Intramolecular Heteroatom and Styryl Diels-Alder Reactions, Asymmetric Cycloadditions of Chiral 3-Phenylallyl Maleic Esters.

Polycyclic aryl naphthalene and tetralin dihydro arylnaphthalene lactone lignans possess anticancer and antibiotic activity. Related furo[3,4-c]pyranones, typified by the sequester-terpenoid isobolivianine, show similar antiproliferative bioactivity. Efficient syntheses of compounds featuring these polycyclic cores have proven challenging due to low yields and poor stereoselectivity. We report the synthesis of chiral cinnamyl but-2-enanoates and 3,3-diphenylallyl-but-2-enoates 1 as new Diels-Alder substrates. These compounds undergo [4 + 2]-cycloadditions to give furo[3,4-c]pyranones 2 in good yield (70%) and diastereoselectivity (7:1), together with naphthyl 3 and dihydronaphthyl tetralins 4 as minor products. Molecular structures and stereochemistries of the major products were verified using X-ray diffraction. Density functional theory calculations revealed that the cycloaddition process involves a bispericyclic/ambimodal process where there is a single transition state that leads to both intramolecular styryl Diels-Alder (ISDA) 3, 4 and intramolecular hetero Diels-Alder (IHDA) cycloadducts 2. With the elevated temperature conditions after cycloaddition, the resulting ISDA cycloadduct either undergoes [3,3]-sigmatropic rearrangement to the more stable major IHDA product or aromatization leading to the phenyltetralin.

Glycosylation in Cervical Cancer: New Insights and Clinical Implications.

Cervical cancer has become the most frequent female malignancy and presents as a general health challenge in many countries undergoing economic development. Various human papillomaviruses (HPV) types have appeared as one of the most critically identifiable causes of widespread cervical cancers. Conventional cervical cytological inspection has limitations of variable sensitivity according to cervical cytology. Glycobiology has been fundamental in related exploration in various gynecologic and reproductive fields and has contributed to our understanding of cervical cancer. It is associated with altered expression of N-linked glycan as well as abnormal expression of terminal glycan structures. The analytical approaches available to determine serum and tissue glycosylation, as well as potential underlying molecular mechanisms involved in the cellular glycosylation alterations, are monitored. Moreover, cellular glycosylation influences various aspects of cervical cancer biology, ranging from cell surface expressions, cell-cell adhesion, cancer signaling, cancer diagnosis, and management. In general, discoveries in glycan profiling make it technically reproducible and affordable to perform serum glycoproteomic analyses and build on previous work exploring an expanded variety of glycosylation markers in the majority of cervical cancer patients.

Applying the British Association of Oral and Maxillofacial Surgeons quality outcomes metrics to a UK oncology and reconstructive surgery service - benchmarking the data.

The British Association of Oral and Maxillofacial Surgery is soon to implement the Quality Outcomes in Oral in Maxillofacial Surgery (QOMS) to provide a platform for quality management across the specialty in the UK. The initial oncology and reconstruction audits for QOMS involves data collection on specific procedures and metrics. The aim of this report is to determine their appropriateness using extant audit datasets in our institution that overlap substantially with the QOMS audits. Pre-existing datasets comprising information on patients treated for oral cavity SCC with curative intent were analysed. Data on surgical margins, lymphadenectomy lymph node yield, delay between surgery and adjuvant radiotherapy, duration of hospital stay, and complications including flap failures were analysed. All statistical analyses were performed with SPSS 25. Run charts describing longitudinal data were generated using SPC for Excel version 6. Twenty three patients (3.1%) of 701 resections had a positive surgical margin reported. Seventeen (4.3%) of patients had less than 18 LNs in the ND specimen analysed. Mean time to start date of adjuvant therapy was 62 days. Only 9% of patients commenced adjuvant therapy within 6 weeks. The median duration of stay was 18 days. In 1153 free flaps a failure rate of 4.3% was identified. A total of 1349 complications (CD I-V) were recorded in the 1111 patients undergoing major surgery with free flap reconstruction. The QOMS selected metrics for oncology and reconstruction are clinically relevant, readily measurable, and likely to be actionable by the surgical team.

HucMSC-exosomes carrying miR-326 inhibit neddylation to relieve inflammatory bowel disease in mice.

