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Single- and double-strand breaks in solid pBR322 DNA induced by ultrasoft X-rays at photon energies of 388, 435 and 573 eV.

We measured strand breaks of pBR322 plasmid DNA irradiated with ultrasoft X-rays using monochromatic synchrotron radiation as a light source. Three photon energies, 388, 435 and 573 eV, a value below and above the nitrogen K-edge and above the oxygen K-edge, respectively, were chosen for the irradiation experiments as they have an equivalent photon transmittance of the sample. Irradiated DNA was analyzed by agarose gel electrophoresis and the numbers of single- and double-strand breaks (ssb and dsb) were determined by measuring the band intensity on the gel after ethidium bromide staining. The action cross-sections for the ssb and dsb slightly increased with the photon energy. The ratio between 388 and 573 eV was about 1.5 for both forms of strand breaks. The absorbed energy required for a strand break was about 60 eV for ssb and 1 keV for dsb, less than one fifth of the values obtained previously in the 2 keV region. On the other hand, the absorbed energies per strand break, as well as the ratio of the action cross-section for the ssb to that for the dsb, were constant regardless of the photon energy used. The K-shell photoabsorption on carbon, nitrogen and oxygen atoms in the DNA molecule, followed by an Auger cascade, induced DNA strand breaks with a constant efficiency in terms of the absorbed energy. These results indicate that the strand breaks of the DNA molecule in the solid state are mainly caused by the photo- and Auger-electrons and the efficiency of the strand breaks little depends on the atoms ejecting these secondary electrons.

DNA↗

8-hydroxy-2'-deoxyguanosine, a major mutagenic oxidative DNA lesion, and DNA strand breaks in nasal respiratory epithelium of children exposed to urban pollution.

Southwest metropolitan Mexico City children are repeatedly exposed to high levels of a complex mixture of air pollutants, including ozone, particulate matter, aldehydes, metals, and nitrogen oxides. We explored nasal cell 8-hydroxy-2'-deoxyguanosine (8-OHdG), a major mutagenic lesion producing G-->T transversion mutations, using an immunohistochemical method, and DNA single strand breaks (ssb) using the single cell gel electrophoresis assay as biomarkers of oxidant exposure. Nasal biopsies from the posterior inferior turbinate were examined in children in grades one through five, including 12 controls from a low-polluted coastal town and 87 Mexico City children. Each biopsy was divided for the 8-OHdG and DNA ssb assays. There was an age-dependent increase in the percentage of nasal cells with DNA tails > 10 microm in Mexico City children: 19 +/- 9% for control cells, and 43 +/- 4, 50 +/- 16, 56 +/- 17, 60 +/- 17 and 73 +/- 14%, respectively, for first through fifth graders (p < 0.05). Nasal ssb were significantly higher in fifth graders than in first graders (p < 0.05). Higher levels (2.3- to 3-fold) of specific nuclear staining for 8-OHdG were observed in exposed children as compared to controls (p < 0.05). These results suggest that DNA damage is present in nasal epithelial cells in Mexico City children. Persistent oxidative DNA damage may ultimately result in a selective growth of pr eneoplastic nasal initiated cells in this population and the potential for nasal neoplasms may increase with age. The combination of 8-OHdG and DNA ssb should be useful for monitoring oxidative damage in people exposed to polluted atmospheres.

8-Hydroxy-2'-Deoxyguanosine↗

Attributional style, defensive functioning and persecutory delusions: symptom-specific or general coping strategy?

