DNA-PKcs kinase activity stabilizes the transcription factor Egr1 in activated immune cells.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is known primarily for its function in DNA double-stranded break repair and nonhomologous end joining (NHEJ). However, DNA-PKcs also has a critical yet undefined role in immunity impacting both myeloid and lymphoid cell lineages spurring interest in targeting DNA-PKcs for therapeutic strategies in immune-related diseases. To gain insight into the function of DNA-PKcs within immune cells, we performed a quantitative phosphoproteomic screen in T cells to identify phosphorylation targets of DNA-PKcs. Our results indicate that DNA-PKcs phosphorylates the transcription factor Egr1 (early growth response protein 1) at serine 301. Expression of Egr1 is induced early upon T cell activation and dictates T cell response by modulating expression of cytokines and key costimulatory molecules such as IL (interleukin) 2, IL6, IFNγ, and NFκB. Inhibition of DNA-PKcs by treatment with a DNA-PKcs specific inhibitor NU7441 or shRNA knockdown increased proteasomal degradation of Egr1. Mutation of serine 301 to alanine via CRISPR-Cas9 reduced EGR1 protein expression and decreased Egr1-dependent transcription of IL2 in activated T cells. Our findings identify DNA-PKcs as a critical intermediary link between T cell activation and T cell fate and a novel phosphosite involved in regulating Egr1 activity.

Correction to: Oxygen Transport to Tissue XL.

The chapters "Changes in Cytochrome-C-Oxidase Account for Changes in Attenuation of Near-Infrared Light in the Healthy Infant Brain" and "Fibreless Multiwavelength NIRS System for Imaging Localised Changes in Cerebral Oxidised Cytochrome C Oxidase" are made as open access as per the author's request in this revised version of the book.

Heterogeneity in tissue oxygenation: from physiological variability in normal tissues to pathophysiological chaos in malignant tumours.

Heterogeneity is a feature of both normal oxygen supply to tissue and of a supply that is disturbed due to a wide range of different pathologies. Here, the physiological importance of heterogeneity of tissue oxygenation is revisited. The anatomical and functional basis for heterogeneity of blood flow, local and regional regulatory mechanisms in normal tissues and the pathophysiology of the failure of regulation will be examined.Under physiological conditions, regulation of blood flow distributions at global, regional and microregional levels play coordinated roles in ensuring adequate O2 supply to all tissue cells. How this is achieved may be organ-/organ layer-specific, depending on its function and priorities to match local O2 delivery to consumption. Examples where these regulatory mechanisms break down under conditions of ischaemia and shock will also be given.In contrast, pathologic heterogeneity in tissue oxygenation resulting from uncontrolled, chaotic growth as seen in malignant tumours represents a pathophysiological status that is not predictable which, in general, is associated with chronic and acute hypoxia. This can have fatal consequences due to hypoxia- induced (mal-)adaptive processes, malignant tumour progression and treatment resistance.

Simultaneous monitoring of brain and skin oxygenation during haemorrhagic shock in piglets.

Phosphorescence quenching and visible lightguide spectrophotometry were used to measure brain cortex oxygen partial pressure and skin oxygen saturation, respectively, during stepwise haemorrhage and re-transfusion in four 4-7-day-old anaesthetised piglets. In three cases, the effect of administration of adrenalin (epinephrine) was investigated. Brain cortex partial pressure was measured using a conventional phosphorescence pO2 probe (bclocpO2) and using a self-contained phosphorescence microsensor (bcmicropO2). Peripheral tissue oxygen saturation was measured on the skin of the abdomen (abSsO2) and the distal right foreleg (flSsO2) using visible lightguide spectrophotometry. Haemorrhage of 65 ml reduced mean arterial blood pressure (MABP) from 75.5 ± 11.0 mmHg (mean ± standard deviation) to 42 ± 2.6 mmHg. Mean bclocpO2 fell from 30.1 ± 3.1 to 13.1 ± 2.5 mmHg and mean bcmicropO2 fell from 33.8 ± 11.4 to 13.3 ± 9.5 mmHg. abSsO2 and flSsO2 values fell from 47.4 ± 8.1 % and 43.6 ± 10.9 %, respectively, to 21.9 ± 5.5 % and 23.8 ± 14.0 %. Infusion of adrenalin produced a mean transient increase in MABP to 137 ± 2.6 mmHg, falling to 75.7 ± 16.3 mmHg within 3 min. bclocpO2 also increased to 24.1 ± 14.6 mmHg, but there were no significant changes in bcmicropO, abSsO2 or flSsO2. Following reinfusion all parameters returned to values that were not statistically different from their pre-haemorrhage values. The dynamic recordings of all the oxygenation parameters indicated that they were sensitive indicators of the degree of haemorrhage during the experiments.

