Dissecting the conformational complexity and mechanism of a bacterial heme transporter.

Iron-bound cyclic tetrapyrroles (hemes) are redox-active cofactors in bioenergetic enzymes. However, the mechanisms of heme transport and insertion into respiratory chain complexes remain unclear. Here, we used cellular, biochemical, structural and computational methods to characterize the structure and function of the heterodimeric bacterial ABC transporter CydDC. We provide multi-level evidence that CydDC is a heme transporter required for functional maturation of cytochrome bd, a pharmaceutically relevant drug target. Our systematic single-particle cryogenic-electron microscopy approach combined with atomistic molecular dynamics simulations provides detailed insight into the conformational landscape of CydDC during substrate binding and occlusion. Our simulations reveal that heme binds laterally from the membrane space to the transmembrane region of CydDC, enabled by a highly asymmetrical inward-facing CydDC conformation. During the binding process, heme propionates interact with positively charged residues on the surface and later in the substrate-binding pocket of the transporter, causing the heme orientation to rotate 180°.

Prosthetic Management of Severe Mandibular Deviation in a Hemi-Mandibulectomy Patient Following Osteoradionecrosis.

Rehabilitation of a patient after hemi-mandibulectomy without reconstruction represents a prosthodontic challenge. Indeed, mandibular deviation and decreased occlusal contacts are a common presentation post-surgery. This paper reports on a patient who presented with these challenges and where chronic osteoradionecrosis has resulted in significant mandibular deviation. A maxillary cobalt chrome mandibular deviation device, designed with palatal bite plane and constructed using 3D printing methods, resulted in a successful outcome. The authors aim to show how restorative management of similar patients can be successful using a modern approach.

Swiss neonatal caregivers express diverging views on parental involvement in shared decision-making for extremely premature infants.

AIM: Due to scarce available national data, this study assessed current attitudes of neonatal caregivers regarding decisions on life-sustaining interventions, and their views on parents' aptitude to express their infant's best interest in shared decision-making. METHODS: Self-administered web-based quantitative empirical survey. All 552 experienced neonatal physicians and nurses from all Swiss NICUs were eligible. RESULTS: There was a high degree of agreement between physicians and nurses (response rates 79% and 70%, respectively) that the ability for social interactions was a minimal criterion for an acceptable quality of life. A majority stated that the parents' interests are as important as the child's best interest in shared decision-making. Only a minority considered the parents as the best judges of what is their child's best interest. Significant differences in attitudes and values emerged between neonatal physicians and nurses. The language area was very strongly associated with the attitudes of neonatal caregivers. CONCLUSION: Despite clear legal requirements and societal expectations for shared decision-making, survey respondents demonstrated a gap between their expressed commitment to shared decision-making and their view on parental aptitude to formulate their infant's best interest. National guidelines need to address these barriers to shared decision-making to promote a more uniform nationwide practice.

Phase I study of PF-03446962, a fully human monoclonal antibody against activin receptor-like kinase-1, in patients with hepatocellular carcinoma.

BACKGROUND: This expansion cohort of a multicenter, dose-escalation, phase I study (NCT00557856) evaluated safety, tolerability, antitumor activity, pharmacokinetics, and pharmacodynamic effects of the anti-activin receptor-like kinase-1 (ALK-1) monoclonal antibody PF-03446962 in advanced hepatocellular carcinoma (HCC). PATIENTS AND METHODS: Patients with HCC and disease progression after prior antiangiogenic therapy or intolerance to treatment received PF-03446962 7 mg/kg intravenously biweekly, as recommended in the dose-escalation part of the study. RESULTS: Twenty-four patients received PF-03446962. The most frequent treatment-related adverse events (AEs) were thrombocytopenia (33.3%), asthenia (29.2), and chills (16.7%). Two patients experienced treatment-related telangiectasia, suggesting an in vivo knockout of ALK-1 function through ALK-1 pathway inhibition. Overall, treatment-related grade 3-4 AEs were reported in eight patients (33.3%). Treatment-related grade 3-4 thrombocytopenia was noted in four patients. No complete or partial responses were reported. Twelve (50%) patients achieved stable disease, which lasted ≥12 weeks in seven (29.2%) patients. The median time to progression was 3 months. Biomarker analyses showed higher mean tumor expression of c-tumor mesenchymal-epithelial transition factor and higher mean serum levels of bone morphogenetic protein-9 in patients with disease control (DC) for ≥12 weeks versus patients with disease progression. Conversely, lower mean serum transforming growth factor-ß and vascular endothelial growth factor receptor-3 levels were detected in patients with DC versus patients with progression. CONCLUSIONS: The observed safety, tolerability, pharmacokinetic profile, and clinical activity support further evaluation of PF-03446962 in patients with HCC and other solid malignancies, as single agent or in combination with other antiangiogenic, chemotherapeutic, or immunotherapeutic agents. TRIAL REGISTRATION NUMBER: NCT00557856.

