Chloroplastic ascorbate modifies plant metabolism and may act as a metabolite signal regardless of oxidative stress.

Ascorbate (Asc) is a major plant metabolite that plays crucial roles in various processes, from reactive oxygen scavenging to epigenetic regulation. However, to what extent and how Asc modulates metabolism is largely unknown. We investigated the consequences of chloroplastic and total cellular Asc deficiencies by studying chloroplastic Asc transporter mutant lines lacking PHOSPHATE TRANSPORTER 4; 4 and the Asc-deficient vtc2-4 mutant of Arabidopsis (Arabidopsis thaliana). Under regular growth conditions, both Asc deficiencies caused minor alterations in photosynthesis, with no apparent signs of oxidative damage. In contrast, metabolomics analysis revealed global and largely overlapping alterations in the metabolome profiles of both Asc-deficient mutants, suggesting that chloroplastic Asc modulates plant metabolism. We observed significant alterations in amino acid metabolism, particularly in arginine metabolism, activation of nucleotide salvage pathways, and changes in secondary metabolism. In addition, proteome-wide analysis of thermostability revealed that Asc may interact with enzymes involved in arginine metabolism, the Calvin-Benson cycle, and several photosynthetic electron transport components. Overall, our results suggest that, independent of oxidative stress, chloroplastic Asc modulates the activity of diverse metabolic pathways in vascular plants and may act as an internal metabolite signal.

Effects of Aging on Successful Object Encoding: Enhanced Semantic Representations Compensate for Impaired Visual Representations.

Although episodic memory and visual processing decline substantially with healthy aging, semantic knowledge is generally spared. There is evidence that older adults' spared semantic knowledge can support episodic memory. Here, we used functional magnetic resonance imaging (fMRI) combined with representational similarity analyses (RSAs) to examine how novel visual and preexisting semantic representations at encoding predict subjective memory vividness at retrieval. Eighteen young and seventeen older adults (female and male participants) encoded images of objects during fMRI scanning and recalled these images while rating the vividness of their memories. After scanning, participants discriminated between studied images and similar lures. RSA based on a deep convolutional neural network and normative concept feature data were used to link patterns of neural activity during encoding to visual and semantic representations. Relative to young adults, the specificity of activation patterns for visual features was reduced in older adults, consistent with dedifferentiation. However, the specificity of activation patterns for semantic features was enhanced in older adults, consistent with hyperdifferentiation. Despite dedifferentiation, visual representations in early visual cortex (EVC) predicted high memory vividness in both age groups. In contrast, semantic representations in lingual gyrus (LG) and fusiform gyrus (FG) were associated with high memory vividness only in the older adults. Intriguingly, data suggests that older adults with lower specificity of visual representations in combination with higher specificity of semantic representations tended to rate their memories as more vivid. Our findings suggest that memory vividness in aging relies more on semantic representations over anterior regions, potentially compensating for age-related dedifferentiation of visual information in posterior regions.SIGNIFICANCE STATEMENT Normal aging is associated with impaired memory for events while semantic knowledge might even improve. We investigated the effects of aging on the specificity of visual and semantic information in the brain when viewing common objects and how this information enables subsequent memory vividness for these objects. Using functional magnetic resonance imaging (fMRI) combined with modeling of the stimuli we found that visual information was represented with less specificity in older than young adults while still supporting memory vividness. In contrast semantic information supported memory vividness only in older adults and especially in those individuals that had the lowest specificity of visual information. These findings provide evidence for a spared semantic memory system increasingly recruited to compensate for degraded visual representations in older age.

The effect of tinzaparin on biomarkers in FIGO stages III-IV ovarian cancer patients undergoing neoadjuvant chemotherapy - the TABANETOC trial: study protocol for a randomized clinical multicenter trial.