Binding by the hepatitis C virus NS3 helicase partially melts duplex DNA.

Binding of NS3 helicase to DNA was investigated by footprinting with KMnO(4), which reacts preferentially with thymidine residues in single-stranded DNA (ssDNA) compared to those in double-stranded DNA (dsDNA). A distinct pattern of reactivity was observed on ssDNA, which repeated every 8 nucleotides (nt) and is consistent with the known binding site size of NS3. Binding to a DNA substrate containing a partial duplex was also investigated. The DNA contained a 15 nt overhang made entirely of thymidine residues adjacent to a 22 bp duplex that contained thymidine at every other position. Surprisingly, the KMnO(4) reactivity pattern extended from the ssDNA into the dsDNA region of the substrate. Lengthening the partial duplex to 30 bp revealed a similar pattern extending from the ssDNA into the dsDNA, indicating that NS3 binds within the duplex region. Increasing the length of the ssDNA portion of the partial duplex by 4 nt resulted in a shift in the footprinting pattern for the ssDNA by 4 nt, which is consistent with binding to the 3'-end of the ssDNA. However, the footprinting pattern in the dsDNA region was shifted by only 1-2 bp, indicating that binding to the ssDNA-dsDNA region was preferred. Footprinting performed as a function of time indicated that NS3 binds to the ssDNA rapidly, followed by slower binding to the duplex. Hence, multiple molecules of NS3 can bind along a ssDNA-dsDNA partial duplex by interacting with the ssDNA as well as the duplex DNA.

A structural feature of Dda helicase which enhances displacement of streptavidin and trp repressor from DNA.

Helicases are molecular motors with many activities. They use the energy from ATP hydrolysis to unwind double-stranded nucleic acids while translocating on the single-stranded DNA. In addition to unwinding, many helicases are able to remove proteins from nucleic acids. Bacteriophage T4 Dda is able to displace a variety of DNA binding proteins and streptavidin bound to biotinylated oligonucleotides. We have identified a subdomain of Dda that when deleted, results in a protein variant that has nearly wild type activity for unwinding double-stranded DNA but exhibits greatly reduced streptavidin displacement activity. Interestingly, this domain has little effect on displacement of either gp32 or BamHI bound to DNA but does affect displacement of trp repressor from DNA. With this variant, we have identified residues which enhance displacement of some proteins from DNA.

Skin SO2 measurement using visible lightguide spectrophotometry in a black population: a feasibility study.

The aim of the study was to investigate the influence of melanin content on the visible wavelength range spectrophotometric measurement of SO(2) in the skin of normal healthy black and white volunteers. The reactive hyperaemia induced by a 5-minute period of tourniquet occlusion of the brachial artery, as manifested in the change in skin SO(2), was compared with the reactive hyperaemia index (RHI) and arterial stiffness index (AI) as measured using the Endo-PAT2000® peripheral arterial tonometry device. Further measurements were carried out on a diabetic patient with critical ischaemia. The measurements in the normal volunteers and the patient showed that there that there was no correlation between SO(2) and melanin index (r(2) = 0.02). There was a poor correlation between the degree of reactive hyperaemia as assessed using tissue SO(2) measurement and the parameters derived using the Endo-PAT2000® device. Measurements on the critically ischaemic lower limb of the diabetic patient revealed a mean medial/lateral SO(2) of 26.3% and a degree of tissue hypoxia (the percentage of recordings with an SO(2) of 15% or less) of 16.2%. This pilot study demonstrated that the measurement of tissue SO(2) in the skin of black subjects is feasible, not only under conditions of normal perfusion, but also in critical limb ischaemia.

