Inactivation of Clostridium botulinum type A spores by high-pressure processing at elevated temperatures.

The effects of high-pressure treatments at various temperature-time combinations on the inactivation of spores of Clostridium botulinum type A strains 62-A and BS-A in phosphate buffer (0.067 M, pH 7.0) and in a crabmeat blend were investigated. The log unit reduction of strain 62-A spores increased significantly as the processing pressure increased from 417 to 827 MPa (from 60,000 to 120,000 lb/in2) at 75 degrees C. The reduction of BS-A and 62-A spores in either medium increased as processing temperatures increased from 60 to 75 degrees C and processing times increased from 5 to 15 or 20 min at a maximum pressure of 827 MPa. Approximately 2- and 3-log reductions of BS-A and 62-A spores, respectively, in phosphate buffer were obtained at the maximum pressure-maximum temperature combination of 827 MPa and 75 degrees C for a processing time of 20 min. Processing for 15 min at the maximum pressure-maximum temperature combination resulted in maximum reductions of 3.2 and 2.7 log units for BS-A and 62-A spores, respectively, in the crabmeat blend. Results obtained in this study indicate that the crabmeat blend did not protect BS-A and 62-A spores against inactivation by high-pressure processing.

The Autosow raised miniature swine as a model for assessing the effects of dietary soy trypsin inhibitor.

Toxicological effects of dietary soy trypsin inhibitor (TI) were assessed in male miniature swine, a model chosen for its similarities to human digestive physiology and anatomy. The TI preparation was extracted from defatted raw soy flour. From 1 through 5 weeks of age, piglets were automatically fed either a TI liquid diet [Autosow TI group (ASTI)] or a control liquid diet [Autosow control group (ASC)]. From 6 to 39 weeks of age, these animals received either swine chow and TI or swine chow and control article. The TI diets were formulated to contain a TI activity of approximately 500 mg TI/100 g dry matter. A sow control (SC) group suckled from birth to 6 weeks of age and then fed as the ASC group with swine chow plus control article from 6 to 39 weeks of age. The SC piglets grew faster than ASC piglets during postnatal weeks 1 and 2; however, the ASC piglets were significantly heavier than the SC piglets (P=0.001) at 6 weeks of age. Compared with the ASC group, TI caused a moderate decrease in feed consumption and a moderate but reversible decrease in growth from 2 to 5 weeks of age, but not thereafter. Some control and TI-fed Autosow-reared piglets had loose stools until 6 weeks of age; the effect was significantly greater in the TI-fed group. Otherwise, all swine were active and had normal appearance and behavior.

Origins of the ethical attitude.

This paper seeks to explore the genesis of the capacity for an ethical attitude, personally and professionally. As analysts working in intimate clinical settings, ethics is at the foundation of our professional lives, as it is at the foundation of our humanity and what it is we struggle towards in our own personal development. The ethical attitude presupposes special responsibilities that we choose to adopt in relation to another. Thus, a parallel situation pertains between caregiver and child and between analyst and patient: they are not equal partners, but nevertheless are in a situation of mutuality, shared subjectivity, and reciprocal influence. The basic premise of this paper is that the analytic attitude is an ethical attitude, and that the ethical attitude is a developmental achievement, and as such it may reach beyond the depressive position.

An LC-MS-MS method for the comprehensive analysis of cocaine and cocaine metabolites in meconium.

A sensitive, precise, and accurate liquid chromatography-mass spectrometry (LC-MS-MS) method was developed to quantitate cocaine and cocaine metabolites, which were simultaneously extracted from suspected drug-positive meconium samples using solid-phase extraction. The ability to analyze cocaine and multiple cocaine metabolites in meconium makes this method a powerful tool for the study of cocaine exposure and metabolism in neonates. Of 22 samples, only 1 did not show the presence of cocaine or any metabolite of cocaine. The identified metabolites varied both qualitatively and quantitatively between samples. Ecgonine appears to hold the most promise as a diagnostic marker compound for neonatal cocaine exposure as this metabolite was present in 21 of 21 of the positive samples tested, and at a relatively high median concentration. However, a core group of eight metabolites (present in at least 20 of 21 positive samples) was identified that appears to possess the greatest utility for determining cocaine exposure. Finally, the use of this method for assessment of the magnitude of fetal cocaine exposure was demonstrated.

