tricarboxylic acid (TCA) cycle via reactions catalyzed by three enzymes: glutamate decarboxylase, GABA transaminase, and succinic semialdehyde dehydrogenase. The GABA shunt plays a major role in primary carbon and nitrogen metabolism and is an integral part of the TCA cycle under stress and non-stress conditions. Tomato is one of the major crops that accumulate a relatively high level of GABA in its fruits. The GABA levels in tomato fruits dramatically change during fruit development; the GABA levels increase from flowering to the mature green stage and then rapidly decrease during the ripening stage. Although GABA constitutes up to 50% of the free amino acids at the mature green stage, the molecular mechanism of GABA accumulation and the physiological function of GABA during tomato fruit development remain unclear. In this review, we summarize recent studies of GABA accumulation in tomato fruits and discuss the potential biological roles of GABA in tomato fruit development.

Tomato Glutamate Decarboxylase Genes SlGAD2 and SlGAD3 Play Key Roles in Regulating γ-Aminobutyric Acid Levels in Tomato (Solanum lycopersicum).

Tomato (Solanum lycopersicum) can accumulate relatively high levels of γ-aminobutyric acid (GABA) during fruit development. However, the molecular mechanism underlying GABA accumulation and its physiological function in tomato fruits remain elusive. We previously identified three tomato genes (SlGAD1, SlGAD2 and SlGAD3) encoding glutamate decarboxylase (GAD), likely the key enzyme for GABA biosynthesis in tomato fruits. In this study, we generated transgenic tomato plants in which each SlGAD was suppressed and those in which all three SlGADs were simultaneously suppressed. A significant decrease in GABA levels, i.e. 50-81% compared with wild-type (WT) levels, was observed in mature green (MG) fruits of the SlGAD2-suppressed lines, while a more drastic reduction (up to <10% of WT levels) was observed in the SlGAD3- and triple SlGAD-suppressed lines. These findings suggest that both SlGAD2 and SlGAD3 expression are crucial for GABA biosynthesis in tomato fruits. The importance of SlGAD3 expression was also confirmed by generating transgenic tomato plants that over-expressed SlGAD3. The MG and red fruits of the over-expressing transgenic lines contained higher levels of GABA (2.7- to 5.2-fold) than those of the WT. We also determined that strong down-regulation of the SlGADs had little effect on overall plant growth, fruit development or primary fruit metabolism under normal growth conditions.

Suppression of γ-aminobutyric acid (GABA) transaminases induces prominent GABA accumulation, dwarfism and infertility in the tomato (Solanum lycopersicum L.).

Tomatoes accumulate γ-aminobutyric acid (GABA) at high levels in the immature fruits. GABA is rapidly converted to succinate during fruit ripening through the activities of GABA transaminase (GABA-T) and succinate semialdehyde dehydrogenase (SSADH). Although three genes encoding GABA-T and both pyruvate- and α-ketoglutarate-dependent GABA-T activities have been detected in tomato fruits, the mechanism underlying the GABA-T-mediated conversion of GABA has not been fully understood. In this work, we conducted loss-of-function analyses utilizing RNA interference (RNAi) transgenic plants with suppressed pyruvate- and glyoxylate-dependent GABA-T gene expression to clarify which GABA-T isoforms are essential for its function. The RNAi plants with suppressed SlGABA-T gene expression, particularly SlGABA-T1, showed severe dwarfism and infertility. SlGABA-T1 expression was inversely associated with GABA levels in the fruit at the red ripe stage. The GABA contents in 35S::SlGABA-T1(RNAi) lines were 1.3-2.0 times and 6.8-9.2 times higher in mature green and red ripe fruits, respectively, than the contents in wild-type fruits. In addition, SlGABA-T1 expression was strongly suppressed in the GABA-accumulating lines. These results indicate that pyruvate- and glyoxylate-dependent GABA-T is the essential isoform for GABA metabolism in tomato plants and that GABA-T1 primarily contributes to GABA reduction in the ripening fruits.

An E8 promoter-HSP terminator cassette promotes the high-level accumulation of recombinant protein predominantly in transgenic tomato fruits: a case study of miraculin.