DNA-PKcs Inhibition Extends Allogeneic Skin Graft Survival.

BACKGROUND: Organ transplantation is life-saving and continued investigations into immunologic mechanisms that drive organ rejection are needed to improve immunosuppression therapies and prevent graft failure. DNA-dependent protein kinase catalytic subunit, DNA dependent-protein kinase catalytic subunit (DNA-PKcs), is a critical component of both the cellular and humoral immune responses. In this study, we investigate the contribution of DNA-PKcs to allogeneic skin graft rejection to potentially highlight a novel strategy for inhibiting transplant rejection. METHODS: Fully MHC mismatched murine allogeneic skin graft studies were performed by transplanting skin from BalbC mice to C57bl6 mice and treating with either vehicle or the DNA-PKcs inhibitor NU7441. Graft rejection, cytokine production, immune cell infiltration, and donor-specific antibody formation were analyzed. RESULTS: DNA-PKcs inhibition significantly reduced necrosis and extended graft survival compared with controls (mean survival 14 d versus 9 d, respectively). Inhibition reduced the production of the cytokines interleukin (IL)-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ and the infiltration of CD3+ lymphocytes into grafts. Furthermore, DNA-PKcs inhibition reduced the number of CD19+ B cells and CD19+ CD138+ plasma cells coinciding with a significant reduction in donor-specific antibodies. At a molecular level, we determined that the immunosuppressive effects of DNA-PKcs inhibition were mediated, in part, via inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells signaling through reduced expression of the p65 subunit. CONCLUSIONS: Our data confirm that DNA-PKcs contributes to allogeneic graft rejection and highlight a novel immunologic function for DNA-PKcs in the regulation of nuclear factor kappa-light-chain-enhancer of activated B cells and concomitant cytokine production.

Laser Polishing of Additive Manufactured Aluminium Parts by Modulated Laser Power.

In this study a new approach to laser polishing with periodic modulated laser power in the kilohertz regime is introduced. By varying the modulation frequency and modulation time, different periodic laser power curves with varying minimum, peak and average laser power can be created. The feasibility of the method is shown by polishing of vertical built AlSi10Mg L-PBF parts with an initial roughness of Ra = 12.22 µm. One polishing pass revealed a decreasing surface roughness with increasing energy density on the surface up to Ra = 0.145 µm. An increasing energy density results in a rising remelting depth between 50 and 255 µm and a rising relative porosity of 0.3% to 4.6%. Furthermore, the thermal process stability, analysed by the melt pool length in scanning direction, reveals a steadily increasing melt pool dimension due to component heating. Multiple laser polishing passes offers a further reduced surface roughness, especially at higher modulation frequencies and provides an improved orientation independent roughness homogeneity. The process stability regarding varying initial surface roughness revealed an almost constant relative roughness reduction rate with an achievable roughness variation after two polishing passes between Ra = 0.13-0.26 µm from an initial state of Ra = 8.0-19.2 µm.

Use of visible spectrophotometry to assess tissue oxygenation in the colostomy stoma.

The aims of this study were to determine the normal range of tissue oxygenation (SO(2)) in the "mature" colostomy stomacolostomy stoma and to investigate whether there were any diurnal variationsdiurnal variations in the SO(2) values. Ten patients with an end colostomy for a minimum duration of three months and using conventional colostomy bags were included in this study. Tissue SO(2) Tissue SO(2) was measured on the stoma using visible wavelength spectroscopy (Whitland RM 200, Whitland Research, Whitland, UK) The measurements were carried out on each patient on three occasions: the first early in the morning (designated "baseline"), a second after 6 h and the third on the next day at 24 h. The results showed that the mean baseline SO(2) in the colostomy stoma was 77.6 +/- 6.8 and there were no differences in the SO(2) measurements between the baseline, 6 h and the 24 h values. There were also no differences in the SO(2) values between the four quadrants of the stomas. In conclusion, visible wavelength spectrophotometry can reliably measure stomal SO(2) in a non-invasive way. No significant diurnal variations in the stomal SO(2) values were detected.

Intraoperative measurement of colonic oxygenation during bowel resection.

