Correlative near-infrared light and cathodoluminescence microscopy using Y2O3:Ln, Yb (Ln = Tm, Er) nanophosphors for multiscale, multicolour bioimaging.

This paper presents a new correlative bioimaging technique using Y2O3:Tm, Yb and Y2O3:Er, Yb nanophosphors (NPs) as imaging probes that emit luminescence excited by both near-infrared (NIR) light and an electron beam. Under 980 nm NIR light irradiation, the Y2O3:Tm, Yb and Y2O3:Er, Yb NPs emitted NIR luminescence (NIRL) around 810 nm and 1530 nm, respectively, and cathodoluminescence at 455 nm and 660 nm under excitation of accelerated electrons, respectively. Multimodalities of the NPs were confirmed in correlative NIRL/CL imaging and their locations were visualized at the same observation area in both NIRL and CL images. Using CL microscopy, the NPs were visualized at the single-particle level and with multicolour. Multiscale NIRL/CL bioimaging was demonstrated through in vivo and in vitro NIRL deep-tissue observations, cellular NIRL imaging, and high-spatial resolution CL imaging of the NPs inside cells. The location of a cell sheet transplanted onto the back muscle fascia of a hairy rat was visualized through NIRL imaging of the Y2O3:Er, Yb NPs. Accurate positions of cells through the thickness (1.5 mm) of a tissue phantom were detected by NIRL from the Y2O3:Tm, Yb NPs. Further, locations of the two types of NPs inside cells were observed using CL microscopy.

Evaluation of postoperative function in patients undergoing reconstruction following resection of superior and lateral oropharyngeal cancer: long-term outcomes of reconstruction with the Gehanno method.

Resection of the superior or lateral wall of the cancer-affected oropharynx can often lead to disturbed nasal breathing, dysphagia, and dysarthria. The authors used the Gehanno method to reconstruct these surgical defects and achieved favourable functional recovery soon after surgery. The present study was undertaken to analyze the long-term outcome and usefulness of this method. Reconstruction was carried out using the Gehanno method in 36 patients during the 10-year period between 1997 and 2007. Both short-term and long-term evaluations of the postoperative function were performed in 12 of the 36 cases. The postoperative function was favourably maintained in all 12 cases, but gradual deterioration was noted in some cases in which the forearm flap had been used for reconstruction. Conventionally, the forearm flap is considered suitable for the reconstruction of complex structures such as the oropharynx because of its excellent flexibility. The results suggest that if the forearm flap is used for reconstruction using the Gehanno method, the surrounding tissue is likely to undergo change over time. The rectus abdominis myocutaneous flap should be considered as the first-choice flap for reconstruction using the Gehanno method.

Isolation, evaluation and use of two strong, carbon source-inducible promoters from Corynebacterium glutamicum.

AIMS: To obtain strong, carbon source-inducible promoters useful for industrial applications of Corynebacterium glutamicum. METHODS AND RESULTS: DNA microarray and qRT-PCR enabled identification of the promoters of cgR_2367 (malE1) and cgR_2459 (git1) as strong, maltose- and gluconate-inducible promoters, respectively, in C. glutamicum. Promoter probe assays revealed that in the presence of the inducing sugars, PmalE1 and Pgit1, respectively, facilitated 3.4- and 4.2-fold increased beta-galactosidase activities compared to the same activity induced by glucose. In addition, PmalE1 was not functional in Escherichia coli, in which Pgit1 function was repressible, which enabled the cloning of a hitherto 'difficult-to-clone' heterologous gene of a lignocellulolytic enzyme, whose secretion was consequently induced by the carbon sources. CONCLUSIONS: PmalE1 and Pgit1 are strong, carbon source-inducible promoters of C. glutamicum whose characteristics in E. coli are integral to the secretion ability of C. glutamicum to secrete lignocellulolytic enzyme. SIGNIFICANCE AND IMPACT OF THE STUDY: Corynebacterium glutamicum, like its counterpart industrial workhorses E. coli and Bacillus subtilis, does exhibit strong, carbon source-inducible promoters, and the functionality of two of which was demonstrated in this study. While this study may be most relevant in the ongoing efforts to establish technologies of the biorefinery, it should also be of interest to general microbiologists exploring the versatility of industrial micro-organisms. In so doing, the study should impact future advances in industrial microbiology.