Benzofuranyl ureas with potent cardiovascular teratogenicity in rats.

Studies of embryo-fetal development in rats were conducted with two 5-lipoxygenase inhibitors. SB-202235 (1,000 mg/kg/day) or SB-210661 (50, 100, or 500 mg/kg/day) was administered orally by gavage to female rats on days 6-17 postcoitus (pc) or days 7-16 pc. SB-202235 (1,000 mg/kg/day) and SB-210661 (100 mg/kg/day) reduced maternal body weight gain for the treatment period by 16% and 21%, respectively, relative to controls. SB-202235 (1,000 mg/kg/day) or SB-210661 (50 or 100 mg/kg/day), did not affect numbers of resorptions, dead or live fetuses/litter, but 500 mg/kg/day of SB-210661 caused 100% embryo lethality. SB-202235 (1,000 mg/kg/day) and SB-210661 (50 and 100 mg/kg/day) reduced fetal body weight by 15-30% and produced extensive cardiovascular malformations, as well as diaphragmatic hernias. SB-210661 also caused thymic abnormalities and cryptorchidism. Cardiovascular defects included abnormalities in aorticopulmonary septation, the aortic arch, pulmonary trunk, and ventricular septal defects are discussed relative to comparable human syndromes of cardiovascular malformation.

Indinavir crystalluria: identification of patients at increased risk of developing nephrotoxicity.

OBJECTIVE: To determine whether the protease inhibitor indinavir sulfate, which is extremely insoluble at physiologic pH levels and which is known to be associated with nephrolithiasis, is associated with crystalluria at a usual therapeutic dose. METHODS: Freshly voided urine from 27 male human immunodeficiency virus patients being treated with indinavir at a dose of 800 mg, tid, in an outpatient setting and from 20 healthy subjects undergoing routine physical examination was subjected to dipstick urinalysis and microscopic examination of urinary sediments. RESULTS: Three (11%) of 27 patients treated with indinavir developed highly characteristic crystalluria during the course of therapy. No such crystals were observed in the urine of the 20 healthy subjects. CONCLUSION: Indinavir crystalluria was identified in asymptomatic patients treated with usual therapeutic doses of the drug. Screening urines of patients taking indinavir may be useful in identifying patients at risk for developing nephrotoxicity.

Growth and toxin production by Clostridium botulinum on sliced raw potatoes in a modified atmosphere with and without sulfite.

The ability of Clostridium botulinum type A or B spores to grow and produce toxin on fresh raw potatoes in a modified atmosphere with or without sulfite was investigated at 22 degrees C. Fresh, peeled, sliced potatoes, untreated or dipped for 2 min into 0.7% sulfite solution and drained, were surface-inoculated at several concentration levels with a mixture of C. botulinum spores, either type A or B. They were placed in a modified atmosphere (30% N/70% CO2) within oxygen-impermeable bags (200 g/bag) and incubated at room temperature (22 degrees C). Toxicity was tested on days 0, 3, 4, 5, 6, and 7. After incubation, the potatoes were blended and centrifuged, and the Millipore-filtered supernatant fluid was injected intraperitoneally into mice. Sensory evaluation, except taste, was also performed. Potatoes inoculated with C. botulinum type A spores but untreated with NaHSO3 became toxic in 4 to 5 days, which coincided with the sensory evaluation "unfit for human consumption". Potatoes treated with NaHSO3 regardless of inoculum size or residual SO2 levels appeared acceptable for human consumption through day 7, even though they were toxic after 4 days of incubation. Although toxicity from type B spores occurred later and in fewer test samples than toxicity from type A, some potatoes again appeared acceptable but were toxic. Thus, although NaHSO3 markedly extended the consumer acceptability of peeled, sliced, raw potatoes at the abuse temperature, it did not inhibit outgrowth and toxin production by C. botulinum under these conditions.

A large outbreak of botulism: the hazardous baked potato.