OBJECTIVE: Previous research has suggested that individuals with persecutory delusions use an exaggerated self-serving bias to protect themselves from real or delusional threats to their underlying self-concept. However, the research to date has been inconsistent thereby limiting the conclusion that can be inferred. A possible explanation for these discrepant findings may be due to the use of measures with questionable validity and reliability such as the Attributional Style Questionnaire and the Rosenberg Self-Esteem Inventory. The present study aims to re-examine this theory using an improved methodology to determine whether defensive functioning is a defining feature of persecutory delusions. DESIGN AND METHODS: Thirty-five participants suffering from recent onset psychosis were assessed in a cross-sectional design. Three tests of attributional style were used, two overt measures (Attributional Style Questionnaire, ASQ; and the Internal Personal and Situational Attributions Questionnaire, IPSAQ) and one covert measure (Pragmatic Inference Test). Self-esteem was measured using the Rosenberg Self-Esteem Inventory (SEI) and a semi-structured interview (Self-Evaluation and Social Support interview - schizophrenia version). RESULTS: A self-serving bias (SSB) was found in the total sample using the ASQ but not specifically in participants with persecutory delusions. In addition, the SSB was unrelated to levels of paranoia on any measure of attributional style. An underlying depressive attributional style was found in both subjects with and without persecutory delusions, but was not associated with levels of paranoia. Lower positive and higher negative self-esteem was associated with increased paranoia and was associated with a SSB. CONCLUSION: The results indicate that the attributional biases observed in individuals with persecutory delusions are not symptom-specific as previously suggested. The presence of an attributional, SSB was associated with lower psychotic experiences. The theoretical implications of the results will be discussed.

Adaptation, Psychological↗

Effect of different bur grinding on the bond strength of self-etching adhesives.

This study compared the microtensile bond strength (MTBS) of three all-in-one adhesive systems and a two-step system using two types of burs to prepare the dentin surfaces. Flat coronal surfaces of 24 extracted human molars were produced using either regular-grit or superfine-grit diamond burs. Resin composite was then bonded to equal numbers of these surfaces using one of the four adhesives: Clearfil SE Bond (CSE), G-Bond (GB), SSB-200 (SSB) or Prompt L-Pop (PLP). After storage for 24 hours in 37 degrees C distilled water, the bonded teeth were sectioned into slices (0.7-mm thick) perpendicular to the bonded surface. The specimens were then subjected to microtensile testing and the bond strengths were calculated at failure. Bond strength data were analyzed by two-way ANOVA and the GamesHowell test for interaction between adhesive and type of cut dentin. The fractured surfaces were observed by SEM to determine the failure mode. In addition, to observe the effect of conditioning, equal numbers of the two bur-cut dentin surfaces of eight additional teeth were conditioned with the adhesives and observed by SEM. Based on the results, when CSE and SSB were bonded to dentin cut with a regular-grit diamond bur, the MTBS values were significantly lower than that of superfine bur-cut dentin; whereas, GB and PLP showed no significant differences in MTBS between the two differently cut surfaces. SEM observation of the fractured surfaces revealed a mixed mode (adhesive in some areas and cohesive in others in the same sample) of failure in all specimens except PLP, which showed cohesive failure within the adhesive for both types of bur preparation. Generally, SEMs of the conditioned surfaces using both types of burs showed partial removal of the smear layer for CSE, minimal for GB and SSB and complete removal for PLP. In conclusion, when cutting dentin, selecting the proper bur type is important for improving the bond strength of some self-etching adhesive systems.

Adhesiveness↗

Effectiveness of two cooking systems in destroying Escherichia coli O157:H7 and Listeria monocytogenes in ground beef patties.

A rapid, high-temperature double-sided grilling-broiling (DGB) system was compared to a single-sided broiling (SSB) system for cooking of foodservice ground beef patties to reduce microbial numbers and maintain textural quality. Patties (110 g) containing either Escherichia coli O157:H7 or Listeria monocytogenes (10(6-7) CFU/g) were cooked to target internal temperatures of 60 or 68 degrees C on each cooking system and immediately removed from the grills without the additional holding time at 60 or 68 degrees C that is recommended for foodservice cooking of ground beef patties. Actual final internal temperature attained, position on the grill, degree of doneness, cooking time, after-cook weight, texture characteristics, and bacterial counts of the patties were monitored. The DGB reduced E. coli O157:H7 and L. monocytogenes populations in ground beef patties by 5.7 log10 and 5.4 log10 CFU/g, respectively, when cooked to a target temperature of 60 degrees C (actual final internal temperature of 71.2 degrees C) and by 6.1 log10 and 5.6 log10 CFU/g, respectively, when cooked to a target temperature of 68 degrees C (actual final internal temperature of 75.8 degree C). The SSB reduced E. coli O157:H7 and L. monocytogenes populations by 1.3 log10 and 1.8 log10 CFU/g, respectively, when cooked to a target temperature of 60 degrees C (actual final internal temperature of 62.7 degrees C) and by 2.9 log10 and 3.6 log10 CFU/g, respectively, when cooked to a target temperature of 68 degrees C (actual final internal temperature of 69.3 degrees C). The DGB system effected a higher, more rapid temperature increase in patties cooked to either target temperature compared to the SSB system. This higher temperature was more effective in destroying pathogens in beef patties. Texture analyses determined that patties cooked on the DGB system had significantly higher values for springiness, adhesiveness, and product height as compared to the SSB system, and patties cooked on either system had significantly higher hardness, gumminess, chewiness, and product height values at the target temperature of 68 degrees C as compared to 60 degrees C.