Risk factors for dietary variety decline among Japanese elderly in a rural community: a 8-year follow-up study from TMIG-LISA.

OBJECTIVE: To examine the factors related to the decline of dietary variety among the rural community-dwelling Japanese elderly people and the implication on the planning of elderly people's nutritional improvement program in the future. DESIGN: A prospective cohort study during 8-year follow-up from 1992 to 2000. SETTING: This study was conducted in Nangai Village, a rural and mainly agricultural area of Akita Prefecture in the northern part of Honshu, one of four main islands in Japan. SUBJECTS: A total of 417 elderly people (160 men, 257 women) who completed interviews and food intake frequency surveys conducted in 1992, 1994, 1996, 1998, and 2000 were studied. METHODS: Dietary variety and variables potentially associated with dietary variety decline were identified from a face-to-face interview at the baseline and 8-year follow-up surveys. The dietary variety was measured using the dietary variety score (DVS), which covers the 10 main food groups in Japanese meals. RESULTS: During the 8-year follow-up, 36.2% of the subjects showed a decline in dietary variety. Health characteristics also change among the 8-year follow-up and these changes have an effect on the decline of dietary variety. Significant predictors for decline in dietary variety included loss of spouse, deterioration in self-perceived chewing ability, and decrease in intellectual activity score. CONCLUSIONS: Loss of spouse, deterioration in chewing ability, and decline in intellectual activity may increase the risk of decline in dietary variety in community-dwelling Japanese elderly people.

Acute effects of glucocorticoids on ATP-induced Ca2+ mobilization and nitric oxide production in cochlear spiral ganglion neurons.

Rapid, non-genomic effects of glucocorticoids on extracellular adenosine 5'-triphosphate (ATP)-induced intracellular Ca(2+) concentration ([Ca(2+)](i)) changes and nitric oxide (NO) production were investigated in type I spiral ganglion neurons (SGNs) of the guinea-pig cochlea using the Ca(2+)-sensitive dye fura-2 and the NO-sensitive dye 4,5-diaminofluorescein (DAF-2). Pretreatment of SGNs with 1 microM dexamethasone for 10 min, a synthetic glucocorticoid hormone, enhanced the ATP-induced [Ca(2+)](i) increase in SGNs. RU 38486, a competitive glucocorticoid receptor antagonist eliminated the effects of dexamethasone on the ATP-induced [Ca(2+)](i) increase in SGNs. These acute effects of dexamethasone were dependent on the presence of extracellular Ca(2+), thereby suggesting that dexamethasone may rapidly enhance the Ca(2+) influx through the activation of ionotropic P2X receptors which may interact with glucocorticoid-mediated membrane receptors. Extracellular ATP increased the intensity of DAF-2 fluorescence, indicating NO production in SGNs. The ATP-induced NO production was mainly due to the Ca(2+) influx through the activation of P2 receptors. S-nitroso-N-acetylpenicillamine, a NO donor, enhanced the ATP-induced [Ca(2+)](i) increase in SGNs while L-N(G)-nitroarginine methyl ester (L-NAME), a NO synthesis inhibitor, inhibited it. Dexamethasone enhanced the ATP-induced NO production in SGNs. The augmentation of dexamethasone on ATP-induced NO production was abolished in the presence of l-NAME. It is concluded that the ATP-induced [Ca(2+)](i) increase induces NO production which enhances a [Ca(2+)](i) increase in SGNs by a positive-feedback mechanism. Dexamethasone enhances the ATP-induced [Ca(2+)](i) increase in SGNs which results in the augmentation of NO production. The present study suggests that NO may play an important role in auditory signal transduction. Our results also indicate that glucocorticoids may rapidly affect auditory neurotransmission due to a novel non-genomic mechanism.

Inhibitor-associated transposition events in Corynebacterium glutamicum.