In April 1994, the largest outbreak of botulism in the United States since 1978 occurred in El Paso, Texas. Thirty persons were affected; 4 required mechanical ventilation. All ate food from a Greek restaurant. The attack rate among persons who ate a potato-based dip was 86% (19/22) compared with 6% (11/176) among persons who did not eat the dip (relative risk [RR] = 13.8; 95% confidence interval [CI], 7.6-25.1). The attack rate among persons who ate an eggplant-based dip was 67% (6/9) compared with 13% (241189) among persons who did not (RR = 5.2; 95% CI, 2.9-9.5). Botulism toxin type A was detected from patients and in both dips. Toxin formation resulted from holding aluminum foil-wrapped baked potatoes at room temperature, apparently for several days, before they were used in the dips. Consumers should be informed of the potential hazards caused by holding foil-wrapped potatoes at ambient temperatures after cooking.

The diagnostic accuracy of bedside and laboratory coagulation: procedures used to monitor the anticoagulation status of patients treated with heparin.

We evaluated the diagnostic accuracy of three bedside coagulation procedures, the Hemochron activated whole-blood clotting time (ACT) (International Technidyne, Edison, NJ), the CoaguChek Plus (Boehringer Mannheim, Indianapolis, Ind) activated partial thromboplastin time (APTT) and the TAS (Cardiovascular Diagnostics, Raleigh, NC) APTT, before removal of arterial sheaths, in patients who received heparin therapy during percutaneous coronary angioplasty. As part of the postprocedure care, nurses performed bedside coagulation tests, removed the sheaths when appropriate coagulation criteria were met, and collected samples for laboratory APTT determinations and heparin assays performed with an automated chromogenic anti-Xa assay. Patients with heparin concentrations of 0.3 U/mL or more were classified as anticoagulated and those with concentrations less than 0.3 U/mL, as not anticoagulated. Analysis of the receiver operator characteristic (ROC) curve was used to rank the performance of the methods. Areas under the ROC curves +/- SE for the laboratory APTT, CoaguChek Plus APTT, Hemochron ACT, and TAS APTT were 0.978 +/- 0.016, 0.872 +/- 0.044, 0.797 +/- 0.039, and 0.795 +/- 0.048, respectively. At cutoff values for the tests that provide greatest safety for the patients (no false-negative results), the false-positive rates for the laboratory, CoaguChek Plus, Hemochron, and TAS methods were 15%, 27%, 62%, and 100%, respectively. The laboratory APTT demonstrated the highest diagnostic accuracy in this application; however, turnaround time for the test (50% of the results were reported in excess of 77 minutes) was inadequate for clinical decision making. To meet this requirement, we developed a point-of-care program using the whole blood APTT performed on the CoaguChek Plus analyzer.

PAPNET testing for HSILs. The few cell/small cell challenge.

OBJECTIVE: To define and evaluate the effectiveness of the PAPNET Testing System for high grade squamous cervical intraepithelial lesions (HSIL). STUDY DESIGN: The literature was reviewed, defining the "few cell/small cell challenge" associated with HSIL. Several recently published investigations demonstrate the effectiveness of PAPNET testing, especially the effectiveness of PAPNET testing in reducing false negatives for these lesions. RESULTS: The results of several independent investigations demonstrate that the PAPNET Testing System is sensitive to the types of abnormalities typically not detected by conventional screening (e.g., abnormal cells that are small and few in number). In clinical practice, the PAPNET Testing System is effective in reducing false negatives. CONCLUSION: The PAPNET Testing System helps solve the "unfortunate paradox of Pap smear diagnosis," as noted by DeMay, that high grade serious lesions may be more difficult to detect and diagnose by cytology than low grade lesions, which are clinically less significant and often regress without therapy. The accuracy of detection of HSIL is improved with the PAPNET Testing System.

Effect of prazosin on sperm transport in male rats.

Our objective was to investigate ejaculation and transport of sperm in the reproductive tract of male rats treated with an alpha-adrenergic receptor antagonist. Males were dosed (s.c.) with vehicle or 1.4 mg/kg prazosin. Sperm recovered in utero and ex vivo from the vas deferens and cauda epididymis were evaluated. Mating behavior and sperm motility were unaffected by prazosin. Prazosin treated males ejaculated fewer sperm (12.58 +/- 8.12 vs. 110.5 +/- 29.15 million), and the distal vas deferens contained fewer sperm (2.72 +/- 0.84 vs. 24.42 +/- 3.25 million) relative to controls. Prazosin-treated males had more sperm in the cauda epididymis relative to controls indicating inhibition of sperm transport to the vas deferens. These data demonstrate that inhibition of sperm transport from the cauda epididymis to the distal vas deferens is related to low ejaculate sperm counts in prazosin treated rats.

