MSTO1 mutations cause mtDNA depletion, manifesting as muscular dystrophy with cerebellar involvement.

MSTO1 encodes a cytosolic mitochondrial fusion protein, misato homolog 1 or MSTO1. While the full genotype-phenotype spectrum remains to be explored, pathogenic variants in MSTO1 have recently been reported in a small number of patients presenting with a phenotype of cerebellar ataxia, congenital muscle involvement with histologic findings ranging from myopathic to dystrophic and pigmentary retinopathy. The proposed underlying pathogenic mechanism of MSTO1-related disease is suggestive of impaired mitochondrial fusion secondary to a loss of function of MSTO1. Disorders of mitochondrial fusion and fission have been shown to also lead to mitochondrial DNA (mtDNA) depletion, linking them to the mtDNA depletion syndromes, a clinically and genetically diverse class of mitochondrial diseases characterized by a reduction of cellular mtDNA content. However, the consequences of pathogenic variants in MSTO1 on mtDNA maintenance remain poorly understood. We present extensive phenotypic and genetic data from 12 independent families, including 15 new patients harbouring a broad array of bi-allelic MSTO1 pathogenic variants, and we provide functional characterization from seven MSTO1-related disease patient fibroblasts. Bi-allelic loss-of-function variants in MSTO1 manifest clinically with a remarkably consistent phenotype of childhood-onset muscular dystrophy, corticospinal tract dysfunction and early-onset non-progressive cerebellar atrophy. MSTO1 protein was not detectable in the cultured fibroblasts of all seven patients evaluated, suggesting that pathogenic variants result in a loss of protein expression and/or affect protein stability. Consistent with impaired mitochondrial fusion, mitochondrial networks in fibroblasts were found to be fragmented. Furthermore, all fibroblasts were found to have depletion of mtDNA ranging from 30 to 70% along with alterations to mtDNA nucleoids. Our data corroborate the role of MSTO1 as a mitochondrial fusion protein and highlight a previously unrecognized link to mtDNA regulation. As impaired mitochondrial fusion is a recognized cause of mtDNA depletion syndromes, this novel link to mtDNA depletion in patient fibroblasts suggests that MSTO1-deficiency should also be considered a mtDNA depletion syndrome. Thus, we provide mechanistic insight into the disease pathogenesis associated with MSTO1 mutations and further define the clinical spectrum and the natural history of MSTO1-related disease.

Eotaxin-1 is involved in parasite clearance during chronic filarial infection.

Eosinophil migration as key feature of helminth infection is increased during infection with filarial nematodes. In a mouse model of filariasis, we investigated the role of the eosinophil-attracting chemokine Eotaxin-1 on disease outcome. BALB/c and Eotaxin-1(-/-) mice were infected with the rodent filaria Litomosoides sigmodontis, and parasitic parameters, cellular migration to the site of infection, and cellular responsiveness were investigated. We found increased parasite survival but unaffected eosinophil migration to the site of infection in Eotaxin-1(-/-) mice. Expression of CD80 and CD86 was reduced on eosinophils from Eotaxin-1(-/-) mice after in vitro TLR2 stimulation and exposure to filarial antigen, respectively, suggesting a potential reduced activation state of eosinophils in Eotaxin-1 deficient mice. We further demonstrated that macrophages from Eotaxin-1(-/-) mice produce decreased amounts of IL-6 in vitro, a cytokine found to be associated with parasite containment, suggesting possible mechanisms by which Eotaxin-1 regulates activation of inflammatory cells and thus parasite survival.

Differential expression of toll-like receptor 2 on dendritic cells from asymptomatic and symptomatic atopic donors.