KEY MESSAGE: The E8 promoter-HSP terminator expression cassette is a powerful tool for increasing the accumulation of recombinant protein in a ripening tomato fruit. Strong, tissue-specific transgene expression is a desirable feature in transgenic plants to allow the production of variable recombinant proteins. The expression vector is a key tool to control the expression level and site of transgene and recombinant protein expression in transgenic plants. The combination of the E8 promoter, a fruit-ripening specific promoter, and a heat shock protein (HSP) terminator, derived from heat shock protein 18.2 of Arabidopsis thaliana, produces the strong and fruit-specific accumulation of recombinant miraculin in transgenic tomato. Miraculin gene expression was driven by an E8 promoter and HSP terminator cassette (E8-MIR-HSP) in transgenic tomato plants, and the miraculin concentration was the highest in the ripening fruits, representing 30-630 µg miraculin of the gram fresh weight. The highest level of miraculin concentration among the transgenic tomato plant lines containing the E8-MIR-HSP cassette was approximately four times higher than those observed in a previous study using a constitutive 35S promoter and NOS terminator cassette (Hiwasa-Tanase et al. in Plant Cell Rep 30:113-124, 2011). These results demonstrate that the combination of the E8 promoter and HSP terminator cassette is a useful tool to increase markedly the accumulation of recombinant proteins in a ripening fruit-specific manner.

Accumulation mechanism of γ-aminobutyric acid in tomatoes (Solanum lycopersicum L.) under low O2 with and without CO2.

The storage of ripe tomatoes in low-O(2) conditions with and without CO(2) promotes γ-aminobutyric acid (GABA) accumulation. The activities of glutamate decarboxylase (GAD) and α-ketoglutarate-dependent GABA transaminase (GABA-TK) were higher and lower, respectively, following storage under hypoxic (2.4 or 3.5% O(2)) or adjusted aerobic (11% O(2)) conditions compared to the activities in air for 7 days at 25 °C. GAD activity was consistent with the expression level of mRNA for GAD. The GABA concentration in tomatoes stored under hypoxic conditions and adjusted aerobic conditions was 60-90% higher than that when they are stored in air on the same day. These results demonstrate that upregulation of GAD activity and downregulation of GABA-TK activity cause GABA accumulation in tomatoes stored under low-O(2) conditions. Meanwhile, the effect of CO(2) on GABA accumulation is probably minimal.

Molecular determinants for small Maf protein control of platelet production.

MafG and p45 possess basic region-leucine zipper (bZip) domains and form a heterodimer called NF-E2, a key regulator of megakaryopoiesis. NF-E2 binds to the Maf recognition element (MARE) and activates transcription of many platelet genes. Since the bZip domain, which mediates DNA binding and heterodimerization, is the only functional domain established for MafG, it has been assumed that MafG is required only for p45 binding to MARE and to facilitate p45-mediated transcriptional activation. Analysis of the C-terminal region of MafG, which is distinct from the bZip domain, revealed that this region contains a nuclear matrix-targeting signal. We used a transgenic complementation rescue assay to delineate the function of the MafG C terminus in vivo. Transgenic mice expressing a mutant MafG protein lacking the C terminus (MafGΔC) were crossed into a MafG-null background. The compound mutant mice displayed severe thrombocytopenia and splenomegaly, which phenocopied p45-null mice. The MafG C terminus is essential for proplatelet formation and platelet gene activation but not for p45 binding to MARE. These results demonstrate that the MafG C terminus is required for NF-E2 function and suggest that efficient targeting of NF-E2 to a specific nuclear scaffold is important to achieve high-level activity.

[Two cases of methanol poisoning with chronological measurements of blood concentrations of methanol, ethanol and formate].