Animals↗

The soluble eumelanin precursor 5,6-dihydroxyindole-2-carboxylic acid enhances oxidative damage in human keratinocyte DNA after UVA irradiation.

Soluble melanin precursors are present in serum and may act as skin chromophores contributing to UVR-induced oxidative damage. Our study aimed to determine whether the soluble eumelanin precursor 5,6-dihydroxy-indole-2-carboxylic acid (DHICA) photosensitizes DNA damage in human keratinocytes exposed to UVA irradiation. The HaCaT keratinocytes were incubated with and without DHICA, before irradiation with broadband UVA (320-400 nm). The DNA photodamage was assessed using the comet assay that detects frank single-strand breaks (SSB) and specific oxidative lesions with the addition of endonuclease III. Without DHICA incubation, there was no significant increase in SSB, compared to unirradiated cells, for doses up to 48.5 J/cm2 (< 1 minimum erythemal dose). Preincubation with 0.5 microM DHICA caused an increase in SSB at every UVA dose (significant from 12.1 to 48.5 J/cm2), while varying the DHICA concentration (0.125-2 microM) showed this effect to be concentration dependent such that SSB increased and endonuclease III-sensitive sites decreased with increasing DHICA concentration. The irradiation of cells in the presence of antioxidants (catalase, mannitol and histidine) suggests that DHICA-induced photosensitization is mediated via singlet oxygen and, to a lesser extent, hydroxyl radicals. These results indicate that DHICA can induce strand breaks with UVA at clinically relevant doses via a mechanism involving reactive oxygen species.

Antioxidants↗

Use of single cell gel electrophoresis (comet assay) modifications for analysis of DNA damage.

Single cell gel electrophoresis (SCGE) or comet assay is a rapid and sensitive fluorescent microscopic method which allows measurement of DNA strand breaks in individual cells. Modifications of SCGE conditions permitted to detect different types of DNA damage. In order to characterize DNA damage induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and hydrogen peroxide (H2O2) in Chinese hamster V79 cells, two approaches were used: (1) two pH values of unwinding and electrophoresis solutions (pH > or = 13.0 and pH = 12.1) to specify the type of DNA lesions [the alkali-labile sites and true DNA single-strand breaks (ssb)] and (2) DNA glycosylases [endonuclease III (EndoIII) and formamidopyrimidine-DNA glycosylase (FaPy)] or DNA inhibitors [hydroxyurea (HU) + 1-(beta-D-arabinofuranosyl)cytosine (AraC)] to characterize the types of DNA damage. Our results showed that the lesions induced by H2O2 represented mainly the true DNA ssb, while MNNG formed predominantly alkali-labile sites, which were converted to DNA ssb under strong alkaline conditions (pH > or = 13.0). The effects of DNA repair enzymes and DNA inhibitors were more significant under lower pH (pH = 12.1) of unwinding and electrophoresis solution. Both, DNA glycosylases and DNA inhibitors increased the level of DNA ssb.

Animals↗

Three-shell head model constructed from scalp geometry for electroencephalogram dipole localization.

One of the most sophisticated methods of dipole localization from (electroencephalograms) is based on a multi-compartment head model, which consists of the scalp, skull, cerebrospinal fluid (CSF) and brain cortex. The CSF layer in normal subjects is thin and can be ignored in dipole localization to good approximation. This multi-shell model is called the SSB (scalp-skull-brain) head model. The SSB head model is constructed from magnetic resonance imaging (MRI) or X-ray computed tomography slices of the head. These image slices, however, are not easy to obtain. The present paper presents a simple method for constructing the SSB head model only from the geometrical shape of the scalp, which is easily measured with a three-dimensional digitizer. We have derived a relationship between the head structure and the geometry of the head from statistical analysis of MRI slices of 12 normal subjects. The average estimation errors of the outer and inner boundaries of the skull were 1.2 and 2.2 mm, respectively. The head model thus estimated is called the sSSB (statistical SSB) model.