A New Systemic Disease Mouse Model for Glioblastoma Capable of Single-Tumour-Cell Detection.

BACKGROUND: Glioblastoma is characterised by extensive infiltration into the brain parenchyma, leading to inevitable tumor recurrence and therapeutic failure. Future treatments will need to target the specific biology of tumour recurrence, but our current understanding of the underlying mechanisms is limited. Significantly, there is a lack of available methods and models that are tailored to the examination of tumour recurrence. METHODS: NOD-SCID mice were orthotopically implanted with luciferase-labelled donor U87MG or MU20 glioblastoma cells. Four days later, an unlabelled recipient tumor was implanted on the contralateral side. The mice were euthanised at a humane end-point and tissue and blood samples were collected for ex vivo analyses. RESULTS: The ex vivo analyses of the firefly-labelled MU20 tumours displayed extensive invasion at the primary tumour margins, whereas the firefly-labelled U87MG tumours exhibited expansive phenotypes with no evident invasions at the tumour margins. Luciferase signals were detected in the contralateral unlabelled recipient tumours for both the U87MG and MU20 tumours compared to the non-implanted control brain. Remarkably, tumour cells were uniformly detected in all tissue samples of the supratentorial brain region compared to the control tissue, with single tumour cells detected in some tissue samples. Circulating tumour cells were also detected in the blood samples of most of the xenografted mice. Moreover, tumour cells were detected in the lungs of all of the mice, a probable event related to haematogenous dissemination. Similar results were obtained when the U87MG cells were alternatively labelled with gaussian luciferase. CONCLUSIONS: These findings describe a systemic disease model for glioblastoma which can be used to investigate recurrence biology and therapeutic efficacy towards recurrence.

Interface engineering of cellobiose dehydrogenase improves interdomain electron transfer.

Cellobiose dehydrogenase (CDH) is a bioelectrocatalyst that enables direct electron transfer (DET) in biosensors and biofuel cells. The application of this bidomain hemoflavoenzyme for physiological glucose measurements is limited by its acidic pH optimum and slow interdomain electron transfer (IET) at pH 7.5. The reason for this rate-limiting electron transfer step is electrostatic repulsion at the interface between the catalytic dehydrogenase domain and the electron mediating cytochrome domain (CYT). We applied rational interface engineering to accelerate the IET for the pH prevailing in blood or interstitial fluid. Phylogenetic and structural analyses guided the design of 17 variants in which acidic amino acids were mutated at the CYT domain. Five mutations (G71K, D160K, Q174K, D177K, M180K) increased the pH optimum and IET rate. Structure-based analysis of the variants suggested two mechanisms explaining the improvements: electrostatic steering and stabilization of the closed state by hydrogen bonding. Combining the mutations into six combinatorial variants with up to five mutations shifted the pH optimum from 4.5 to 7.0 and increased the IET at pH 7.5 over 12-fold from 0.1 to 1.24 s-1 . While the mutants sustained a high enzymatic activity and even surpassed the IET of the wild-type enzyme, the accumulated positive charges on the CYT domain decreased DET, highlighting the importance of CYT for IET and DET. This study shows that interface engineering is an effective strategy to shift the pH optimum and improve the IET of CDH, but future work needs to maintain the DET of the CYT domain for bioelectronic applications.

Efficacy of bevacizumab plus erlotinib for advanced hepatocellular carcinoma and predictors of outcome: final results of a phase II trial.