Recently lightguide spectrophotometry (LGS) has been investigated for assessing bowel mucosal oxygenation and may prove helpful in the diagnosis of bowel ischaemia. This pilot study explores the use of LGS and laser Doppler flowmetry (LDF) to measure SO2 and perfusion in the bowel during key stages of colon surgery. SO2 and perfusion in the mucosal and serosal layers of the rectum, sigmoid and descending colon were measured in 7 patients by LGS (Whitland Research, UK) and LDF (Moor Instruments, UK) respectively at four stages (baseline, after mobilisation of the sigmoid, after ligation of the inferior mesenteric artery (IMA) and after complete devascularisation of the sigmoid). The sigmoid mucosal SO2 and LDF values were significantly lower than the baseline after the ligation of IMA and devascularisation. Mean (SD) baseline sigmoid mucosal SO2 (73%) decreased to 55% after ligation of IMA and to 39% after complete devascularisation. The sigmoid serosal SO2 did not show any change after ligation of IMA and showed only 7% decrease after devascularisation. There was no difference in baseline SO2 and LDF values in different parts of the bowel but the mean mucosal baseline SO2 (75%) was significantly lower than that in the serosa (87%). In conclusion, mucosal SO2 measurements can accurately diagnose bowel ischaemia but serosal SO2 does not reflect mucosal ischaemia.

Amputation level viability in critical limb ischaemia: setting new standards.

We have previously demonstrated the successful use of skin oxygen saturation (SO2) measurements to predict the healing viability in lower limb amputations for critical limb ischaemia. The measurements are quick and easy to perform, but the instrument that has been used to date is now obsolete and a new, lightweight, portable instrument has recently been introduced. However, fundamental differences between the two instruments could influence the criteria used for determining amputation level viability. The purpose of this study was to compare the in vivo measurements using the two instruments in order to validate amputation level viability criteria using the RM200. Skin SO2 measurements were carried out on critically ischaemic lower limbs of patients, and on the forearms of normal volunteers during before, during and after a 5 minute period of tourniquet ischaemia. A linear correlation (r2 = 0.91) was found between the values obtained from the two instruments within the range of interest (0 to 40% SO2). Differences between the instruments lay within 1 standard deviation of the mean, demonstrating a high degree of agreement between the two methods. The RM200 is thus an acceptable replacement for the MCPD instrument for amputation level viability assessments.

Cytochrome redox states and respiratory control in mouse and beef heart mitochondria at steady-state levels of hypoxia.

Mitochondrial control of cellular redox states is a fundamental component of cell signaling in the coordination of core energy metabolism and homeostasis during normoxia and hypoxia. We investigated the relationship between cytochrome redox states and mitochondrial oxygen consumption at steady-state levels of hypoxia in mitochondria isolated from beef and mouse heart (BHImt, MHImt), comparing two species with different cardiac dynamics and local oxygen demands. A low-noise, rapid spectrophotometric system using visible light for the measurement of cytochrome redox states was combined with high-resolution respirometry. Monophasic hyperbolic relationships were observed between oxygen consumption, JO2, and oxygen partial pressure, Po2, within the range <1.1 kPa (8.3 mmHg; 13 µM). P50j (Po2 at 0.5·Jmax) was 0.015 ± 0.0004 and 0.021 ± 0.003 kPa (0.11 and 0.16 mmHg) for BHImt and MHImt, respectively. Maximum oxygen consumption, Jmax, was measured at saturating ADP levels (OXPHOS capacity) with Complex I-linked substrate supply. Redox states of cytochromes aa3 and c were biphasic hyperbolic functions of Po2. The relationship between cytochrome oxidation state and oxygen consumption revealed a separation of distinct phases from mild to severe and deep hypoxia. When cytochrome c oxidation increased from fully reduced to 45% oxidized at 0.1 Jmax, Po2 was as low as 0.002 kPa (0.02 µM), and trace amounts of oxygen are sufficient to partially oxidize the cytochromes. At higher Po2 under severe hypoxia, respiration increases steeply, whereas redox changes are small. Under mild hypoxia, the steep slope of oxidation of cytochrome c when flux remains more stable represents a cushioning mechanism that helps to maintain respiration high at the onset of hypoxia.