Adult↗

Enhanced strand break induction of DNA by resonant metal-innershell photoabsorption.

We determined the number of single and double strand breaks (ssb and dsb) in a DNA-chloroterpyridine platinum complex induced by resonant photoabsorption in the L(III) innershell of a platinum atom. The number of ssb and dsb were measured in supercoiled plasmids (AG30) versus the chloroterpyridine platinum concentration, i.e., the ratio of intercalated molecules to the number of phosphate sites in DNA. A significant increase in the number of ssb and dsb was observed when the DNA contained intercalated molecules. This technique is an efficient way to induce ssb and dsb triggered by the atomic Auger effect.

Algorithms↗

Kinetics of Ionizing Irradiation-induced DNA Strand Breaks.

By measuring the dose effect of ionizing irradiation-induced single- and double-strand breaks (SSB and DSB) of pBR322, the responses of the yields of SSB and DSB to DNA concentrations were obtained. It was found that, in the presence of mannitol, the reciprocals of G(SSB) and G(alphaDSB) had a linear relationship to the reciprocal of DNA concentration so that the competition reaction of DNA and mannitol to.OH radicals could be described by a second-order kinetics. Moreover, the rate coefficients and the efficiencies of the.OH radical inducing SSB and alphaDSB were deduced.

Journal Article↗

[The regulation of the DNA repair process in mammalian cells. IV. The role of DNA polymerases in the epidermal growth factor regulation of the repair of single-stranded DNA breaks induced by ionizing radiation in Swiss 3T6 mouse cells].

A study was made of the repair of ionizing radiation-induced DNA single-strand breaks (SSB) in proliferating and quiescent mouse Swiss 3T6 cells and in those stimulated from the quiet status by epidermal growth factor in combination with insulin, in the presence of specific inhibitors of DNA polymerase alpha and delta (aphidicolin) and DNA polymerase beta (2', 3'-dideoxythymidine-5'-triphosphate). The repair of DNA SSB induced by X-ray-irradiation (10 Gr) or by gamma-ray irradiation (150 Gr) is more sensitive to aphidicolin independently of cell proliferating status. Aphidicolin inhibits the recovery of single-strand DNA in quiescent and mitogen-stimulated cells three times stronger than in proliferating cells. The influence of 2', 3'-dideoxythymidine-5'-triphosphate on the rate of DNA SSB repair in cells of all the three types does not differ. Thus, the decrease in DNA repair efficiency in quiescent cells is connected with a decrease in the activity of aphidicolin-sensitive DNA polymerase, apparently DNA polymerase alpha. It is suggested that the regulation action of mitogens on the DNA SSB repair may be determined by qualitative changes of this enzyme or of some conditions in which it functions. The involvement of DNA polymerase delta in this process is not excluded.

Animals↗

[European multicenter cross-sectional study on exposure to low doses of benzene].

A cross-sectional multicenter european study has been carried out to evacuate the relations between exposure to low level of benzene and biological markers of internal dose (t,t-MA, S-PMA) and early biological effect (DNA-SSB). The research has shown significantly increased levels (adjusted for smoking habits) of the urinary excretion of t,t-MA, S-PMA and DNA-SSB in petrochemical workers (mean benzene level = 5,694 micrograms/m3) but not in filling station attendants, traffic police officers, and bus drivers compared to referent workers. Dose-response relations were detected between benzene air levels, t,t-MA, S-PMA and DNA-SSB in petrochemical workers, with significantly increased levels of DNA-SSB detected for benzene exposure levels in the range 391-1,800 micrograms/m3 (0.12-0.58 ppm).

Benzene↗

Sensitivity of fresh acute myeloid leukemia cells to etoposide: relationship with cell growth characteristics and DNA single-strand breaks.