BACKGROUND: Inflammatory bowel disease (IBD) is a group of chronic intestinal inflammation that is a risk factor for many gastrointestinal cancers. Exosomes are gradually gaining attention as an emerging treatment method for IBD due to their important biological characteristics. NF-κB is an important pro-inflammatory transcription factor kept inactive by IκB protein in the cytoplasm by masking the nuclear localization signal of NF-κB. The deterioration of IκB is mainly ubiquitination, and this depends on neddylation. METHODS: In this study, we established a dextran sulfate sodium (DSS)-induced IBD model in BABL/C mice to evaluate the effect of human umbilical cord mesenchymal stem cell-derived exosomes (hucMSC-exosomes, hucMSC-Ex) on the repair of IBD. At the same time, human colorectal mucosa cells (FHC) were stimulated by LPS (lipopolysaccharide) in vitro to activate the inflammatory environment to study the mechanism of hucMSC-Ex regulating neddylation. The microRNA (miRNA) obtained by sequencing and transfection with hucMSC-Ex was used to verify the role of miR-326/neddylation/IκB/NF-κB signaling pathway in IBD repair. RESULTS: HucMSC-Ex inhibited the process of neddylation in relieving DSS-induced IBD in mice. The binding of NEDD8 (neural precursor cell-expressed, developmentally downregulated gene 8) to cullin 1 and the activation of NF-κB signaling pathway were suppressed along with reduced expression levels of neddylation-related enzyme molecules. The same phenomenon was observed in FHC cells. The miRNA comparison results showed that miR-326 was highly expressed in hucMSC-Ex and played an important role in inhibiting the neddylation process. The therapeutic effect of hucMSC-Ex with high expression of miR-326 on IBD mice was significantly stronger than that of ordinary hucMSC-Ex. CONCLUSIONS: HucMSC-Ex relieves DSS-induced IBD in a mouse model by inhibiting neddylation through miR-326.

Targeted laser therapy synergistically enhances efficacy of antibiotics against multi-drug resistant Staphylococcus aureus and Pseudomonas aeruginosa biofilms.

The growing prevalence of biofilm-associated multi-drug resistant (MDR) bacteria necessitates the innovation of non-traditional approaches to improve the effectiveness of mainstay antibiotics. Here, we evaluated the use of gold nanoparticle (GNP)-targeted pulsed laser therapy to enhance antibiotic efficacy against in vitro methicillin-resistant Staphylococcus aureus (MRSA) and MDR Pseudomonas aeruginosa biofilms. Treatment with antibody-conjugated GNPs followed by nanosecond-pulsed laser irradiation at 532 nm (~1.0 J/cm2) dispersed 96-99% of the biofilms relative to controls. GNP-targeted laser therapy combined with gentamicin or amikacin caused a synergistic 4- and 5-log reduction in the viability of MRSA and P. aeruginosa biofilms, respectively, whereas GNP-targeted laser therapy or antibiotics alone decreased biofilm viability by only ~1 log. Notably, GNP-targeted laser therapy was able to increase the antibiotic susceptibility of the biofilms to the level of drug sensitivity observed in planktonic MRSA and P. aeruginosa cultures, further indicating effective biofilm dispersal via this novel approach.

Mild hyperthermia enhances transport of liposomal gemcitabine and improves in vivo therapeutic response.

Obstructive biological barriers limit the transport and efficacy of cancer nanotherapeutics. Creative manipulation of tumor microenvironment provides promising avenues towards improving chemotherapeutic response. Such strategies include the use of mechanical stimuli to overcome barriers, and increase drug delivery and therapeutic efficacy. The rational use of gold nanorod-mediated mild hyperthermia treatment (MHT) alters tumor transport properties, increases liposomal gemcitabine (Gem Lip) delivery, and antitumor efficacy in pancreatic cancer CAPAN-1 tumor model. MHT treatment leads to a threefold increase in accumulation of 80-nm liposomes and enhances spatial interstitial distribution. I.v. injection of Gem Lip and MHT treatment lead to a threefold increase in intratumor gemcitabine concentration compared to chemotherapeutic infusion alone. Furthermore, combination of MHT treatment with infusion of 12 mg kg(-1) Gem Lip leads to a twofold increase in therapeutic efficacy and inhibition of CAPAN-1 tumor growth when compared to equimolar chemotherapeutic treatment alone. Enhanced therapeutic effect is confirmed by reduction in tumor size and increase in apoptotic index where MHT treatment combined with 12 mg kg(-1) Gem Lip achieves similar therapeutic efficacy as the use of 60 mg kg(-1) free gemcitabine. In conclusion, improvements in vivo efficacy are demonstrated resulting from MHT treatment that overcome transport barriers, promote delivery, improve efficacy of nanomedicines.