BACKGROUND: Tinzaparin, a low-molecular weight heparin (LMWH), has shown anti-neoplastic properties in animal models and in in vitro studies of human cancer cell lines. The reduction of CA-125 levels during neoadjuvant chemotherapy (NACT) in patients with epithelial ovarian cancer (EOC) co-varies with the prognosis; the larger the decrease in CA-125, the better the prognosis. PURPOSE: This study aims to evaluate the potential anti-neoplastic effects of tinzaparin by investigating changes in serum CA-125 levels in advanced EOC patients who receive NACT. MATERIAL AND METHODS: This is an open randomized multicenter pilot trial. Forty patients with EOC selected to receive NACT will be randomized 1:1 to receive daily addition of tinzaparin or no tinzaparin. The processing and treatment of the patients will otherwise follow the recommendations in the Swedish National Guidelines for Ovarian Cancer. Before every cycle of chemotherapy, preoperatively, and 3 weeks after the last cycle of chemotherapy, a panel of biomarkers, including CA-125, will be measured. PATIENTS: Inclusion criteria are women aged 18 years or older, World Health Organization performance status 0-1, histologically confirmed high-grade serous, endometrioid or clear cell EOC, International Federation of Gynecology and Obstetrics (FIGO) stages III-IV. In addition, a CA-125 level of ≥ 250 kIE/L at diagnosis. Exclusion criteria are contraindications to LMWH, ongoing or recent treatment with unfractionated heparin, LMWH, warfarin or non-vitamin K antagonist oral anticoagulants. INTERPRETATION: This study will make an important contribution to the knowledge of the anti-neoplastic effects of tinzaparin in EOC patients and may thus guide the planning of a future study on the impact of tinzaparin on survival in EOC.

Altered alpha/beta desynchronization during item-context binding contributes to the associative deficit in older age.

It is proposed that older adults have difficulties to bind item and context and to recruit deep, elaborative processing during encoding. Senescent changes in the oscillatory foundations of these processes are currently unclear. We recorded electroencephalography during item-context memory formation in younger (n = 57) and older (n = 55) adults. At test, we assessed memory for the items and the item-context pairs and examined encoding-related activity based on how much information was recovered at retrieval (miss < item-only < pair). Item memory was comparable between age groups while pair memory was reduced in the older adults. Theta synchronization and alpha/beta desynchronization increased linearly with the amount of information available. Single-trial theta power could not predict subsequent item memory, but predicted pair memory in an age-invariant manner, in line with a mechanism supporting associative memory. In contrast, single-trial alpha/beta power predicted both item and pair memory, in line with a mechanism reflecting the depth of information processing, and predicted pair memory less well in the older than the younger adults. Thus, theta and alpha/beta oscillations contribute differently in shaping the contents of memories and reduced processing capacity contributes to episodic memory decline in older age.

Ethical challenges in residential care facilities during COVID-19: Leaders' perspective.

BACKGROUND: Person-centred care is based on ethical principles, and it is regarded as high-quality care. Care of older persons should embrace person-centredness. During the pandemic, older persons were highlighted as a vulnerable group at risk of developing serious illness and/or suffering death from COVID-19. Several pandemic-related measures were introduced in residential care facilities (RCFs) to reduce this risk, which influenced the possibilities to lead and provide a person-centred care. AIM: This study's aim was to explore ethical challenges in relation to person-centredness during the COVID-19 pandemic, from the perspective of leaders in RCFs. RESEARCH DESIGN: The study had a qualitative descriptive design. PARTICIPANTS AND RESEARCH CONTEXT: Semi-structured interviews were conducted with 26 leaders working in RCFs in Sweden. Data were analysed using conventional content analysis. ETHICAL CONSIDERATIONS: The study was approved by the Swedish Ethical Review Authority. The participants received oral and written information about the study and gave written consent. The study was conducted in accordance with the Declaration of Helsinki. FINDINGS: The overarching ethical challenge was Having to disregard the individual needs of the person in order to protect the group and society. This included (a) Protecting the group versus promoting the older person's autonomy; (b) Being forced to lead care based on uncertainty instead of evidence; (c) Striving to provide dignified care but lacking opportunities; and (d) Going far beyond ordinary duty and endangering one's own and the staff's health. DISCUSSION: The ethical challenges meant being torn between the person's individual needs and protecting the group and society, with clashing ethical principles as a consequence. CONCLUSIONS: The leaders faced ethical situations resulting in undignified and compromised person-centred care, which has implications for stakeholders and management who need to address the work conditions in RCFs.

Out of Rhythm: Compromised Precision of Theta-Gamma Coupling Impairs Associative Memory in Old Age.