The not-so-silent couple in the individual.

This paper aims to examine two often separate areas of analytical enquiry, the nature of the self, with its foundation in the concept of a primary self which may achieve a sense of coherence over time, and the nature of internal objects, a concept that forms the basis of theories concerning part selves and sub-personalities. It is argued that these concepts might be integrated to provide a unified model of the self, thereby integrating theoretically disparate aspects of mental structure and functioning. Through an examination of clinical material, the archetype of the coniunctio is evoked to offer an understanding of how, in the absence of a stable conjunction of (maternal) reverie and (paternal) thinking functions, a series of linked but oppositional internal couples may be created which lends to the self the experience of a combined and sustaining inner couple. The internal couple creates a source of psychological survival for the self, with the function of providing a reliable structure in which the processes of the self may unfold, but equally requiring strict adherence to a pernicious system of internal defences that allows for very little interaction with others outside the self. At the same time, these defences inevitably become blocks to further development, and the work of transformation is thwarted. For transformation to occur, it is necessary for the self to find another, often the analyst, who may be allowed to partake in the internal conjunction, thereby promoting a better grounding for the self.

Terminology of developmental abnormalities in common laboratory mammals (version 1).

This paper presents the first version of an internationally-developed glossary of terms for structural developmental abnormalities in common laboratory animals. The glossary is put forward by the International Federation of Teratology Societies (IFTS) Committee on International Harmonization of Nomenclature in Developmental Toxicology, and represents considerable progress toward harmonization of terminology in this area. The purpose of this effort is to provide a common vocabulary that will reduce confusion and ambiguity in the description of developmental effects, particularly in submissions to regulatory agencies worldwide. The glossary contains a primary term or phrase, a definition of the abnormality, and notes, where appropriate. Selected synonyms or related terms, which reflect a similar or closely related concept, are noted. Nonpreferred terms are indicated where their usage may be incorrect. Modifying terms used repeatedly in the glossary (e.g., absent, branched) are listed and defined separately, instead of repeating their definitions for each observation. Syndrome names are generally excluded from the glossary, but are listed separately in an appendix. The glossary is organized into broad sections for external, visceral, and skeletal observations, then subdivided into regions, structures, or organs in a general overall head to tail sequence. Numbering is sequential, and not in any regional or hierarchical order. Uses and misuses of the glossary are discussed. Comments, questions, suggestions, and additions from practitioners in the field of developmental toxicology are welcomed on the organization of the glossary as well as on the specific terms and definitions. Updates of the glossary are planned based on the comments received.

Spontaneous and induced alterations in the cardiac membranous ventricular septum of fetal, weanling, and adult rats.

Alterations of the cardiac membranous ventricular septum were studied using macrodissection, scanning electron and light microscopy of fetal, weanling, and adult Sprague-Dawley rats. Membranous ventricular septal defects (VSDs) were observed in 2.0% of fetuses on day 21 postcoitus (pc) but not in weanling or adult rats. The most common observation was a nonpatent depression in the membranous septum with an incidence of 38.1, 10.5, 4.3% for fetuses on days 17, 19, or 21 pc, respectively, 11.8% for weanlings, and 9.1% for adults. VSDs were characterized by a split in the endocardial cushion cells in the interventricular component of the membranous septum. Nonpatent depressions were characterized by a split in the endocardial cushion cells in the atrioventricular component of the septum, and they persisted postnatally as a blind-ended diverticulum directed above the tricuspid valve. The cardiovascular teratogens, trimethadione and trypan blue, produced in fetuses nonpatent depressions and VSDs morphologically similar to untreated fetuses. Maternal diet restriction (25% of controls) lowered fetal (day 21 pc) body weight by 47% but did not affect the incidence of ventricular septal alterations, suggesting that intrauterine growth retardation is not necessarily associated with alterations in the development of the ventricular septum. We conclude that neither VSDs nor nonpatent depressions in Sprague-Dawley rats affect postnatal survival and that VSDs close spontaneously during neonatal life.

Shelf Life and Toxin Development by Clostridium botulinum during Storage of Modified-Atmosphere- Packaged Fresh Aquacultured Salmon Fillets.