Human equilibrative nucleoside transporter-1 knockdown tunes cellular mechanics through epithelial-mesenchymal transition in pancreatic cancer cells.

We report cell mechanical changes in response to alteration of expression of the human equilibrative nucleoside transporter-1 (hENT1), a most abundant and widely distributed plasma membrane nucleoside transporter in human cells and/or tissues. Modulation of hENT1 expression level altered the stiffness of pancreatic cancer Capan-1 and Panc 03.27 cells, which was analyzed by atomic force microscopy (AFM) and correlated to microfluidic platform. The hENT1 knockdown induced reduction of cellular stiffness in both of cells up to 70%. In addition, cellular phenotypic changes such as cell morphology, migration, and expression level of epithelial-mesenchymal transition (EMT) markers were observed after hENT1 knockdown. Cells with suppressed hENT1 became elongated, migrated faster, and had reduced E-cadherin and elevated N-cadherin compared to parental cells which are consistent with epithelial-mesenchymal transition (EMT). Those cellular phenotypic changes closely correlated with changes in cellular stiffness. This study suggests that hENT1 expression level affects cellular phenotype and cell elastic behavior can be a physical biomarker for quantify hENT1 expression and detect phenotypic shift. Furthermore, cell mechanics can be a critical tool in detecting disease progression and response to therapy.

Transient mild hyperthermia induces E-selectin mediated localization of mesoporous silicon vectors in solid tumors.

BACKGROUND: Hyperthermia treatment has been explored as a strategy to overcome biological barriers that hinder effective drug delivery in solid tumors. Most studies have used mild hyperthermia treatment (MHT) to target the delivery of thermo-sensitive liposomes carriers. Others have studied its application to permeabilize tumor vessels and improve tumor interstitial transport. However, the role of MHT in altering tumor vessel interfacial and adhesion properties and its relationship to improved delivery has not been established. In the present study, we evaluated effects of MHT treatment on tumor vessel flow dynamics and expression of adhesion molecules and assessed enhancement in particle localization using mesoporous silicon vectors (MSVs). We also determined the optimal time window at which maximal accumulation occur. RESULTS: In this study, using intravital microscopy analyses, we showed that temporal mild hyperthermia (∼1 W/cm(2)) amplified delivery and accumulation of MSVs in orthotopic breast cancer tumors. The number of discoidal MSVs (1000×400 nm) adhering to tumor vasculature increased 6-fold for SUM159 tumors and 3-fold for MCF-7 breast cancer tumors. By flow chamber experiments and Western blotting, we established that a temporal increase in E-selectin expression correlated with enhanced particle accumulation. Furthermore, MHT treatment was shown to increase tumor perfusion in a time-dependent fashion. CONCLUSIONS: Our findings reveal that well-timed mild hyperthermia treatment can transiently elevate tumor transport and alter vascular adhesion properties and thereby provides a means to enhance tumor localization of non-thermally sensitive particles such as MSVs. Such enhancement in accumulation could be leveraged to increase therapeutic efficacy and reduce drug dosing in cancer therapy.

Tumor vascular permeabilization using localized mild hyperthermia to improve macromolecule transport.

The abnormal tumor vasculature presents a major challenge to the adequate delivery of chemotherapeutics, often limiting efficacy. We developed a nanoparticle-based technique to deliver localized mild hyperthermia (MHT) used to transiently alter tumor vascular transport properties and enhance transport of macromolecules into tumor interstitium. The strategy involved administering and localizing accumulation of stealth gold nanorods (GNRs, 103 µg of GNRs/g of tumor), and irradiating tumor with a low-photon laser flux (1 W/cm(2)) to generate MHT. The treatment increased vascular permeability within 24 h after treatment, allowing enhanced transport of macromolecules up to 54 nm in size. A mathematical model is used to describe changes in tumor mass transport properties where the rate of macromolecular exchange between interstitial and vascular region (R) and maximum dye enhancement (Ymax) of 23-nm dextran dye is analytically solved. During enhanced permeability, R increased by 200% while Ymax increased by 30% relative to untreated group in pancreatic CAPAN-1 tumors. MHT treatment also enhanced transport of larger dextran dye (54 nm) as assessed by intravital microscopy, without causing occlusive cellular damage. Enhanced vascular transport was prolonged for up to 24 h after treatment, but reversible with transport parameters returning to basal levels after 36 h. This study indicates that localized mild hyperthermia treatment opens a transient time-window with which to enable and augment macromolecule transport and potentially improve therapeutic efficacy. From the clinical editor: In this study, local intra-tumor mild hyperthermia is induced using a nanoparticle-based approach utilizing stealth gold nanorods and irradiating the tumor with low-photon laser flux, resulting in locally increased vascular permeability enabling enhanced delivery of therapeutics, including macromolecules up to 54 nm in size. Similar approaches would be very helpful in addressing treatment-resistant malignancies in clinical practice.