OBJECTIVE: A phase II study was performed to evaluate the efficacy and tolerability of bevacizumab and erlotinib in advanced hepatocellular carcinoma (HCC) patients, and to investigate clinical and molecular predictors of outcome. METHODS: 59 patients with advanced HCC received 10 mg/kg i.v. of bevacizumab every 14 days and 150 mg p.o. of erlotinib daily. The primary endpoint was progression-free survival (PFS) at 16 weeks. Clinical characteristics and plasma biomarkers expression levels were analyzed. RESULTS: PFS at 16 weeks was 64% (95% CI 51-76): 14 patients achieved partial response (24%), 33 had stable disease (56%), 6 progressed (10%), and 6 were not evaluable (10%). Median overall survival was 13.7 months (95% CI 9.6-19.7), and median PFS was 7.2 months (95% CI 5.6-8.3). Grade 3-4 adverse events included fatigue (30%), diarrhea (17%), hypertension (14%), elevated transaminases (12%), and gastrointestinal hemorrhage (10%). High plasma angiopoietin-2, epidermal growth factor receptor, and endothelin-1, and lack of acneiform rash were associated with poor outcome. CONCLUSIONS: The combination of bevacizumab with erlotinib achieved encouraging results in patients with advanced HCC. Current correlatives may help to guide future HCC studies.

Electrochemical alkene azidocyanation via 1,4-nitrile migration.

An electrochemical method for the azidocyanation of alkenes via 1,4-nitrile migration has been developed. This organic oxidant free method is applicable across various alkene containing cyanohydrins, and provides access to a broad range of synthetically useful 1,2-azidonitriles (28 examples). This methodology was extended to an electrochemical alkene sulfonylcyanation procedure, as well as to access a trifunctionalized hexanenitrile from a malononitrile starting material. The orthogonal derivatization of the products was also demonstrated through chemoselective transformations.

A cost analysis of postoperative management in endometrial cancer patients treated by robotics versus laparoscopic approach.

OBJECTIVE: The purpose of this study is to compare postoperative pain management and costs in endometrial cancer patients who had a robotic-assisted or laparoscopic-assisted hysterectomy. METHODS: This is a retrospective cohort study of all endometrial cancer patients from 9/2005 to 6/2010 who had a completed robotic-assisted or laparoscopic-assisted hysterectomy. All surgeries were performed by gynecologic oncologists on the da Vinci S surgical system. Demographic data, patient-recorded pain scores, pain-management interventions, and postoperative pain medication costs were compared. Data was analyzed using Student's t-tests and Pearson's χ(2) tests in SPSS. RESULTS: Two-hundred fifteen (101 robotic and 114 laparoscopic) patients met the inclusion criteria. There were no significant differences between the groups in age, BMI, clinical stage, comorbidities, lymph nodes retrieved, and the number of narcotic vs. non-narcotic drug interventions administered. Robotic patients had a lower number of initial drug interventions (21 vs. 52; P<.001) and total drug interventions (162 vs. 219; P<.001) than laparoscopic patients. Robotics had a lower initial pain score (2.1 vs. 3.0; P=.012). There was a 50% reduction in the pain medication cost on the day of surgery for robotic patients ($12.24 vs. $24.45; P<.01), and a 56% cost reduction for the rest of their length of stay ($3.63 vs. $8.17; P<.01). CONCLUSION: Endometrial cancer patients who have robotic surgery experience less initial postoperative pain and have fewer drug interventions. The cost associated for their pain management represents a savings of greater than 50%. These factors demonstrate the value of robotic surgery in regard to postoperative pain management by delivering higher quality care at a lower cost.

Engineering the Turnover Stability of Cellobiose Dehydrogenase toward Long-Term Bioelectronic Applications.

Cellobiose dehydrogenase (CDH) is an attractive oxidoreductase for bioelectrochemical applications. Its two-domain structure allows the flavoheme enzyme to establish direct electron transfer to biosensor and biofuel cell electrodes. Yet, the application of CDH in these devices is impeded by its limited stability under turnover conditions. In this work, we aimed to improve the turnover stability of CDH by semirational, high-throughput enzyme engineering. We screened 13 736 colonies in a 96-well plate setup for improved turnover stability and selected 11 improved variants. Measures were taken to increase the reproducibility and robustness of the screening setup, and the statistical evaluation demonstrates the validity of the procedure. The selected CDH variants were expressed in shaking flasks and characterized in detail by biochemical and electrochemical methods. Two mechanisms contributing to turnover stability were found: (i) replacement of methionine side chains prone to oxidative damage and (ii) the reduction of oxygen reactivity achieved by an improved balance of the individual reaction rates in the two CDH domains. The engineered CDH variants hold promise for the application in continuous biosensors or biofuel cells, while the deduced mechanistic insights serve as a basis for future enzyme engineering approaches addressing the turnover stability of oxidoreductases in general.