Episodic memory declines with advancing adult age. This decline is particularly pronounced when associations between items and their contexts need to be formed. According to theories of neural communication, the precise coupling of gamma power to the phase of the theta rhythm supports associative memory formation. To investigate whether age differences in associative memory are related to compromised theta-gamma coupling, we took EEG recordings during the encoding phase of an item-context association task. Fifty-eight younger (33 females) and 55 older (24 females) adults studied pictures of objects superimposed on background scenes. In a recognition test, objects were presented on old or new backgrounds, and participants responded if they had seen (1) the object and (2) the object/scene pair. Theta-gamma coupling supported pair memory formation in both age groups. Whereas pair memory was associated with coupling closer to the peak of the theta rhythm, item-only memory was associated with a deviation in phase angle relative to pair memory. Furthermore, a stable relation between coupling phase and pair memory performance demonstrated that coupling closer to the peak is beneficial for associative memory. Critically, older adults' lower pair memory was accompanied by a shift in coupling phase relative to that of younger adults. In concert, the present results are consistent with the hypothesis that decrements in the temporal precision with which gamma power is coupled to a specific theta phase underlie the decline of associative memory in normal cognitive aging.SIGNIFICANCE STATEMENT According to prominent theories of neural communication, the precise coordination of oscillatory activity enables the formation of associative memories. We propose that normal cognitive aging impairs associative memory formation by compromising the temporal precision of neural communication. We show that the coupling of high-frequency gamma power to low-frequency theta phase supports associative memory formation in both younger and older adults, with coupling closer to the theta peak benefitting memory performance. However, compared with younger adults, the coupling phase angle is shifted in time and is more variable in the older adults. We conclude that alterations in the precise timing of theta-gamma coupling contribute to adult age differences in associative memory.

Coordinated pyruvate kinase activity is crucial for metabolic adaptation and cell survival during mitochondrial dysfunction.

Deoxyguanosine kinase (DGUOK) deficiency causes mtDNA depletion and mitochondrial dysfunction. We reported long survival of DGUOK knockout (Dguok-/-) mice despite low (<5%) mtDNA content in liver tissue. However, the molecular mechanisms enabling the extended survival remain unknown. Using transcriptomics, proteomics and metabolomics followed by in vitro assays, we aimed to identify the molecular pathways involved in the extended survival of the Dguok-/- mice. At the early stage, the serine synthesis and folate cycle were activated but declined later. Increased activity of the mitochondrial citric acid cycle (TCA cycle) and the urea cycle and degradation of branched chain amino acids were hallmarks of the extended lifespan in DGUOK deficiency. Furthermore, the increased synthesis of TCA cycle intermediates was supported by coordination of two pyruvate kinase genes, PKLR and PKM, indicating a central coordinating role of pyruvate kinases to support the long-term survival in mitochondrial dysfunction.

Pan-cancer application of a lung-adenocarcinoma-derived gene-expression-based prognostic predictor.

Gene-expression profiling can be used to classify human tumors into molecular subtypes or risk groups, representing potential future clinical tools for treatment prediction and prognostication. However, it is less well-known how prognostic gene signatures derived in one malignancy perform in a pan-cancer context. In this study, a gene-rule-based single sample predictor (SSP) called classifier for lung adenocarcinoma molecular subtypes (CLAMS) associated with proliferation was tested in almost 15 000 samples from 32 cancer types to classify samples into better or worse prognosis. Of the 14 malignancies that presented both CLAMS classes in sufficient numbers, survival outcomes were significantly different for breast, brain, kidney and liver cancer. Patients with samples classified as better prognosis by CLAMS were generally of lower tumor grade and disease stage, and had improved prognosis according to other type-specific classifications (e.g. PAM50 for breast cancer). In all, 99.1% of non-lung cancer cases classified as better outcome by CLAMS were comprised within the range of proliferation scores of lung adenocarcinoma cases with a predicted better prognosis by CLAMS. This finding demonstrates the potential of tuning SSPs to identify specific levels of for instance tumor proliferation or other transcriptional programs through predictor training. Together, pan-cancer studies such as this may take us one step closer to understanding how gene-expression-based SSPs act, which gene-expression programs might be important in different malignancies, and how to derive tools useful for prognostication that are efficient across organs.

The Roles of Nicotinamide Adenine Dinucleotide Phosphate Reoxidation and Ammonium Assimilation in the Secretion of Amino Acids as Byproducts of Clostridium thermocellum.