BACKGROUND: Early microbial exposure may reduce the risk for developing allergies on an atopic genetic background. Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns of microbes and modulate innate and adaptive immunity. Different expression of TLRs in symptomatic and asymptomatic atopic donors may contribute to the development of allergic disease. METHODS: Monocytes and monocyte-derived dendritic cells (DCs) from symptomatic (n = 12) and asymptomatic atopic donors (n = 11), healthy nonatopics (n = 14) and from patients with psoriasis (n = 13) were analyzed for their expression of TLR2, TLR4 and TLR9 by real-time PCR. RESULTS: Monocytes did not show any differences in TLR2, TLR4 and TLR9 expression between the 4 groups. In contrast, DCs from asymptomatic donors showed an enhanced expression of TLR2 over DCs from nonatopics (p = 0.038) and just failed to reach significance when compared to symptomatic atopic patients (p = 0.060). TLR2 expression kinetics from monocytes to monocyte-derived DCs showed sustained expression of TLR2 in DCs only from asymptomatic donors but downregulation in the other groups. In DCs from symptomatic atopic donors, the expression of TLR2 correlated significantly with total IgE values in the serum (p = 0.01994). CONCLUSION: Differential expression and functional regulation of TLR2 expression by DCs from symptomatic and asymptomatic atopic donors may be important for the manifestation of allergic disease. Increased and sustained TLR2 expression on DCs, possibly as a result of an increased exposure to microorganisms or as a mechanism enhancing the sensitivity of microbe detection, may be of functional importance for the maintenance of clinical unresponsiveness toward allergens.

Filariasis and lymphoedema.

Among the causes of lymphoedema (LE), secondary LE due to filariasis is the most prevalent. It affects only a minority of the 120 million people infected with the causative organisms of lymphatic filariasis (LF), Wuchereria bancrofti and Brugia malayi/timori, but is clustered in families, indicating a genetic basis for development of this pathology. The majority of infected individuals develop filarial-specific immunosuppression that starts even before birth in cases where mothers are infected and is characterized by regulatory T-cell responses and high levels of IgG4, thus tolerating high parasite loads and microfilaraemia. In contrast, individuals with this pathology show stronger immune reactions biased towards Th1, Th2 and probably also Th17. Importantly, as for the aberrant lymph vessel development, innate immune responses that are triggered by the filarial antigen ultimately result in the activation of vascular endothelial growth factors (VEGF), thus promoting lymph vessel hyperplasia as a first step to lymphoedema development. Wolbachia endosymbionts are major inducers of these responses in vitro, and their depletion by doxycycline in LF patients reduces plasma VEGF and soluble VEGF-receptor-3 levels to those seen in endemic normals preceding pathology improvement. The search for the immunogenetic basis for LE could lead to the identification of risk factors and thus, to prevention; and has so far led to the identification of single-nucleotide polymorphisms (SNP) with potential functional relevance to VEGF, cytokine and toll-like receptor (TLR) genes. Hydrocele, a pathology with some similarity to LE in which both lymph vessel dilation and lymph extravasation are shared sequelae, has been found to be strongly associated with a VEGF-A SNP known for upregulation of this (lymph-)angiogenesis factor.

New insights into the biology of filarial infections.

Recent successes in the control of lymphatic filariasis and onchocerciasis need continuing research in order to sustain the achievements and to develop further tools to tackle the new questions that arise when only reduced infection prevalences prevail. In this regard, in a symposium held at the Xth European Multicolloquium of Parasitology (August 2008, Paris) questions such as the impact of filarial immunosuppression, and its lack following filarial control, on the outcome of co-infections were addressed, as were new approaches to treatment with promising drugs such as moxidectin or the antibiotic chemotherapy against Wolbachia endosymbionts in filariae. In particular, longer treatment courses of doxycycline could be carried out by community-directed treatment at high coverage, thus potentially allowing its use in restricted areas with suboptimal responses to ivermectin against onchocerciasis, or in areas with co-infection by loiasis where onchocerciasis or lymphatic filariasis need to be controlled. New, more potent drugs, or eventually vaccines, will be of importance because in many vector-filarial parasite relationships worldwide, transmission efficacy increases with low numbers of ingested microfilariae, and since ivermectin may render treated hosts more susceptible to new infection.

Selective oxidative dehydrogenation of propane to propene using boron nitride catalysts.

The exothermic oxidative dehydrogenation of propane reaction to generate propene has the potential to be a game-changing technology in the chemical industry. However, even after decades of research, selectivity to propene remains too low to be commercially attractive because of overoxidation of propene to thermodynamically favored CO2 Here, we report that hexagonal boron nitride and boron nitride nanotubes exhibit unique and hitherto unanticipated catalytic properties, resulting in great selectivity to olefins. As an example, at 14% propane conversion, we obtain selectivity of 79% propene and 12% ethene, another desired alkene. Based on catalytic experiments, spectroscopic insights, and ab initio modeling, we put forward a mechanistic hypothesis in which oxygen-terminated armchair boron nitride edges are proposed to be the catalytic active sites.