An age-structured model to evaluate the potential of novel malaria-control interventions: a case study of fungal biopesticide sprays.

It has recently been proposed that mosquito vectors of human diseases, particularly malaria, may be controlled by spraying with fungal biopesticides that increase the rate of adult mortality. Though fungal pathogens do not cause instantaneous mortality, they can kill mosquitoes before they are old enough to transmit disease. A model is developed (i) to explore the potential for fungal entomopathogens to reduce significantly infectious mosquito populations, (ii) to assess the relative value of the many different fungal strains that might be used, and (iii) to help guide the tactical design of vector-control programmes. The model follows the dynamics of different classes of adult mosquitoes with the risk of mortality due to the fungus being assumed to be a function of time since infection (modelled using the Weibull distribution). It is shown that substantial reductions in mosquito numbers are feasible for realistic assumptions about mosquito, fungus and malaria biology and moderate to low daily fungal infection probability. The choice of optimal fungal strain and spraying regime is shown to depend on local mosquito and malaria biology. Fungal pathogens may also influence the ability of mosquitoes to transmit malaria and such effects are shown to further reduce vectorial capacity.

Refuge theory and biological control.

An important question in ecology is the extent to which populations and communities are governed by general rules. Recent developments in population dynamics theory have shown that hosts' refuges from their insect parasitoids predict parasitoid community richness patterns. Here, the refuge theory is extended to biological control, in which parasitoids are imported for the control of insect pests. Theory predicts, and data confirm, that the success of biological control is inversely related to the proportion of insects protected from parasitoid attack. Refuges therefore provide a general mechanism for interpreting ecological patterns at both the community level (their species diversity) and population level (their dynamics).

A splice site single nucleotide polymorphism of the fatty acid binding protein 4 gene appears to be associated with intramuscular fat deposition in longissimus muscle in Australian cattle.

Fatty acid binding protein 4 (FABP4) is a candidate gene affecting fatness traits of mammals. However, its association with fatness traits in cattle and other livestock species is not consistent from one study to another. Here, we sequenced the coding sequence of FABP4 looking for non-synonymous variants. We identified a splice site mutation between the third exon and the third intron of bovine FABP4. We genotyped this SNP, FABP4:g.2502C>G, in 1409 cattle with intramuscular fat measurements from seven breeds. The average allele frequency of the C allele was 0.66 with a range of 0.45 to 0.85. A regression on the number of G alleles shows a statistically significant effect of alpha = 0.11, P = 0.044. This appears to confirm an association between IMF and variation at FABP4, with an effect of 0.3% of the variation in our sample when using this SNP.

A phase II study of UFT with leucovorin administered as a twice daily schedule in the treatment of patients with metastatic colorectal cancer.

Prolonged infusions have been shown to be safer and potentially more effective than bolus regimens of 5-fluorouracil (5-FU) as treatment for metastatic colorectal cancer (mCRC). However, infusional 5-FU requires central venous access and costly infusion pumps. Oral fluoropyrimidines enable longer exposures to 5-FU with increased convenience. Tegafur-uracil (UFT) with leucovorin (LV) given thrice daily has improved safety plus comparable survival and response rates to bolus 5-FU/LV. We conducted a phase II clinical study in 98 patients with mCRC to evaluate if UFT with LV given twice daily provided comparable time to progression (TTP), efficacy and tolerability to that reported for thrice daily in two phase III clinical studies. Secondary objectives included overall response rate (ORR) and overall survival (OS). Median TTP was 3.8 months, when compared with 3.5 months for thrice daily. The ORR (11%) and median OS (12.8 months) with twice daily administration were similar to that of thrice daily administration (12% and 12.4 months). The incidence of grade 3/4 treatment-related diarrhoea was 30% on the twice daily and 21% on the thrice daily schedule. These results suggest that twice daily administration has similar efficacy and tolerability to thrice daily administration and is an acceptable alternative for patients who would benefit from UFT with LV therapy.

The effect of genetic variation of the retinoic acid receptor-related orphan receptor C gene on fatness in cattle.