We report two cases of methanol poisoning and evaluate the kinetics of methanol, ethanol, and formate. The first case was a 48-year-old man (case 1). His initial methanol level was 56.4 mg/dL and serum ethanol level was 2.4 mg/dL. Serum formate was not detected, and ethanol administration was initiated. However, methanol was eliminated slowly, and serum formate increased. His methanol and formate levels decreased rapidly following hemodialysis. He was discharged without any sequelae. The second case was a 35-year-old man (case 2). His serum methanol level was 400 mg/dL, and serum ethanol was not detected. His serum formate level was 13.4 mg/dL, and ethanol and activated folate were administered. He underwent hemodialysis immediately after diagnosis. Methanol and formate decreased rapidly, and he was discharged without any sequelae. Methanol and formate are eliminated slowly if ethanol is administered alone. We suggest that hemodialysis should be considered immediately after ethanol administration.

The exudate of pressure ulcers contains a substantial amount of vascular endothelial growth factor.

Pressure ulcers (PUs) are chronic wounds that occur as areas of tissue necrosis that results from external physical compression, shear forces, and friction. Recently, the efficacy of polyvinylidene film dressing (PVFD) for PUs without any agents promoting wound healing has been reported, suggesting that PUs have their own mechanism of spontaneous healing achieved by vascularization, synthesis of extracellular matrix, and re-epithelization. Since vascular endothelial growth factor (VEGF) 165 and fibroblast growth factor 2 (FGF-2) are released at traumatic or surgical wound sites and play major roles in vascularization and wound healing, we measured the concentrations of VEGF165 and FGF-2 in the exudate and fibrinous sloughs of PUs after PVFD. We collected 10 exudate samples and 3 samples of fibrinous sloughs from 10 PUs of 9 patients immediately after PVFD for 8 h and measured VEGF165 and FGF-2 by ELISA. All 10 exudate samples contained substantial amounts of VEGF165, from 2.79 to 13.27 microg g(-1), irrespective of the severity of the PUs. In contrast, we detected FGF-2 (0.21 and 2.03 microg g(-1)) in only two exudate samples. Similarly, we detected VEGF165 (from 3.14 to 5.93 microg g(-1)) and FGF-2 (less than 0.31 microg g(-1)) in fibrinous sloughs of 3 PUs. These results demonstrate that the exudate and fibrinous sloughs of PUs contain considerable amounts of VEGF, which would contribute to the spontaneous healing of PUs by PVFD. The presence of VEGF165 in the exudate of PUs inspires us to reconsider the treatment strategy of PUs that enhances the spontaneous healing.

Genetic analysis of hierarchical regulation for Gata1 and NF-E2 p45 gene expression in megakaryopoiesis.

GATA1 and NF-E2 p45 are two important regulators of megakaryopoiesis. Whereas GATA1 is known to regulate the p45 gene, details of the GATA1 contribution to the spatiotemporal expression of the p45 gene remain to be elucidated. To clarify the relationship between GATA1 and p45, we performed genetic complementation rescue analysis of p45 function in megakaryocytes utilizing the hematopoietic regulatory domain of the Gata1 gene (G1HRD). We established transgenic mouse lines expressing p45 under G1HRD regulation and crossed the mice with p45-null mice. Compound mutant mice displayed normal platelet counts and no sign of hemorrhage, indicating that G1HRD has the ability to express p45 in a spatiotemporally correct manner. However, deletion of 38 amino acids from the N-terminal region of p45 abrogated the p45 rescue function, suggesting the presence of an essential transactivation activity in the region. We then crossed the G1HRD-p45 transgenic mice with megakaryocyte-specific Gata1 gene knockdown (Gata1(Delta)(neo)(Delta)(HS)) mice. The G1HRD-p45 transgene was insufficient for complete rescue of the Gata1(Delta)(neo)(Delta)(HS) megakaryocytes, suggesting that GATA1 or other factors regulated by GATA1 are required to cooperate with p45 for normal megakaryopoiesis. This study thus provides a unique in vivo validation of the hierarchical relationship between GATA1 and p45 in megakaryocytes.

NF-E2 domination over Nrf2 promotes ROS accumulation and megakaryocytic maturation.