Molecular isoforms of Na+/K(+)-ATPase in the nervous system and kidney of the spontaneously diabetic BB/Wor rat.

Na+/K(+)-ATPase was evaluated in the retina and kidney of the spontaneously diabetic BB/Wor rat after 1 and 4 months of insulin dependency. Retinal synthesis of the Na+/K(+)-ATPase was measured during a 2-h intravitreal pulse of [35S]methionine and analyzed by SDS-PAGE and scintillation counting. Synthesis of the alpha-1 and 'alpha(+)' (includes both alpha-2 and alpha-3) isoforms of the catalytic subunit was increased 123% and 69%, respectively at 4 months. Increases were also suggested at 1 month, but were not significant. The diabetes-dependent peak of synthesis in long-term diabetic rats turned over rapidly and by 3 days after intravitreal labeling, radioactively labeled enzyme was equal in both control and diabetic retinae. The amount of axonally transported, labeled enzyme recovered from endings of the optic nerve in the superior colliculus paralleled retinal labeling. Significant renal hypertrophy (48%) was noted at 4 months, but not at 1 month. The strophanthidin-inhibition constant for diabetes-induced renal enzyme was the same as for control enzyme (approx. 10(-4) M), indicating that diabetic renal hypertrophy does not induce a Na pump isozyme that is more sensitive to cardiotonic steroids. SDS-PAGE of the renal enzyme also failed to indicate more than one isoform of the alpha subunit.

Inhibition of the allograft response by donor specific blood transfusion: association with reduced local TH1 cytokines and nitric oxide but enhanced prostaglandin E2 production.

BACKGROUND: Donor-specific blood transfusion (DST) may improve allograft survival in human and animal models, but the mechanisms for this graft protective effect are incompletely understood. The sponge matrix allograft model was used to determine if DST induces regulatory factors within the allograft. METHODS: C57BL/6 (H-2b) recipients received donor-specific (DBA/2J, H-2d) or syngeneic (C57BL/6) blood 7 days before sponge matrix allograft (DBA/2J) implantation. Fourteen days postgrafting, the sponge infiltrating cells (SIC) were examined for cytotoxic T cell (CTL) and natural killer (NK) activity, and sponge exudate fluid (SEF) was assessed for nitric oxide (.N=O) and prostaglandin E2 (PGE2) content. Interleukin- (IL) 2, IL-4, IL-10, and interferon-gamma (IFN-gamma) production by SIC was also determined. Recipient splenocytes were simultaneously assessed for anti-donor cytotoxic and proliferative responses and .N=O production. RESULTS: SIC from mice receiving syngeneic transfusions (ST) acquired both CTL and NK activity postgrafting, with maximal activity by day 14. DST suppressed both CTL and NK activity throughout the postgrafting period. Limiting dilution analysis (LDA) of SIC to determine precursor and native CTL frequency showed significantly lower responder cell frequency after DST compared with ST. SEF .N=O levels and SIC production of IL-2 and IFN-gamma in grafted DST mice were significantly lower than in grafted mice receiving ST. No significant amounts of IL-4 and very low levels of IL-10 were produced by SIC from grafted mice after either ST or DST. Conversely, PGE2 content of sponge fluid and serum from DST mice was higher than in mice receiving ST. Antigen stimulated splenocyte proliferation and CTL development assessed by LDA were also inhibited by DST. CONCLUSIONS: Reduction in local TH1 cytokines, absence of detectable TH2 cytokines, with enhanced PGE2 and depressed .N=O were observed in the local graft environment after DST. These data support the hypothesis that DST induces donor-specific intragraft suppressor factors, accompanied by reduced local and systemic immune activation.

Graft hyporeactivity induced by immature donor-derived dendritic cells.