Clostridium thermocellum is a cellulolytic thermophile that is considered for the consolidated bioprocessing of lignocellulose to ethanol. Improvements in ethanol yield are required for industrial implementation, but the incompletely understood causes of amino acid secretion impede progress. In this study, amino acid secretion was investigated via gene deletions in ammonium-regulated, nicotinamide adenine dinucleotide phosphate (NADPH)-supplying and NADPH-consuming pathways as well as via physiological characterization in cellobiose-limited or ammonium-limited chemostats. First, the contribution of the NADPH-supplying malate shunt was studied with strains using either the NADPH-yielding malate shunt (Δppdk) or a redox-independent conversion of PEP to pyruvate (Δppdk ΔmalE::Peno-pyk). In the latter, branched-chain amino acids, especially valine, were significantly reduced, whereas the ethanol yield increased from 46 to 60%, suggesting that the secretion of these amino acids balances the NADPH surplus from the malate shunt. The unchanged amino acid secretion in Δppdk falsified a previous hypothesis on an ammonium-regulated PEP-to-pyruvate flux redistribution. The possible involvement of another NADPH-supplier, namely, NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (nfnAB), was also excluded. Finally, the deletion of glutamate synthase (gogat) in ammonium assimilation resulted in the upregulation of NADPH-linked glutamate dehydrogenase activity and decreased amino acid yields. Since gogat in C. thermocellum is putatively annotated as ferredoxin-linked, a claim which is supported by the product redistribution observed in this study, this deletion likely replaced ferredoxin with NADPH in ammonium assimilation. Overall, these findings indicate that a need to reoxidize NADPH is driving the observed amino acid secretion, likely at the expense of the NADH needed for ethanol formation. This suggests that metabolic engineering strategies that simplify the redox metabolism and ammonium assimilation can contribute to increased ethanol yields. IMPORTANCE Improving the ethanol yield of C. thermocellum is important for the industrial implementation of this microorganism in consolidated bioprocessing. A central role of NADPH in driving amino acid byproduct formation was demonstrated by eliminating the NADPH-supplying malate shunt and separately by changing the cofactor specificity in ammonium assimilation. With amino acid secretion diverting carbon and electrons away from ethanol, these insights are important for further metabolic engineering to reach industrial requirements on ethanol yield. This study also provides chemostat data that are relevant for training genome-scale metabolic models and for improving the validity of their predictions, especially considering the reduced degree-of-freedom in the redox metabolism of the strains generated here. In addition, this study advances the fundamental understanding on the mechanisms underlying amino acid secretion in cellulolytic Clostridia as well as on the regulation and cofactor specificity in ammonium assimilation. Together, these efforts aid in the development of C. thermocellum for the sustainable consolidated bioprocessing of lignocellulose to ethanol with minimal pretreatment.

Evaluation of measures related to dosimetric uncertainty of VMAT plans.

Dosimetric uncertainty is most often not included in the process of creating and selecting plans for treatment. Treatment planning and the physician's choice of treatment plan is instead often based only on evaluation of clinical goals of the calculated absorbed dose distribution. Estimation of the dosimetric uncertainty could potentially have impact in the process of comparing and selecting volumetric modulated arc therapy (VMAT) plans. In this study, different measures for estimation of dosimetric uncertainty based on treatment plan parameters for plans with similar dose distributions were evaluated. VMAT plans with similar dose distributions but with different treatment plan designs were created using three different optimization methods for each of ten patient cases (tonsil and prostate cancer). Two plans were optimized in Eclipse, one with and one without the use of aperture shape controller, and one plan was optimized in RayStation. The studied measures related to dosimetric uncertainty of treatment plans were aperture-based complexity metric analysis, investigation of modulation level of multi leaf collimator leaves, gantry speed and dose rate, quasi-3D measurements and evaluation of simulations of realistic delivery variations. The results showed that there can be variations in dosimetric uncertainty for treatment plans with similar dose distributions. Dosimetric uncertainty assessment could therefore have impact on the choice of plan to be used for treatment and lead to a decrease in the uncertainty level of the delivered absorbed dose distribution. This study showed that aperture shape complexity had a larger impact on dosimetric uncertainty compared to modulation level of MLC, gantry speed and dose rate.

Subjective and objective evaluation of masticatory function in patients with bimaxillary implant-supported prostheses.