Shelf life (onset of sensory spoilage) and the potential for toxin production by Clostridium botulinum type E in retail-type packages of fresh aquacultured salmon fillets packaged in high-barrier film bags under selected atmospheres (100% air, a modified atmosphere containing 75% CO2:25% N2, and vacuum) and stored under refrigeration (4°C) and temperature-abuse conditions (8 and 16°C) were investigated. Chemical spoilage indicators (trimethylamine and surface pH) and microbial populations were compared with sensory spoilage characteristics. Storage temperature influenced the time to onset of both sensory spoilage and toxin development in salmon fillets packaged in all atmospheres. The shelf life of fillets packaged in all atmospheres decreased with increase of storage temperature from 4 to 16°C. Trimethylamine content associated with the onset of spoilage for 100% air-packaged fillets increased as storage temperature increased. However, for modified-atmosphere-packaged fillets, the trimethylamine content associated with the onset of spoilage increased as storage temperature decreased from 8 to 4°C. Surface pH was not a good spoilage indicator for modified-atmosphere-packaged fillets. Toxin development preceded sensory spoilage at 16°C storage for fillets packaged in modified atmospheres. Toxin development coincided with sensory spoilage or was slightly delayed for the fillets packaged in all the atmospheres at 8°C storage. At 4°C none of the fillets packaged in either of the atmospheres developed toxin, even 20 days after spoilage as determined by sensory characteristics.

An outbreak of type A botulism associated with a commercial cheese sauce.

BACKGROUND: Although botulism is rare, recognition of a possible case of this illness represents a public health emergency. To prevent more cases, prompt investigation must be done to determine whether illness is linked to commercial product or restaurant. Botulism can masquerade as other illnesses, and seemingly unlikely foods can harbor botulinum toxin. OBJECTIVE: To confirm the diagnosis and determine the cause and extent of an outbreak of botulism associated with food served at a delicatessen. DESIGN: Retrospective cohort study of patrons of the delicatessen; laboratory analysis of food, serum samples, and stool samples; and traceback of implicated food. SETTING: Community in Georgia. PARTICIPANTS: Patrons of the delicatessen. MAIN OUTCOME MEASURES: Botulinum toxin in food, serum, or stool and Clostridium botulinum in food and stools. RESULTS: 8 of 52 patrons (15%) met the case definition for botulism. In 4 of the 8 patrons, and illness other than botulism was initially diagnosed. Five of the 8 were hospitalized, and 1 died. Stool cultures from 4 patrons yielded type AC. botulinum, and two serum samples contained botulinum toxin. All ill persons ate food from the delicatessen on 1 October 1993. Of the 22 persons who ate at the delicatessen that day, all 8 ill persons but none of the 14 well persons ate a potato stuffed with meat and cheese sauce. An open can of cheese sauce contained type A botulinum toxin and yielded C botulinum on culture. Cheese sauce experimentally inoculated with C botulinum spores became toxic after 8 days at a temperature of 22 degrees C (room temperature). CONCLUSIONS: A commercial, canned cheese caused a botulism outbreak. This product readily becomes toxic when contaminated by C botulinum spores and left at room temperature. Mild botulism caused by unusual vehicles may be misdiagnosed. Botulism should be included in the differential diagnosis of persons with signs or symptoms of acute cranial nerve dysfunction.

The male polyurethane condom: a review of current knowledge.

Condoms are one of the oldest form of contraceptive and the best recognized form of protection against sexually transmitted diseases. Their use, however, is limited by both behavioral factors and device-related factors, including complaints about decreased sensitivity and sexual enjoyment. To address these limitations, a male condom made of polyurethane was developed. Polyurethane is a strong impermeable material with good heat transfer characteristics that is less susceptible to deterioration during storage than latex. Because little information is available comparing polyurethane and latex condoms in terms of consumer preferences as well as breakage and slippage, we reviewed four pre-marketing studies of polyurethane condoms, one of which included comparison to latex. No significant differences in slippage and breakage rates between latex and polyurethane condoms were reported in the study that included a latex comparator, and other studies of polyurethane condoms alone resulted in rates in the same range as published for latex condoms. Subjectively, consumers expressed significantly greater preference for the polyurethane condom over latex in regard to appearance, lack of smell, likelihood of slippage, comfort, sensitivity, natural look, natural feel, and overall. While additional testing is needed, these preliminary results suggest that the male polyurethane condom reviewed performed at least as well as latex condoms and is preferred by consumers. If preference translates to greater use, the male polyurethane condom may address important barriers that have been linked with inadequate condom use in the past. These results, however, may not be generalizable to other brands of polyurethane condom currently under development.