In up to 100% of all bacteria grown in the presence of initially inhibitory concentrations of five diverse inhibitors, an extra copy of the resident insertion element IS 31831 was found in specific chromosomal regions, the sites of which apparently depended on the inhibitor used. Thus, in nine out of nine independently isolated cyanide-associated transpositions, the acquired copy was located within an ORF encoding a protein related to the hypothetical but conserved protein YeiH of Escherichia coli. A putative Sox box upstream of the yeiH gene implicates superoxide as a potential regulator of the gene, a possibility further supported by the finding that superoxide dismutase (SodA) is overexpressed in cells cultured in cyanide-containing medium. Neither the cyanide-associated nor any of the other transposition mutations appeared to confer any discernible phenotypic advantage upon cells grown in the presence or absence of the inhibitors, as revealed most stringently by mixed-cell experiments. An alternative, albeit heterodox, explanation for the emergence of the mutants postulates a very high rate of transpositional activity in the presence of inhibitors. The initial emergence of the mutants was found to depend crucially upon the cell density. Thus, when growth medium was supplemented with 50 mM fluoropyruvate and inoculated to a density of 2 x 10(7) cfu/ml, single colonies with heterogeneous restriction fragment length polymorphisms (RFLPs) were routinely isolated at a frequency of 6 to 16% after 1-2 days of incubation. After 3 days, 10-36% of the colonies showed RFLPs, but the type was now dominated by the fluoropyruvate-specific RFLP, which, at higher resolution, invariably proved to be heterogeneous. This heterogeneity proved that these specific mutants were of multiple origin, indicating that clonal enrichment was irrelevant to their emergence. It is suggested that the presence of the inhibitor induces the development of hyper-transpositional activity, which is regulated by a soluble bacterial product.

Effect of calcium antagonist on cerebral blood flow and oxygen metabolism in patients with hypertension and chronic major cerebral artery occlusion: a positron emission tomography study.

To evaluate the effect of a calcium antagonist, nilvadipine, on cerebral blood flow and oxygen metabolism, we prospectively examined five ischaemic stroke patients, with both hypertension and chronic major cerebral artery occlusion, using positron emission tomography. The blood pressure showed a significant decrease after 3 months of nilvadipine treatment, the cerebral blood flow in the affected regions showed a significant increase and the oxygen extraction fraction showed a significant decrease. We conclude that nilvadipine is a safe and effective anti-hypertensive agent for patients with both hypertension and chronic major cerebral artery occlusion.

The ptsI gene encoding enzyme I of the phosphotransferase system of Corynebacterium glutamicum.

The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is widespread among bacteria where it mediates carbohydrate uptake and often serves in carbon control. Here we present cloning and analysis of the monocistronic ptsI gene of Corynebacterium glutamicum R, which encodes PTS Enzyme I (EI). EI catalyzes the first reaction of PTS and the reported ptsI was shown to complement the corresponding defect in Escherichia coli. The deduced 59.2-kDa EI of 564 amino acids shares more than 50% homology with EIs from Bacillus stearothermophilus, Bacillus subtilis, and Lactobacillus sake. Chromosomal inactivation of ptsI demonstrated that EI plays an indispensable role in PTS of C. glutamicum R and this system represents a dominant sugar uptake system. Cellobiose was only transported and utilized in adaptive mutants of C. glutamicum R. Cellobiose transport was also found to be PTS-dependent and repressed by PTS sugar glucose.

[Incidence and prognosis of, and risk factors for the home-bound in a community elderly population].

This longitudinal study examined numbers of homebound and bed bound individuals over time, and risk factors in a rural community elderly populations. A total of 731 older persons who needed no assistance of another person in traveling from their homes participated in the baseline survey in 1992 and were assessed annually for their ability to travel for subsequent six-years. We defined operationally a homebound status as the necessity for assistance of another person to be able to leave home, but without being bedbound. During the follow-up period, 86 homebound (23.7 per 1,000 person-years) and 32 bedbound cases (8.4 per 1,000 person-years) were noted, the former being 2.8 times more likely to occur. Of the newly bound cases 26% returned to a state capable of leaving home by themselves one year later, and maintained this independent living level even two years later. Of the other cases who remained as homebound or bedbound one year later, only one (1/31) had returned to independent living after two years; most of them became bedbound at home, hospital admitted or deceased. Stepwise multiple logistic regression analysis showed that independent risk factors for the homebound include older age, male gender, slower walking speed, history of hospital admission in the previous year, lower masticatory ability, and higher level of serum beta 2-microglobin. These results indicate that health promotion programs for prevention of becoming homebound among community elderly people should target more intensively assistance to maintaining walking and masticatory ability. In addition, public health professionals should pay more attention to elderly people shortly after hospital discharge.