In megakaryocytes, the maturation process and oxidative stress response appear to be closely related. It has been suggested that increased oxygen tension and reactive oxygen species (ROS) promote megakaryopoiesis and that the expression of stress-responsive genes responsible for ROS elimination declines during megakaryocytic maturation. NF-E2 p45 is an essential regulator of megakaryopoiesis, whereas Nrf2 is a key activator of stress-responsive genes. Because p45 and Nrf2 have similar DNA-binding specificities, we hypothesized that p45 competes with Nrf2 to repress stress-responsive genes and achieves favorable intracellular conditions to allow ROS to be efficiently used as signaling molecules. We conducted comprehensive gene expression profiling with wild-type and p45-null megakaryocytes and examined the functional relationship between p45 and Nrf2. We found that 2 characteristic gene clusters are defined within p45 target genes: platelet genes and cytoprotective genes. The former are unique targets activated by p45, whereas the latter are common targets of p45 and Nrf2. Further analysis suggested that, as a less efficacious activator, p45 maintains moderate expression of cytoprotective genes through competing with Nrf2 and promotes ROS accumulation. Increased ROS enhanced platelet gene expression. These results suggest that p45 dominates over Nrf2 to enhance megakaryocytic maturation by promoting ROS accumulation.

[Analysis of bialaphos and its active metabolite L-glufosinate in biological specimens by HPLC].

The symptoms of acute poisoning caused by ingestion of bialaphos (BIAL), an ingredient of herbicide, are supposed to be due to the L-glufosinate (L-GLUF), which is formed by the degradation of bialaphos. To elucidate the pharmacokinetics of BIAL and L-GLUF, we attempted a simultaneous analysis of BIAL and L-GLUF in biological samples by exploiting a reversed phase HPLC method. The derivatization reaction of BIAL and L-GLUF using (+) -1- (9-fluorenyl) ethyl chloroformate was completed in 30 min at 40 degrees C and both derivatives were stable for 48 hr at 25 degrees C. A fluorescence detector were used for HPLC; the exicitation wavelength was set at 265 nm and the emission wavelength at 315 nm. Respective calibration curves prepared by adding BIAL and L-GLUF to serum were linear within ranges of 0.01-10.0 and 0.005-10.0 microg/mL in derivatived liquid samples for introducing into HPLC. The lower limits of detection for BIAL and L-GLUF were 0.005 and 0.001 microg/mL, respectively. An 83-year old male who ingested approximately 350 mL of Herby Liquid, a herbicide containing 18% BIAL and 82% surfactant, in an attempt to commit suicide developed delayed respiratory depression and seizures. L-GLUF was detected in the serum of the patient 2.7 hr after ingestion, but BIAL was not. The change in serum L-GLUF concentration measured over time was consistent with a 2-compartment model, with a distribution half-life of 1.70 hr and an elimination half-life of 6.03 hr.

[Clinical usefulness of the acetaminophen detection kit in treating acute poisoning--data from a survey of 28 cases treated at Niigata City general hospital].

In acute poisoning caused by acetaminophen (N-acetyl-p-aminophenol, APAP), it is critical to predict the onset of delayed liver injury based on the prompt measurement of serum APAP level and to administer the antidote N-acetylcysteine (NAC) without delay as needed. However, all emergency medical facilities are not necessarily equipped with an expensive analytical instrument that allows prompt determination of APAP. Here, we tested the clinical usefulness of the Acetaminophen Detection Kit (Kanto Chemical Co., Ltd.), which claims to rapidly detect APAP in serum using a simple procedure, by spectrophotometrically measuring the APAP concentration in 34 serum samples collected from 28 patients with acute APAP poisoning. The results showed that the correlation coefficient between the APAP value measured by the Acetaminophen Detection Kit and that determined by the HPLC method was, at 0.888, not very high, but that the decision on whether to administer NAC based on the measured APAP level was consistent between the two analytical methods in 23 out of 25 patients. Also, the value obtained by the Acetaminophen Detection Kit was equal to, or larger than, that obtained by the HPLC method, suggesting that it is unlikely that patients requiring NAC would be left untreated. These results indicate that the Acetaminophen Detection Kit, with its ease and simplicity of use, is clinically useful in emergency medical facilities for which an expensive analytical instrument is not affordable.