PIP: A literature review of four pre-marketing studies of the Avanti male polyurethane condom was conducted to compare consumer preferences, breakage, and slippage of polyurethane and latex condoms. The condom manufacturer, London International, recruited participants for these studies throughout the UK. In the only study that had two comparison groups, the polyurethane condom had the highest overall consumer reference rating (7.4 vs. 6.8; p = 0.002) and the lower breakage and slippage rates (0.9% vs. 2.1% and 0.4% vs. 1.1%, respectively). Slippage and breakage rates in the other three studies were similar to those in the literature for latex condoms. Consumers preferred the polyurethane condom to the latex condom because they perceived it as having a natural feel and look with no smell and enhanced sensitivity and comfort. The number of patients lost to follow-up was low for all four studies (no greater than 9%). The leading reason for not using any of the different condoms provided to study participants was lack of opportunity (56%). Additional research is needed, particularly studies with a comparison latex group. Clinical trials of the male polyurethane condom examining contraceptive efficacy, breakage, and slippage data are currently being conducted among 800 couples recruited in many centers. Nevertheless, the findings of the literature review suggest that the Avanti polyurethane condom perform at least as well as the latex condom and that consumers prefer the polyurethane condom. If preference increases use, the Avanti polyurethane condom may overcome barriers that have been associated with poor condom use in the past.

Adaptation of a functional assay for protein S to a Cobas Fara II centrifugal analyzer.

An adaptation of the manual Staclot protein S functional assay (American Bioproducts, Parsippany, NJ) for the Cobas FARA II centrifugal analyzer is described. This automated method is based on determining the clotting time by measuring the change in turbidity of the specimen as clotting progresses. The accurate and precise pipetting of the analyzer combined with its enhanced data acquisition and rapid parallel processing features result in a markedly improved procedure compared to the less accurate and reproducible manual method. The assay is linear over the range of protein S concentrations encountered clinically (0% to 150%); has a limit of detection of 3% protein S; within and between day precision (CV) at a level of 50% protein S is 4.7 and 12.6%, respectively; and results agree closely with those obtained on the STA automated hemostasis analyzer (slope = 0.98, intercept 3.92, and r = 0.98). Protein S values obtained with this assay for specimens from 20 normal males and 20 normal females were 115% +/- 22.31% (mean +/- standard deviation [SD]) and 98% +/- 18.2%, respectively. In 14 patients on chronic stable treatment with Coumadin (DuPont Pharmaceuticals, Wilmington, DE) the mean functional protein S activity was 16.2% and the SD 11.7%. The method for determination of protein S activity on the centrifugal analyzer yields results comparable to data reported with a variety of other techniques.

1-Methyl-2-pyrrolidone (NMP): reproductive and developmental toxicity study by inhalation in the rat.

A two-generation reproduction study with a developmental toxicity component was conducted. For the reproduction phase, male and female rats inhaled 0, 10, 51, or 116 ppm NMP daily for 6 hr/day, 7 days/week from 34 days of age to the end of the mating period for the males (100 exposure days) and till weaning for the females (about 143 exposure days, but interrupted from Day 20 of gestation to Day 4 Postpartum). On Day 70 postpartum, one male and one female selected from each litter later mated with newly obtained, nonexposed adults of the opposite sex to produce an F2 generation. For the developmental phase, rats of both sexes inhaled 0 or 116 ppm NMP as outlined above, but euthanization of the females occurred on Day 21 of gestation followed by fetal examination for structural alterations. The indices of reproductive performance for the NMP-exposed rats did not differ significantly from those obtained for the control rats. Rats exposed to 116 ppm had a detectable decrease in response to sound. No other signs of NMP-related toxicity were detected among the parental rats. An exposure-related but slight decrease in fetal weight was detected only among the F1 offspring whose parents both inhaled NMP at 116 ppm. This slight effect also appeared at birth among the pups of the reproductive phase where it persisted till 21 days after birth when NMP inhalation by the mother ceased. Thereafter, the body weight of the offspring was comparable to the control values. No detectable or developmental effects appeared in the 10 or 51 ppm groups.