Common solvent toxicity: autoxidation of respiratory redox-cyclers enforced by membrane derangement.

Unspecific biological effects of chemically diverse solvents strikingly reveal the unifying motif of oxidant toxicity both in higher organisms and in aerobic bacteria. In a few spectacular cases, solvent metabolites with oxidant properties were demonstrated, which however cannot explain extrahepatic toxicity, e.g. in muscle and nerve cells. A common source of solvent-inducible oxidants, by contrast, is suggested to be located in mitochondria or, more general, in membranes where the respiratory chain operates. Orderly respiration depends on membrane integrity, which is invariably compromised by exposure to most solvents and many other lipophils. In rat mitochondria, toluene-induced membrane derangement has been directly implicated with superoxide production, resulting from autoxidation of the membrane-located respiratory redox-cycler ubisemiquinone. A related mechanism may occur in bacteria: Exposure of Escherichia coli to lipophils such as ethanol, tetralin, indole, chlorpromazine and procaine, or to heat shock, induces anti-oxidant proteins, which are reliable indicators of increased oxidant levels. Although many molecular details remain to be elucidated, this review documents that oxidant toxicity of lipophilic compounds is a common physiological phenomenon correlated with derangement of membranes where respiratory processes take place. Subjective consequences of acute oxidant injury are probably the hangover from alcohol and nicotine consumption, and the sudden death from recreational solvent abuse. Suggestions concerning oxidants as major contributors to ageing remain unchallenged.

Structure, expression and products of the ribosomal RNA operons of Rhodopseudomonas palustris No. 7.

Rhodopseudomonas palustris strains carry one or two ribosomal rRNA operons, and those with duplicated rrn operons grow faster. The two rrn operons in R. palustris No. 7 are virtually identical over a 54,70-bp stretch containing the genes for 16S rRNA, tRNAile, tRNAala, 23S rRNA and 5S rRNA, as well as the intergenic spacers and part of the extragenic spacer. In R. palustris, unlike most bacteria with multiple rrn operons, the putative promoter sequences of the two operons are highly diverged, suggesting possible functional differentiation. By simultaneous primer-extension analysis of both pre-rRNAs, we detected a two-fold higher level of expression from rrnA under photoautotrophic conditions. Alteration of the conditions of growth leads to changes in the relative levels of expression of the two operons. Within the 5,470-bp segment, only two sequence differences are found between the 23S rRNA genes; one is at the center of the 23S rRNA molecule and affects a site of unknown function, and the other is within or immediately adjacent to sequences involved in processing of the 5' 23S rRNA IVS. In vitro processing of 5' IVS-containing 23S rRNA precursors from each operon does not reveal any detectable difference between them. The 5' ends of the mature 16S, 23S, and 5S rRNAs were determined by primer-extension analysis, and the 3' end of 23S rRNA was determined by RNA linker ligation-mediated cDNA cloning. The 5' and 3' ends of the R. palustris 23S rRNA molecule are extensively processed, suggesting that, unlike the situation in the established eubacterial model, these ends cannot basepair.

[Clinical study on prognostic factors in thyroid carcinoma].

We treated 227 patients, 45 men and 182 women, with thyroid carcinoma at our hospital from 1984 to 1998. Of these, 177 had papillary carcinoma and 50 follicular carcinoma. The extent of resection was based on tumor size in papillary carcinoma but not follicular carcinoma, and 70% of carcinoma patients underwent hemithyroidectomy. Neck lymph nodes were resected in 93.2% of papillary carcinoma patients, with D1 neck dissection in 45.7% and D2 or D3 neck dissection in 47.5%. In contrast, 70% of follicular carcinoma patients with lymph node resection had D1 dissection. Locoregional recurrence was noted in 22 patients and distant metastasis in 6 cases. Nonsurvivors numbered 17, 12 papillary and 5 follicular carcinoma patients, died of their primary disease. Almost all deaths were in patients with advanced disease, pT3 in 3, pT4 in 10, N1a in 3 and N1b in 8. The prognostic factors for papillary carcinoma were extracapsular spread, age, and distant metastasis, while the only factor for follicular carcinoma was distant metastasis. The 5-year survival for patients with papillary carcinoma was 93.0% and 10-year survival 88.8%, compared to 5-year survival for 93.5% of follicular carcinoma patients and 10-year survival for 93.5%.