Targeted near-IR hybrid magnetic nanoparticles for in vivo cancer therapy and imaging.

We report the use of immuno-targeted gold-iron oxide hybrid nanoparticles for laser-assisted therapy and for MRI-based imaging as demonstrated in xenograft colorectal cancer tumor model. Immuno-targeted gold-iron oxide nanoparticles selectively accumulate in SW1222 xenograft tumors as compared to the accumulation in non-antigen-expressing tumor xenografts. Effective photothermal treatment using near-IR laser irradiation (808nm, 5W cm(-2)) application is shown where >65% of the antigen-expressing tumor cells presented corrupt extracellular matrix and cytoplasmic acidophilia suggesting effectiveness of nanoparticle-assisted thermal therapy. Cell killing was confirmed by hematoxylin and eosin (H&E) histological staining where scar-like structure containing collagen bundles was observed in the treatment group. Further, systemically injected HNPs were shown to be effective T2 magnetic resonance (MR) imaging contrast agents, localized and detected at the antigen-expressing xenograft tumors. These findings suggest that the new class of bio-conjugated HNPs exhibits great potential for dual-therapy and diagnostics (theranostics) applications. FROM THE CLINICAL EDITOR: This team reports the successful use of immuno-targeted gold-iron oxide hybrid nanoparticles for both laser-assisted therapy and MRI-based imaging in a xenograft colorectal cancer tumor model, demonstrating strong potentials for dual applications in cancer diagnosis and therapy.

PAA-derived gold nanorods for cellular targeting and photothermal therapy.

A single-step LbL procedure to functionalize CTAB-capped GNRs via electrostatic self-assembly is reported. This approach allows for consistent biomolecule/GNR coupling using standard carboxyl-amine conjugation chemistry. The focus is on cancer-targeting biomolecule/GNR conjugates and selective photothermal destruction of cancer cells by GNR-mediated hyperthermia and NIR light. GNRs were conjugated to a single-chain antibody selective for colorectal carcinoma cells and used as probes to demonstrate photothermal therapy. Selective targeting and GNR uptake in antigen-expressing SW 1222 cells were observed using fluorescence microscopy. Selective photothermal therapy is demonstrated using SW 1222 cells, where >62% cell death was observed after cells are treated with targeted A33scFv-GNRs.

Ten year performance evaluation of a field-scale zero-valent iron permeable reactive barrier installed to remediate trichloroethene contaminated groundwater.

The Monkstown zero-valent iron permeable reactive barrier (ZVI PRB), Europe's oldest commercially-installed ZVI PRB, had been treating trichloroethene (TCE) contaminated groundwater for about 10 years on the Nortel Network site in Northern Ireland when cores from the reactive zone were collected in December, 2006. Groundwater data from 2001-2006 indicated that TCE is still being remediated to below detection limits as the contaminated groundwater flows through the PRB. Ca and Fe carbonates, crystalline and amorphous Fe sulfides, and Fe (hydr)oxides have precipitated in the granular ZVI material in the PRB. The greatest variety of minerals is associated with a approximately 1-2 cm thick, slightly cemented crust on top (up-gradient influent entrance) of the ZVI section of the PRB and also with the discontinuous cemented ZVI material ( approximately 23 cm thick) directly below it. The greatest presence of microbial communities also occurred in the up-gradient influent portion of the PRB compared to its down-gradient effluent section, with the latter possibly due to less favorable conditions (i.e., high pH, low oxygen) for microbial growth. The ZVI filings in the down-gradient effluent section of the PRB have a projected life span of >10 years compared with ZVI filings from the continuous to discontinuous cemented up-gradient ZVI section (upper approximately 25 cm) of the PRB, which may have a life span of only approximately 2-5 more years. Supporting Information from applied, multi-tracer testing indicated that restricted groundwater flow is occurring in the upper approximately 25 cm of the ZVI section and preferential pathways have also formed in this PRB over its 10 years of operation.