In this study, we evaluated the individual in vitro sensitivity of fresh acute myeloid leukemia (AML) cells to VP-16, and attempted to correlate VP-16 cytotoxicity with AML cell growth characteristics and drug-induced DNA single-strand breaks (SSB). Primary (PE1) colony inhibition assays allowed us to characterize two distinct groups of AML: group I (patients 1 through 6), which displayed sensitivity to VP-16 similar to that of normal CFU-GM (IC90 of 20.52 +/- 2.44 micrograms/mL v 20.48 +/- 2.23 micrograms/mL after 1 hour drug exposure, respectively); and group II (patients 7 through 11), which was more sensitive to VP-16 (IC90 of 7.26 +/- 2.93 micrograms/mL, P = .004). Subsequently, groups I and II were termed normosensitive and hypersensitive, respectively. This objective VP-16 sensitivity classification, as determined by PE1, remained unaltered when assessed by secondary (PE2) colony inhibition assay (evaluating the self-renewal fraction of AML progenitors), or by cytofluorometric viability assay (evaluating the ultimately differentiated blast cell population). These findings would suggest that individual sensitivity to VP-16 of a particular cell population is maintained throughout CFU-AML differentiation. Finally, we report that sensitivity of AML cells to VP-16 did not correlate either with cell growth characteristics or with SSB generation. Indeed, AML cell sensitivity to VP-16 appeared more closely related to DNA repair kinetics after drug removal, ie, hypersensitivity being essentially characterized by a prolonged retention of SSB during the posttreatment period. Interestingly, the established HL-60 cell line, which presented greater sensitivity to VP-16 cytotoxicity than KG1, HEL, and K562, was also found to exhibit delayed DNA SSB repair kinetics, as compared with the other AML cell lines. These results suggest that hypersensitivity to VP-16 of some AML cells may be related to a deficient DNA-repair mechanism.

Cell Division↗

Single-stranded DNA-binding protein facilitates gel-free analysis of polymerase chain reaction products in capillary electrophoresis.

It has been recently demonstrated that single-stranded DNA-binding protein (SSB) can facilitate quantitative analyses of DNA, RNA, and proteins in gel-free capillary electrophoresis (CE). Here, we report the application of SSB-mediated gel-free CE for analyses of polymerase chain reaction (PCR) products. The unique ability of SSB to bind ssDNA but not double-stranded DNA (dsDNA) allows efficient separation of three types of DNA molecules in the PCR reaction mixture: primers, products (amplified templates), and by-products, which originate from non-specific DNA hybridization. SSB-mediated gel-free CE analysis of PCR products combines simplicity, high sensitivity, and outstanding quantitative capabilities. The ability of the method to distinguish between products and by-products makes this method an indispensable tool in preparative PCR (e.g., in the development of nucleotide aptamers).

DNA Primers↗

Synthesis of DNA by DNA polymerase epsilon in vitro.

The isolation of DNA polymerase (Pol) epsilon from extracts of HeLa cells is described. The final fractions contained two major subunits of 210 and 50 kDa which cosedimented with Pol epsilon activity, similar to those described previously (Syvaoja, J., and Linn, S. (1989) J. Biol. Chem. 264, 2489-2497). The properties of the human Pol epsilon and the yeast Pol epsilon were compared. Both enzymes elongated singly primed single-stranded circular DNA templates. Yeast Pol epsilon required the presence of a DNA binding protein (SSB) whereas human Pol epsilon required the addition of SSB, Activator 1 and proliferating cell nuclear antigen (PCNA) for maximal activity. Both enzymes were totally unable to elongate primed DNA templates in the presence of salt; however, activity could be restored by the addition of Activator 1 and PCNA. Like Pol delta, Pol epsilon formed complexes with SSB-coated primed DNA templates in the presence of Activator 1 and PCNA which could be isolated by filtration through Bio-Gel A-5m columns. Unlike Pol delta, Pol epsilon bound to SSB-coated primed DNA in the absence of the auxiliary factors. In the presence of salt, Pol epsilon complexes were less stable than they were in the absence of salt. In the in vitro simian virus 40 (SV40) T antigen-dependent synthesis of DNA containing the SV40 origin of replication, yeast Pol epsilon but not human Pol epsilon could substitute for yeast or human Pol delta in the generation of long DNA products. However, human Pol epsilon did increase slightly the length of DNA chains formed by the DNA polymerase alpha-primase complex in SV40 DNA synthesis. The bearing of this observation on the requirement for a PCNA-dependent DNA polymerase in the synthesis and maturation of Okazaki fragments is discussed. However, no unique role for human Pol epsilon in the in vitro SV40 DNA replication system was detected.