Genotypes at the retinoic acid receptor-related orphan receptor C (RORC) gene were associated with fatness in 1750 cattle. Ten SNPs were genotyped in RORC and the adjacent gene leucine-rich repeat neuronal 6D (LRRN6D) to map the QTL, 7 of which are in a 4.2-kb sequence around the ligand-binding domain of the RORC gene. Of the 29 inferred haplotypes for these SNPs, 2 have a combined frequency of 54.6% while the top 5 haplotypes have a combined frequency of 85.3%. The average D' value of linkage disequilibrium was 0.92 although the average r2 was a low 0.18. The RORC:g.3290T>G SNP had the strongest association with marbling. The inferred haplotypes were significantly associated with marbling and the difference between the most divergent haplotypes was 0.35 sigma(p) of marbling and 0.28 sigma(p) of rump fat, explaining the previously reported QTL effect. cDNA for RORC were sequenced and 2 new alternative transcripts were found. Fetal tissue shows 40 times greater transcription of RORC than adult tissue. The highest expression in fetal tissue was found in liver and kidney, but in adults the longissimus muscle had the greatest expression of the tissues tested.

A validated whole-genome association study of efficient food conversion in cattle.

The genetic factors that contribute to efficient food conversion are largely unknown. Several physiological systems are likely to be important, including basal metabolic rate, the generation of ATP, the regulation of growth and development, and the homeostatic control of body mass. Using whole-genome association, we found that DNA variants in or near proteins contributing to the background use of energy of the cell were 10 times as common as those affecting appetite and body-mass homeostasis. In addition, there was a genic contribution from the extracellular matrix and tissue structure, suggesting a trade-off between efficiency and tissue construction. Nevertheless, the largest group consisted of those involved in gene regulation or control of the phenotype. We found that the distribution of micro-RNA motifs was significantly different for the genetic variants associated with residual feed intake than for the genetic variants in total, although the distribution of promoter sequence motifs was not different. This suggests that certain subsets of micro-RNA are more important for the regulation of this trait. Successful validation depended on the sign of the allelic association in different populations rather than on the strength of the initial association or its size of effect.

Epistasis between calpain 1 and its inhibitor calpastatin within breeds of cattle.

The calpain gene family and its inhibitors have diverse effects, many related to protein turnover, which appear to affect a range of phenotypes such as diabetes, exercise-induced muscle injury, and pathological events associated with degenerative neural diseases in humans, fertility, longevity, and postmortem effects on meat tenderness in livestock species. The calpains are inhibited by calpastatin, which binds directly to calpain. Here we report the direct measurement of epistatic interactions of causative mutations for quantitative trait loci (QTL) at calpain 1 (CAPN1), located on chromosome 29, with causative mutations for QTL variation at calpastatin (CAST), located on chromosome 7, in cattle. First we identified potential causative mutations at CAST and then genotyped these along with putative causative mutations at CAPN1 in >1500 cattle of seven breeds. The maximum allele substitution effect on the phenotype of the CAPN1:c.947G>C single nucleotide polymorphism (SNP) was 0.14 sigma(p) (P = 0.0003) and of the CAST:c.155C>T SNP was also 0.14 sigma(p) (P = 0.0011) when measured across breeds. We found significant epistasis between SNPs at CAPN1 and CAST in both taurine and zebu derived breeds. There were more additive x dominance components of epistasis than additive x additive and dominance x dominance components combined. A minority of breed comparisons did not show epistasis, suggesting that genetic variation at other genes may influence the degree of epistasis found in this system.

Exposure to brominated and organophosphate ester flame retardants in U.S. childcare environments: Effect of removal of flame-retarded nap mats on indoor levels.