Gold hybrid nanoparticles for targeted phototherapy and cancer imaging.

Gold and iron oxide hybrid nanoparticles (HNPs) synthesized by the thermal decomposition technique are bio-functionalized with a single chain antibody, scFv, that binds to the A33 antigen present on colorectal cancer cells. The HNP-scFv conjugates are stable in aqueous solution with a magnetization value of 44 emu g(-1) and exhibit strong optical absorbance at 800 nm. Here we test this material in targeting, imaging and selective thermal killing of colorectal cancer cells. Cellular uptake studies showed that A33-expressing cells take up the A33scFv-conjugated HNPs at a rate five times higher than cells that do not express the A33 antigen. Laser irradiation studies showed that approximately 53% of the A33-expressing cells exposed to targeted HNPs are killed after a six-minute laser treatment at 5.1 W cm(-2) using a 808 nm continuous wave laser diode while < 5% of A33-nonexpressing cells are killed. At a higher intensity, 31.5 W cm(-2), the thermal destruction increases to 99 and 40% for A33-expressing cells and A33 nonexpressing cells, respectively, after 6 min exposure. Flow cytometric analyses of the laser-irradiated A33 antigen-expressing cells show apoptosis-related cell death to be the primary mode of cell death at 5.1 W cm(-2), with increasing necrosis-related cell death at higher laser power. These results suggest that this new class of bio-conjugated hybrid nanoparticles can potentially serve as an effective antigen-targeted photothermal therapeutic agent for cancer treatment as well as a probe for magnetic resonance-based imaging.

Metabolic indicators in the skeletal muscles of harbor seals (Phoca vitulina).

The goal of this study was to determine the distribution of citrate synthase (CS), beta-hydroxyacyl coenzyme A dehydrogenase (HOAD), and lactate dehydrogenase (LDH) activities and myoglobin (Mb) concentration in the locomotor muscles (epaxial muscles) and heart of harbor seals. The entire epaxial musculature, which produces most of the power for submerged swimming, was removed and weighed, and three transverse sections (cranial, middle, and caudal) were taken along the muscle bundle. Multiple samples were taken along points on a circular grid using a 6-mm biopsy. A single sample was taken from the left ventricle of the heart. Muscle groups of similar function were taken from three dogs as a control. Mean values were calculated for four roughly equal quadrants in each transverse section of the epaxial muscles. There were no significant differences among the quadrants within any of the transverse sections for the three enzymes or Mb. However, there were significant differences in the mean enzyme activities and Mb concentrations along the length of the muscle. The middle and caudal sections had significantly higher mean levels of CS, LDH, and Mb than the cranial section, which may be correlated with power production during swimming. The enzyme ratios CS/HOAD and LDH/CS exhibited no variation within transverse sections or along the length of the epaxial muscles. Relative to the dog, the epaxial muscles and heart of the harbor seal had higher HOAD levels and lower CS/HOAD, which, taken together, indicate an increased capacity for aerobic lipid metabolism during diving.

Localization of specific binding sites for 125I-TGF-beta1 to fenestrated endothelium in bone and anastomosing capillary networks in enamel organ suggests a role for TGF-beta1 in angiogenesis.

Previous studies have shown endothelial cells to be a major target for endocrine TGF-beta in several soft tissues in the normal growing rat. The potent effect of TGF-beta1 on bone formation prompted us to analyze in detail the localization of specific binding sites for endocrine TGF-beta in hard tissues. At 2.5 minutes after injection of 125I-TGF-beta1, specific binding, as demonstrated by quantitative radioautography, was localized to fenestrated endothelium participating in angiogenesis in the vascular invasion region of the growth plate in bone as well as to anatomizing capillary networks in the maturation zone of the enamel organ. At 15 minutes after injection, the bound ligand was internalized into endocytic vesicles of endothelial cells. In bone, quantitation revealed significant differences in receptor density between endothelia undergoing proliferation vs those in a state of elongation and anastomosis with neighboring endothelial cells. In the rat incisor, specific binding of 125I-TGF-beta1 to endothelium correlated with increased formation of anastomotic capillary networks. These studies identify differential specific binding sites of 125I-TGF-beta1 in angiogenically active endothelium, providing an important link between TGF-beta1, the endothelium, and hard tissue development.