Blood flow and oxygen metabolism in a case of Lhermitte-Duclos disease: results of positron emission tomography.

We evaluated blood flow and oxygen metabolism in a patient with Lhermitte-Duclos disease using brain positron emission tomography. The lesion showed increased blood flow, reduced oxygen extraction fraction and normal metabolic rates of oxygen. These findings suggest that lesions of Lhermitte-Duclos disease may constitute hamartoma or hypertrophy rather than neoplasm.

[Dietary calcium and women's health].

Among the many possible contributors to primary osteoporosis, calcium deficiency is currently considered to be most important. The average calcium intake of the Japanese, by national nutrition survey, however, is now around 568 mg/day which is much lower in women than the recommended dietary allowances of calcium proposed by the Ministry of Health and Welfare, and than those in western countries. Milk is a food with a high ratio of nutrients to energy, and it is also primarily identified as an excellent source of calcium. The significance of dietary calcium intake and milk consumption for bone health of women should be provided by mass health education and individual guidance in a practice of public health.

Bacterial phosphotransferase system (PTS) in carbohydrate uptake and control of carbon metabolism.

More than 20 carbohydrates may be transported into the bacterial cell by the phosphoenopyruvate:carbohydrate phosphotransferase system (PTS) that is widely spread among bacteria. The PTS consists of two cytoplasmic energy-coupling proteins (Enzyme I and HPr) and a range of carbohydrate-specific Enzymes II, which catalyze concomitant carbohydrate translocation and phosphorylation. The phosphorylation status of PTS components reflects the availability of carbohydrates and the energy conditions of the cell. In many bacteria, PTS and the associated proteins convert this information to signals, which transduced through different mechanisms lead to phenomena of catabolite repression, inducer control or chemotaxis. These features of PTS provide bacteria with an integrated system, which assures optimal utilization of carbohydrates in complex environments. Furthermore, some bacteria evolved parallel systems that serve a regulatory functions, but apparently do not catalyze the carbohydrate transport. Here we review the findings that recently advanced the understanding of various aspects of PTS-dependent carbohydrate transport and regulation of bacterial catabolism.

[A case of left occipital lobe hemorrhage in a patient with progressive systemic sclerosis: evaluation of cerebral angiography and histology].

Involvement of the central nervous system is uncommon in progressive systemic sclerosis, with only 2 reported cases associated with intracerebral hemorrhage detected by neuroimaging. A 55-year-old woman with a 10-year history of scleroderma presented with left occipital lobe hemorrhage manifesting as headache and vomiting. She had no signs of hypertension, diabetes mellitus and hyperlipidemia. CT and MRI, on admission, showed left occipital lobe hemorrhage with ventricular rupture and acute left subdural hematoma. Serial cerebral angiography was performed on day 0, day 7 and day 14, and found no evidence of aneurysm, arteriovenous multiformation or tumor stain in the left occipital lobe. However, the bilateral anterior cerebral arteries showed increasing segmental narrowing suggestive of vasculitis. Histological examination of a section from the brain cortex adjacent to the hemorrhage revealed no evidence of vasculitis, fibrinoid degeneration or amyloid deposition. Focal vasculitis may have occurred secondary to the homorrhagic lesion.

Walking speed as a good predictor for the onset of functional dependence in a Japanese rural community population.

OBJECTIVE: to investigate and compare the predictive values of four physical performance measures for the onset of functional dependence in older Japanese people living at home. DESIGN: a population-based prospective cohort study. SETTING: Nangai village, Akita Prefecture, Japan. METHODS: out of the population aged 65 years and older living in Nangai (n = 940) in 1992, we measured hand grip-strength, one-leg standing, and usual and maximum walking speeds in 736 subjects who were independent in the five basic activities of daily living. Their functional status was assessed each year for the subsequent 6 years. The outcome event was the onset of functional dependence, defined as a new disability in one or more of the five basic activities of daily living, or death of a subject who had shown no disability at the previous follow-up. RESULTS: even after controlling for age, sex and a number of chronic conditions, lower scores on each baseline performance measure showed increased risk for the onset of functional dependence. Maximum walking speed was most sensitive in predicting future dependence for those aged 65-74 years, while usual walking speed was most sensitive for people aged > or = 75 years. CONCLUSION: walking speed was the best physical performance measure for predicting the onset of functional dependence in a Japanese rural older population.