Immature dendritic cells (DCs) are deficient in surface co-stimulatory molecules and have been shown to exhibit a 'tolerogenic' potential. We investigated the allostimulatory activity of immature DCs in one-way mixed leukocyte reactions and their capacity to inhibit anti-donor cytolytic activity in the sponge matrix allograft model. Immature DCs (CD80 and CD86 deficient) were derived from bone marrow cells propagated in GM-CSF and TGF-beta1. Mature DCs (CD80+ and CD86+) were derived from bone marrow cells propagated in GM-CSF and IL-4. Either 2 x 10(6) DBA/2J (DBA, H-2d) immature DCs or 2 x 10(6) mature DCs were injected intravenously into C57BL/6J (B6, H-2b) mice 7 days prior to sponge matrix allograft implantation. On day 12, the sponge was harvested and the graft-infiltrating cells were tested in vitro for cytotoxic T lymphocyte (CTL) activity. Immature dendritic cell (DC) infused significantly and markedly inhibited intra-graft CTL activity compared to mature DCs and syngeneic bone marrow control cells. The administration of immature DCs directly into the sponge allograft failed to induce hyporeactivity. Thus, the only systemic infusion of immature donor DCs was able to recapitulate the donor-specific transfusion effect, and the capacity of donor bone marrow cells to induce donor-specific hyporeactivity in the sponge allograft model.

Postnatal changes in [3H]fucosyl glycopeptides of hamster optic nerve synaptosomal membrane.

Axonally transported glycopeptides of the optic nerve synaptosomal membrane were labeled in neonatal and adult hamsters by intraocular injection of [3H]fucose. A prominent polypeptide in the 50,000 molecular weight region was heavily labeled after eye-opening in 16-day-old and adult hamsters, but not in 5- or 12-day-olds. The data suggest that either synthesis or fucosylation of a major synaptosomal membrane glycopeptide is under developmental control and expressed primarily after the eye-lids open.

Continuing damage to rat retinal DNA during darkness following light exposure.

The damaging effects of visible light on the mammalian retina can be detected as functional, morphological or biochemical changes in the photoreceptor cells. Although previous studies have implicated short-lived reactive oxygen species in these processes, the termination of light exposure does not prevent continuing damage. To investigate the degenerative processes persisting during darkness following light treatment, rats were exposed to 24 h of intense visible light and the accumulation of DNA damage to restriction fragments containing opsin, insulin 1 or interleukin-6 genes was measured as single-strand breaks (ssb) on alkaline agarose gels. With longer dark treatments all three DNA fragments showed increasing DNA damage. Treatment of rats with the synthetic antioxidant dimethylthiourea prior to light exposure reduced the initial development of alkali-sensitive strand breaks and allowed significant repair of all three DNA fragments. The time course of double-strand DNA breaks was also examined in specific genes and repetitive DNA. Nucleosomal DNA laddering was evident immediately following the 24 h light treatment and increased during the subsequent dark period. The increase in the intensity of the DNA ladder pattern suggests a continuation of enzymatically mediated apoptotic processes triggered during light exposure. The protective effects of antioxidant suggests that the light-induced DNA degradative process includes both early oxidative reactions and enzymatic processes that continue after cessation of light exposure.

Damage to rat retinal DNA induced in vivo by visible light.

Intense visible light can damage retinal photoreceptor cells by photochemical or thermal processes, leading to cell death. The precise mechanism of light-induced damage is unknown; however, oxidative stress is thought to be involved, based on the protective effect of antioxidants on the light-exposed retina. To explore the in vivo effects of light on retinal DNA, rats were exposed to intense visible light for up to 24 h and the time courses of single-strand breaks in restriction fragments containing the opsin, insulin 1 and interleukin-6 genes were measured. All three gene fragments displayed increasing single-strand modifications with increasing light exposure. Treatment with the antioxidant dimethylthiourea prior to light exposure delayed the development of net damage. The time course of double-strand DNA damage was also examined in specific genes and in repetitive DNA. The appearance of discrete 140-200 base-pair DNA fragments after 20 h of light exposure implicated a nonrandom, possibly enzymatic damaging mechanism. The generation of nucleosome core-sized DNA fragments, in conjunction with single-strand breaks, suggests two phases of light-induced retinal damage, with random attack on DNA by activated oxygen species preceding enzymatic degradation.

Histamine plays a major role for drinking elicited by spontaneous eating in rats.