Poly(A) polymerase and the regulation of cytoplasmic polyadenylation.

Translational activation in oocytes and embryos is often regulated via increases in poly(A) length. Cleavage and polyadenylation specificity factor (CPSF), cytoplasmic polyadenylation element binding protein (CPEB), and poly(A) polymerase (PAP) have each been implicated in cytoplasmic polyadenylation in Xenopus laevis oocytes. Cytoplasmic polyadenylation activity first appears in vertebrate oocytes during meiotic maturation. Data presented here shows that complexes containing both CPSF and CPEB are present in extracts of X. laevis oocytes prepared before or after meiotic maturation. Assessment of a variety of RNA sequences as polyadenylation substrates indicates that the sequence specificity of polyadenylation in egg extracts is comparable to that observed with highly purified mammalian CPSF and recombinant PAP. The two in vitro systems exhibit a sequence specificity that is similar, but not identical, to that observed in vivo, as assessed by injection of the same RNAs into the oocyte. These findings imply that CPSFs intrinsic RNA sequence preferences are sufficient to account for the specificity of cytoplasmic polyadenylation of some mRNAs. We discuss the hypothesis that CPSF is required for all polyadenylation reactions, but that the polyadenylation of some mRNAs may require additional factors such as CPEB. To test the consequences of PAP binding to mRNAs in vivo, PAP was tethered to a reporter mRNA in resting oocytes using MS2 coat protein. Tethered PAP catalyzed polyadenylation and stimulated translation approximately 40-fold; stimulation was exclusively cis-acting, but was independent of a CPE and AAUAAA. Both polyadenylation and translational stimulation required PAPs catalytic core, but did not require the putative CPSF interaction domain of PAP. These results demonstrate that premature recruitment of PAP can cause precocious polyadenylation and translational stimulation in the resting oocyte, and can be interpreted to suggest that the role of other factors is to deliver PAP to the mRNA.

PMP22 carrying the trembler or trembler-J mutation is intracellularly retained in myelinating Schwann cells.

Missense mutations in the murine peripheral myelin protein-22 gene (Pmp22) underly the neuropathies in the trembler (Tr) and trembler-J (Tr-J) mice and in some humans with Charcot-Marie-Tooth disease. We have generated replication-defective adenoviruses containing epitope-tagged, wild-type-, Tr-, or Tr-J-PMP22 bicistronic with the Lac-Z reporter gene. These viruses were microinjected into the sciatic nerves of 10-day-old Sprague-Dawley rats and, later, analyzed by immunohistochemistry to determine the distribution of mutant protein in infected myelinating Schwann cells. We found that epitope-tagged, wild-type PMP22 is successfully transported to compact myelin, whereas the Tr and the Tr-J mutant proteins are retained in cytoplasmic compartment, colocalizing with the endoplasmic reticulum. These results provide in vivo evidence that the pathogenesis of the Tr and Tr-J mutations are most likely a function of abnormal retention within the endoplasmic reticulum of myelinating Schwann cells.

Determination of the free-energy change for repair of a DNA phosphodiester bond.

The repair of phosphodiester bonds in nicked DNA is catalyzed by DNA ligases. Ligation is coupled to cleavage of a phosphoanhydride bond in a nucleotide cofactor resulting in a thermodynamically favorable process. A free energy value for phosphodiester bond formation was calculated using the reversibility of the T4 DNA ligase reaction. The relative number of DNA nicks to phosphodiester bonds in a circular plasmid DNA, formed during this reaction at fixed concentrations of ATP to AMP and PP(i), was quantified. At 25 degrees C, pH 7, the equilibrium constant (K(eq)) for the ligation reaction is 3.89 x 10(4) m. This value corresponds to a standard free energy (DeltaG degrees ') of -6.3 kcal mol(-1). By subtracting the known energy contribution due to hydrolysis of ATP to AMP and PP(i), DeltaG degrees ' for the hydrolysis of a DNA phosphodiester bond is -5.3 kcal mol(-1).