Autoantigens↗

Comparative DNA strand breakage induced by FUra and FdUrd in human ileocecal adenocarcinoma (HCT-8) cells: relevance to cell growth inhibition.

Although several mechanisms of 5-fluorouracil (FUra) and 5-fluoro-2'-deoxyuridine (FdUrd) cytotoxicity have been postulated, their effects on cellular DNA integrity have not been fully investigated. Cytotoxicity and the induction of DNA single and double strand breaks (SSB and DSB) were evaluated in the human ileocecal adenocarcinoma cell line, HCT-8, following 2 hr of exposure to these agents. Alkaline and neutral elution techniques were utilized to quantitate the amounts of DNA SSB and DSB induced by FdUrd and FUra. FdUrd, but not FUra, induced a high level of DNA SSB and DSB. These effects were concentration and time dependent, reaching a maximum at 10 microM FdUrd and at 12 hr post-treatment. Minimal amounts of DNA damage were observed in HCT-8 cells exposed to FUra, even at a concentration of 300 microM that produced greater than 99% inhibition of cell growth. In order to delineate the mechanisms associated with the effects observed following FUra and FdUrd treatment of HCT-8 cells, the effects of thymidine and leucovorin were evaluated. DNA SSB and DSB induced by FdUrd were reversed by thymidine. The effects of thymidine were time dependent. Complete reversal of DNA damage and cytotoxicity was achieved when 10 microM thymidine was added at up to 12 hr after treatment, the time of appearance of maximum DNA damage. These results suggest that the extensive DNA damage induced by FdUrd (but not by FUra) was an important determinant of FdUrd cytotoxicity in HCT-8 cells.

Adenocarcinoma↗

Mechanism of stimulation of DNA replication by bacteriophage phi 29 single-stranded DNA-binding protein p5.

Protein p5 is a Bacillus subtilis phage phi 29-encoded protein required for phi 29 DNA replication in vivo. Protein p5 has single-stranded DNA binding (SSB) capacity and stimulates in vitro DNA replication severalfold when phi 29 DNA polymerase is used to replicate either the natural phi 29 DNA template or primed M13 single-stranded DNA (ssDNA). Furthermore, other SSB proteins, including Escherichia coli SSB, T4 gp32, adenovirus DNA-binding protein, and human replication factor A, can functionally substitute for protein p5. The stimulatory effect of phi 29 protein p5 is not due to an increase of the DNA replication rate. When both phi 29 DNA template and M13 competitor ssDNA are added simultaneously to the replication reaction, phi 29 DNA replication is strongly inhibited. This inhibition is fully overcome by adding protein p5, suggesting that protein p5-coated M13 ssDNA is no longer able to compete for replication factors, probably phi 29 DNA polymerase, which has a strong affinity for ssDNA. Electron microscopy demonstrates that protein p5 binds to M13 ssDNA forming saturated complexes with a smoothly contoured appearance and producing a 2-fold reduction of the DNA length. Protein p5 also binds to ssDNA in the phi 29 replicative intermediates produced in vitro, which are similar in structure to those observed in vivo. Our results strongly suggest that phi 29 protein p5 is the phi 29 SSB protein active during phi 29 DNA replication.

Bacillus subtilis↗

Hydroxyl radical quenching agents protect against DNA breakage caused by both 365-nm UVA and by gamma radiation.

The ability of hydroxyl radical (.OH) scavengers to reduce DNA breakage in isolated DNA from Bacillus subtilis by either gamma radiation or monochromatic radiation in the UVA region (365 nm) was examined by comparing dose reduction factors (the ratio of dose required to induce n DNA breaks in the absence to the presence of quencher). Previous data have demonstrated that acetate, formate, azide, and mannitol protect supercoiled DNA against gamma-radiation-induced ssb (single-strand breaks-relaxation of supercoil by first nick) in close agreement with the rate at which their solutions quench .OH. Here we show that these quenchers also protect against 365-nm-induced ssb. The ratios for protection against 365-nm induced DNA ssb in isolated B. subtilis DNA by the four quenchers are also in proportion to their ability to quench .OH. In view of the diverse chemical nature of the quenchers and the wide range of concentrations involved, these findings are evidence that both these radiations may induce ssb in DNA via a common step that might involve .OH.

Acetates↗