BACKGROUND: People perform poorly in masticatory function tests despite well-functioning prostheses. However, it is unclear whether there is an agreement between subjective and objective measures of mastication. OBJECTIVES: To investigate the association between subjective and objective measures of masticatory function in patients with bimaxillary implant-supported prostheses. MATERIALS AND METHODS: An experimental group (n = 25, age = 70.6 ± 7.5 years, eight women) with bimaxillary implant-supported fixed prostheses and a control group (n = 25, age = 69.0 ± 5.3, 13 women) with natural dentition were recruited. The participants in the experimental group were included if they had been using the prosthesis for at least a year and had no obvious complaints with their prostheses. The control group was people with natural dentition and without any prostheses or complaints related to the masticatory system. The masticatory function was evaluated objectively with food comminution and mixing ability tests, and subjectively with jaw function limitation scale (JLFS) and oral health impact profile (OHIP). RESULTS: The experimental group performed poorly in both objective tests (p < .001). However, there was no significant differences between the two groups in total JFLS (p = .114) and OHIP (p = .312) scores. Though, there were certain positive correlations between the food comminution test and JFLS subdomains in the control group, and a positive correlation between food comminution test and specific subdomains of OHIP in the experimental group indicating poor correlation between the subjective and objective measures. CONCLUSION: Although patients with implant prostheses show poor masticatory performance, there is no agreement in the objective and subjective measures of mastication.

Meanings and functions of different types of heart images in the communication of doctors and patients.

Images in medical communication are appreciated by both professionals and patients, but we know little about how they are actually used in clinical practice and in the learning processes of patients. This qualitative study investigates the use and the meaning potential of two different types of heart images: the hand-drawn doctor's sketch and the digital illustration from the web. The analysis starts with how these are recontextualised in social media, tracks them back to their original contexts and finally explores their material resources. The analytical perspective is that of social semiotics and multimodal discourse and interaction analysis. While the hand-drawn sketch is recontextualised as a witness of the specific consultation, the digital illustration is used to focus on the heart defect as such. It is also shown how the act of drawing works as a means for framing and structuring the consultation, slowing down the pace and reducing context and detail and thus focussing on what is uniquely relevant. While the digital and more realistic illustration is technically neutral and objective and is free from unique context, the drawing on paper is physically tied to its context of origin, which is also its main resource.

Dosimetric effects of respiratory motion during stereotactic body radiation therapy of lung tumors.

BACKGROUND: Respiratory-induced lung tumor motion may affect the delivered dose in stereotactic body radiation therapy (SBRT). Previous studies are often based on phantom studies for one specific treatment technique. In this study, the dosimetric effect of tumor motion was quantified in real patient geometries for different modulated treatments and tumor motion amplitudes for lung-SBRT. MATERIAL AND METHODS: A simulation method using deformable image registrations and 4-dimensional computed tomographies (4DCT) was developed to assess the dosimetric effects of tumor motion. The method was evaluated with ionization chamber and Gafchromic film measurements in a thorax phantom and used to simulate the effect for 15 patients with lung tumors moving 7.3-27.4 mm. Four treatment plans with different complexities were created for each patient and the motion-induced dosimetric effect to the gross tumor volume (GTV) was simulated. The difference between the planned dose to the static tumor and the simulated delivered dose to the moving tumor was quantified for the near minimum (D98%), near maximum (D2%) and mean dose (Dmean) to the GTV as well as the largest observed local difference within the GTV (Maxdiff). RESULTS: No correlation was found between the dose differences and the tumor motion amplitude or plan complexity. However, the largest deviations were observed for tumors moving >15.0 mm. The simulated delivered dose was within 2.5% from the planned dose for D98% (tumors moving <15 mm) and within 3.3% (tumors moving >15 mm). The corresponding values were 1.7% vs. 6.4% (D2%); 1.7% vs. 2.4% (Dmean) and 8.9% vs. 35.2% (Maxdiff). Using less complex treatment techniques minimized Maxdiff for tumors moving >15.0 mm. CONCLUSION: The dosimetric effects of respiratory-induced motion during lung SBRT are patient and plan specific. The magnitude of the dosimetric effect cannot be assessed solely based upon tumor motion amplitude or plan complexity.

Head and neck cancer patient positioning using synthetic CT data in MRI-only radiation therapy.