We assessed exposure to 39 brominated and 16 organophosphate ester flame retardants (FRs) from both dust and indoor air at seven childcare centres in Seattle, USA, and investigated the importance of nap mats as a source of these chemicals. Many childcare centres serving young children use polyurethane foam mats for the children's naptime. Until recently, the vast majority of these mats sold in the United States contained flame-retarded polyurethane foam to meet California Technical Bulletin 117 (TB117) requirements. With the 2013 update of TB117, allowing manufacturers to meet flammability standards without adding FRs to filling materials, FR-free nap mats have become widely available. We conducted an intervention study by actively switching out FR-treated nap mats with FR-free nap mats and measuring FR levels in indoor air and dust before and after the switch-out. The predominant FRs found in dust and indoor air were 2-ethylhexyl tetrabromobenzoate (EHTBB) and tris(1-chloro-2-propyl) phosphate (TCIPP), respectively. Nap mat samples analysed from four of the six centres contained a Firemaster® mixture, while one mat was predominantly treated with tris(1,3-dichloroisopropyl) phosphate (TDCIPP) and the other contained no detectable target FRs. After replacement, there was a significant decrease (p = 0.03-0.09) in median dust concentrations for bis(2-ethylhexyl) tetrabromophthalate (BEHTBP), EHTBB, tris(4-butylphenyl) phosphate (TBPP), and TDCIPP with reductions of 90%, 79%, 65%, and 42%, respectively. These findings suggest that the nap mats were an important source of these FRs to dust in the investigated childcare environments and that a campaign of swapping out flame-retarded mats for FR-free ones would reduce exposure to these chemicals. While calculated exposure estimates to the investigated FRs via inhalation, dust ingestion, and dermal absorption were below established reference dose values, they are likely underestimated when considering the toddlers' direct contact to the mats and personal cloud effects.

Live Cell Imaging of the TGF- ß/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System.

Transforming Growth Factor ß (TGF-ß) signaling regulates many important functions required for cellular homeostasis and is commonly found overexpressed in many diseases, including cancer. TGF-ß is strongly implicated in metastasis during late stage cancer progression, activating a subset of migratory and invasive tumor cells. Current methods for signaling pathway analysis focus on endpoint models, which often attempt to measure signaling post-hoc of the biological event and do not reflect the progressive nature of the disease. Here, we demonstrate a novel adenovirus reporter system specific for the TGF-ß/Smad3 signaling pathway that can detect transcriptional activation in live cells. Utilizing an Ad-CAGA12-Td-Tom reporter, we can achieve a 100% infection rate of MDA-MB-231 cells within 24 h in vitro. The use of a fluorescent reporter allows for imaging of live single cells in real-time with direct identification of transcriptionally active cells. Stimulation of infected cells with TGF-ß displays only a subset of cells that are transcriptionally active and involved in specific biological functions. This approach allows for high specificity and sensitivity at a single cell level to enhance understanding of biological functions related to TGF-ß signaling in vitro. Smad3 transcriptional activity can also be reported in vivo in real-time through the application of an Ad-CAGA12-Luc reporter. Ad-CAGA12-Luc can be measured in the same manner as traditional stably transfected luciferase cell lines. Smad3 transcriptional activity of cells implanted in vivo can be analyzed through conventional IVIS imaging and monitored live during tumor progression, providing unique insight into the dynamics of the TGF-ß signaling pathway. Our protocol describes an advantageous reporter delivery system allowing for quick high-throughput imaging of live cell signaling pathways both in vitro and in vivo. This method can be expanded to a range of image based assays and presents as a sensitive and reproducible approach for both basic biology and therapeutic development.

Cyanide poisoning in cattle from Dysphania glomulifera (red crumbweed): using the internet for rapid plant identification and diagnostic advice.

A 300-strong Angus-Brahman cattle herd near Springsure, central Queensland, was being fed Acacia shirleyi (lancewood) browse during drought and crossed a 5-hectare, previously burnt area with an almost pure growth of Dysphania glomulifera subspecies glomulifera (red crumbweed) on their way to drinking water. Forty cows died of cyanide poisoning over 2 days before further access to the plant was prevented. A digital image of a plant specimen made on a flat-bed scanner and transmitted by email was used to identify D glomulifera. Specific advice on the plant's poisonous properties and management of the case was then provided by email within 2 hours of an initial telephone call by the field veterinarian to the laboratory some 600 km away. The conventional method using physical transport of a pressed dried plant specimen to confirm the identification took 5 days. D glomulifera was identified in the rumen of one of two cows necropsied. The cyanogenic potential of D glomulifera measured 4 days after collection from the site of cattle deaths was 18,600 mg HCN/kg in dry matter. The lethal dose of D glomulifera for a 420 kg cow was estimated as 150 to 190 g wet weight. The plant also contained 4.8% KNO3 equivalent in dry matter, but nitrate-nitrite poisoning was not involved in the deaths.