The effects of combined antagonism of H1 (using 1 mg/kg dexbrompheniramine IP) and H2 (using 16 mg/kg cimetidine IP) receptors for histamine prior to (a) drinking after 2.5 mg/kg histamine SC, (b) drinking after 1-hr water deprivation, and (c) drinking during spontaneous eating were examined at 1 hr into the dark phase of a 12:12'-hr light/dark cycle for 14 Sprague-Dawley male rats. Such antagonism of histamine receptors abolished drinking elicited by exogenous histamine without affecting drinking after water deprivation. Histaminergic antagonism did not affect spontaneous eating, but it appeared to abolish drinking prior to a meal (for only those 3 rats which exhibited such drinking), delayed the latency to initiate drinking after initiating a meal, and inhibited drinking which occurred during and after eating but prior to postprandial resting (i.e., satiety for food). Because antagonism of peripheral histamine receptors inhibited food-related drinking by over 60%, these results provide indirect support for the hypothesis that the preabsorptive food-contingent vagally-mediated release of gastric mucosal histamine plays a major role in spontaneous food-related drinking in the rat.

Histamine-elicited drinking in weanling and adult rats.

A total of 260 male and female adult (60-70 days of age) and weanling (22-25 days of age) Sprague-Dawley derived rats were used in these experiments. Subcutaneous administration of histamine (HA) elicited drinking in a dose-dependent manner for both ages tested, although the threshold dose varied with age. A dose of 5.0 mg/kg HA elicited significant increases in water intake for adults, whereas for weanlings a dose of 20 mg/kg HA was necessary. Adult rats exhibited decreased latency to drink after all doses of HA tested, whereas for weanlings, decreased latency was evident only after doses of HA sufficient to elicit increases in water intake. Combined antagonism of H1 and H2 receptors for HA, using dexbrompheniramine and cimetidine, respectively, inhibited HA-elicited drinking in adults and weanlings. Further investigation of the ontogeny of histamine- and food-related drinking may provide a useful approach to examine the physiological mechanisms underlying fluid consumption in adult animals and as they are gradually elaborated during ontogeny.

p53 protein expression in human breast carcinoma: lack of prognostic potential for recurrence of the disease.

Mutations of the p53 gene are now known to be one of the most commonly detected genetic defects among human cancers. Because of its stability, the mutant p53 protein can be detected by immunohistochemical methods. Overexpression of the mutant p53 protein has been suggested as a prognostic indicator for the recurrence of breast cancer. Using a monoclonal antibody to p53, formalin-fixed, paraffin embedded breast cancer tissues retrieved from up to 10 years storage in the archival files were processed for staining. A total of 125 cases was examined p53 overexpression was identified by brown nuclear staining. Clinical parameters studied included estrogen and progesterone receptors, tumor size, nodal status, obesity, stage, and histopathological grade. The only significant association seen for p53 overexpression was with negative estrogen and progesterone receptors. All other clinical parameters studied were independent of p53 overexpression. Thus, p53 overexpression does not appear to be a useful prognostic indicator for recurrence and survival in human breast cancer.

Feasibility of testing DNA repair inhibitors for mutagenicity by a simple method.

A simple screening methodology for the determination of mutagenicity of DNA repair inhibitors has been tested in this laboratory. Radiation-resistant E. coli B/r and WP2 hcr+ and hcr- are suitable strains for mutagenicity testing. In these strains irradiated with 40-60 ergs/mm2, chemicals which interfere with repair of ultraviolet-induced pre-mutational lesions can be shown to enhance significantly the frequency of mutations to streptomycin resistance. This phenomenon is termed "mutational synergism" [18,20]. We have attempted to apply the procedure for securing data for "mutational synergism" between ultraviolet (UV) radiation and a number of antimalarial drugs including quinine hydrochloride (50 microgram/ml), quinine hydrobromide (50 microgram/ml), primaquine diphosphate (50 microgram/ml), chloroquine (50 microgram/ml), quinine (50 microgram/ml) and quinacrine dihydrochloride (25 microgram/ml). All drugs tested give synergistic effects with UV light. The synergistic activity ranges from 3- to 35-fold. Quinine and quinacrine dihydrochloride have been found to be much more efficient enhancers of the mutagenic effect of UV than caffeine. In general, we have found that the expression of synergistic action occurs at a concentration well below the minimum inhibitory concentration (MIC) with the drugs tested. The implication of these observations in the establishment of a screening method for the evaluation of the mutagenicity of DNA repair inhibitors is discussed.

Effect of high ionic strength and inhibitors of H,K-ATPase on the ouabain-sensitive K-p-nitrophenylphosphatase activity in the sea anemone Stichodactyla helianthus.