Divergent mechanisms of 5' 23S rRNA IVS processing in the alpha-proteobacteria.

Widespread occurrence of a separate small RNA derived from the 5'-end of 23S rRNA and of an intervening sequence (IVS) which separates this domain from the main segment of 23S rRNA in the alpha-proteobacteria implies that processing reactions which act to excise the IVS are also maintained in this group. We previously characterized the first example of processing of this IVS in Rhodopseudomonas palustris, which is classified with the Bradyrhizobia In this case, IVS excision occurs by a multistep process and RNase III appears to act at an early step. Here, we characterize in vivo and in vitro IVS processing in two other related, but phenotypically distinct, Bradyrhizobia We also examine in vivo and in vitro processing of rRNA precursors from a more distantly related alpha-proteobacterium, Rhodobacter sphaeroides which produces a separate 5' 23S rRNA domain but has different sequences in the 5' 23S rRNA IVS. The details of the in vivo processing of all of the Bradyrhizobial rRNAs closely resemble the R. palustris example and in vitro studies suggest that all of the Bradyrhizobia utilize RNase III in the first step of IVS cleavage. Remarkably, in vivo and in vitro studies with R.sphaeroides indicate that initial IVS cleavage uses a different mechanism. While the mechanism of IVS cleavage differs among these alpha-proteobacteria, in all of these cases the limits of the internal segments processed in vivo are almost identical and occur far beyond the initial cleavage sites within the IVSs. We propose that these bacteria possess common secondary maturation pathways which enable them to generate similarly processed 23S rRNA 5'- and 3'-ends.

Selenium and glutathione peroxidase mRNA in rat glioma.

The purpose of current study was to determine the step at which dietary selenium (Se) regulates the transcriptional expression of the gene for Se-glutathione peroxidase (Se-GPx) in rat brain and transplanted glioma tissue. Wistar rats were fed a Se-free diet or the same diet supplemented with 0.5, 2.0, and 4.0 mg Se/kg as sodium selenite for at least 3 wk. Then, the rats were transplanted with C6 rat glioma cells into the right frontal lobe parenchyma. All rats were observed for 30 d, then tumor and contralateral brain tissue were excised and divided into three portions for purification of selenium content, for the assay of Se concentration, Se-GPx activity, and for Se-GPx mRNA. Se concentration and Se-GPx activity are increased with Se supplementation both in tumor tissue and contralateral brain tissue, and Se concentration in tumor is higher than that in contralateral brain tissue at each dietary Se content. Se-GPx mRNA of brain and tumor were probed with fragments from a rat Se-GPx cDNA in Northern blot analysis. There was significant differences of Se-GPx mRNA transcription in brain tumor tissue among each dietary group of the Se content, and the steady-state level of Se-GPx mRNA was markedly reduced by Se deficiency. These results suggest that dietary Se exerts its augmenting effect on Se-GPx gene transcription.

Analysis of oxidation sensitivity of maleate cis-trans isomerase from Serratia marcescens.

The maleate cis-trans isomerase gene (maiA) from Serratia marcescens IFO3736 was cloned and sequenced. Serratia MaiA has 62.4% amino acid identity with Alcaligenes faecalis IFO13111 MaiA and 64.9% with Bacillus stearothermophilus MI-102 MaiA. All known ten amino acid sequences of MaiA had significant conserved regions containing cysteine residues, which were previously suggested to be involved in an active site of the enzyme. The maiA gene was expressed in Escherichia coli, and expressed products MaiA was purified and characterized. The purified enzyme of strain IFO3736 showed high activity at room temperature and high heat stability. It also showed higher activity in the presence of high concentration of aspartic acid than the enzyme of A. faecalis IFO13111, but it was also sensitive to chemical oxidation. By amino acid composition analysis, cysteine, methionine, and tyrosine residues were suggested to be oxidized to inactivate the enzyme by chemical oxidation. To investigate the mechanism of chemical oxidation of the enzyme, six methionine residues in the conserved regions of S. marcescens MaiA were replaced with cysteine residues by site-directed mutagenesis. The analysis of the constructed mutants suggested that the Met201 residue near the Cys198 residue is involved in the sensitivity of the enzyme to chemical oxidation.