The ouabain-sensitive, K-stimulated p-nitrophenyl phosphatase (K-pNPPase) activity, an associated activity of the Na,K-ATPase, was assayed in tentacles of the sea anemone Stichodactyla helianthus to investigate the possibility that the sea anemone Na,K-ATPase activity is an associated activity of an H,K-ATPase. Activity was maximal at pH 6.5-7.0, decreasing only slightly in acidic medium but falling abruptly in alkaline medium to 60% of maximum at pH 7.4. The pH of maximum activity was not remarkably altered in high ionic strength medium (560 mM choline chloride), but ouabain-sensitive K-pNPPase activity of both rat and sea anemone was strongly inhibited. Inhibitors of the gastric H,K-ATPase, 100 microM omeprazole and 10 microM SCH 28080, did not inhibit the ouabain-sensitive K-pNPPase activity. Activity of the sea anemone enzyme was inhibited by 10 microM ammonium vanadate, an inhibitor of P-type ATPase, and not by 2.5 mM sodium azide, an inhibitor of both F-type and V-type ATPase. Because the sea anemone K-pNPPase activity was previously found to be more sensitive to ouabain than the Na,K-ATPase activity, K(+)-ouabain antagonism was investigated and found to be relatively muted, whereas K(+)-Na+ competition was stronger than in the rat kidney.

An ouabain-sensitive Na+,K(+)-ATPase in tentacles of the sea anemone Stichodactyla helianthus.

Tentacles of Stichodactyla helianthus contain an ouabain-inhibitable, (Na+,K+)-stimulated ATPase. The K0.5 for Na+ was 24 mM and for K+, 3.2 mM. The apparent affinity for ouabain was low, I50 = 10(-4) M. The order of cation affinities was Rb+ > K+ > NH4+ = Cs+. The catalytic subunit of the enzyme comprised a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, M(r) = 105 kDa, that was phosphorylated by [32P]ATP in the presence of NaCl and dephosphorylated by the addition of KCl. The alpha subunit was weakly reactive with antibodies directed against the rat alpha subunit.

Immunoperoxidase staining for estrogen and progesterone receptors in archival formalin fixed, paraffin embedded breast carcinomas after microwave antigen retrieval.

Immunoperoxidase staining was performed for estrogen and progesterone receptors in 93 cases of primary breast carcinoma. Breast tumor samples were fixed in formalin and embedded in paraffin. Antigen retrieval was performed by microwave heating in citrate buffer, pH 6.0, using precisely defined and reproducible conditions. The cases studied included material from the current year and from paraffin blocks retrieved from archival storage dating back to 1981. In all cases, estrogen and progesterone receptor values determined by biochemical assay were available for comparison with the immunohistochemical results. We found 94% agreement of results between the two methods.

Carcass composition of "bob" and "special-fed" veal and its prediction.

Percentage of lean, fat, and bone were determined in 18 bob veal (BV) and 28 special-fed veal (SFV) carcasses. Carcasses were subjected to a set of visual conformation scores and a variety of physical measurements. No significant differences were found regarding carcass percentage of lean, fat, and bone within the three BV weight groups (P > .05). On average, SFV were 12% fatter than BV and did not have a greater percentage of lean (P > .05), except for SFV carcasses weighing 88.2 to 97.7 kg. Bob veal had less fat (internal, external, and intermuscular) and a higher bone percentage than SFV (P < .05). The round and shoulder primals had the greatest proportion of lean in both the BV and SFV carcasses. Bob veal carcasses had an average conformation score of average Good and SFV carcasses had an average conformation score of average Choice. In addition, a parsimonious subset of variables was identified for predicting total percentage of lean (TPLEAN) for both BV and SFV separately, using "stepwise" regression model building procedures. For BV, all four identified predictor variables were subjective conformation scores (i.e., muscling, appearance, leg thickness, loin-back plumpness) (R = .73, P < .03). For SFV, four predictor variables were also identified: kidney and pelvic fat, fat thickness, carcass length, and lateral thickness (R = .61, P < .03). Although both regression equations were significant predictors of TPLEAN, confidence limits for predicting future TPLEAN value were wide relative to the variation in the actual TPLEAN values. Thus, the practical utility of the regression equations is limited.