PURPOSE: The accuracy and precision of patient positioning is crucial in radiotherapy; however, there are no publications available using synthetic computed tomography (sCT) that evaluate rotations in head and neck (H&N) patients positioning or the effect of translation and rotation combined. The aim of this work was to evaluate the differences between using sCT with the CT for 2D- and 3D-patient positioning in a magnetic resonance imaging (MRI)-only workflow. METHODS: This study included 14 H&N cancer patients, with generated sCT data (MRI Planner v2.2) and the CT deformably registered to the MRI. Patient positioning was evaluated by comparing sCT against CT data: 3D cone beam CT (CBCT) was registered to the deformed CT (dCT) and sCT in six degrees of freedom (DoF) with a rigid auto-registration algorithm and bone threshold, and 2D deformed digital reconstructed radiographs (dDRR) and synthetic DRRs (sDRR) were manually registered to orthogonal projections in five DoF by six blinded observers. The difference in displacement in all DoF were calculated for dCT and sCT, as well as for dDRR and sDRR. The interobserver variation was evaluated by separate application of the paired dDRR and sDRR registration matrices to the original coordinates of the planning target volume (PTV) structures and calculation of the Euclidean distance between the corresponding points. The Dice similarity coefficient (DSC) was calculated between dDRR/sDRR-registered PTVs. RESULTS: The mean difference in patient positioning using CBCT was <0.7 mm and <0.3° and using orthogonal projections <0.4 mm and <0.2° in all directions. The maximum Euclidean distance was 5.1 mm, the corresponding mean (1SD) Euclidean distance and mean DSC were 3.5 ± 0.7 mm and 0.93, respectively. CONCLUSIONS: This study shows that the sCT-based patient positioning gives a comparable result with that based on CT images, allowing sCT to replace CT as reference for patient treatment positioning.

A gene expression-based single sample predictor of lung adenocarcinoma molecular subtype and prognosis.

Disease recurrence in surgically treated lung adenocarcinoma (AC) remains high. New approaches for risk stratification beyond tumor stage are needed. Gene expression-based AC subtypes such as the Cancer Genome Atlas Network (TCGA) terminal-respiratory unit (TRU), proximal-inflammatory (PI) and proximal-proliferative (PP) subtypes have been associated with prognosis, but show methodological limitations for robust clinical use. We aimed to derive a platform independent single sample predictor (SSP) for molecular subtype assignment and risk stratification that could function in a clinical setting. Two-class (TRU/nonTRU=SSP2) and three-class (TRU/PP/PI=SSP3) SSPs using the AIMS algorithm were trained in 1655 ACs (n = 9659 genes) from public repositories vs TCGA centroid subtypes. Validation and survival analysis were performed in 977 patients using overall survival (OS) and distant metastasis-free survival (DMFS) as endpoints. In the validation cohort, SSP2 and SSP3 showed accuracies of 0.85 and 0.81, respectively. SSPs captured relevant biology previously associated with the TCGA subtypes and were associated with prognosis. In survival analysis, OS and DMFS for cases discordantly classified between TCGA and SSP2 favored the SSP2 classification. In resected Stage I patients, SSP2 identified TRU-cases with better OS (hazard ratio [HR] = 0.30; 95% confidence interval [CI] = 0.18-0.49) and DMFS (TRU HR = 0.52; 95% CI = 0.33-0.83) independent of age, Stage IA/IB and gender. SSP2 was transformed into a NanoString nCounter assay and tested in 44 Stage I patients using RNA from formalin-fixed tissue, providing prognostic stratification (relapse-free interval, HR = 3.2; 95% CI = 1.2-8.8). In conclusion, gene expression-based SSPs can provide molecular subtype and independent prognostic information in early-stage lung ACs. SSPs may overcome critical limitations in the applicability of gene signatures in lung cancer.

Severe mtDNA depletion and dependency on catabolic lipid metabolism in DGUOK knockout mice.

Deoxyguanosine kinase (DGUOK) provides guanosine and adenosine nucleotides for mitochondrial DNA (mtDNA) replication, and its deficiency in humans leads to hepatocerebral mtDNA depletion syndrome or to isolated hepatic disease. There are poor treatment options for DGUOK deficiency and the aim of this study was to generate a model for further studies of the disease that could reveal novel treatment strategies. We report a Dguok-deficient mouse strain that, similar to humans, is most severely affected in the liver. The Dguok complete knockout mice (Dguok-/-) were born normal, but began to lose weight at week 6. A change of fur color from black to blueish grey started at week 16 and was complete at week 20. The movements and behavior were indistinguishable compared to wild-type (wt) mice. A decrease of mtDNA copy number occurred in multiple tissues, with the liver being the most severely affected. The mtDNA-encoded protein cytochrome c oxidase was much lower in Dguok-/- liver tissue than in the wt, whereas the expression of the nuclear-encoded succinate dehydrogenase complex subunit A was unaffected. Histopathology showed severe alterations and immunohistochemistry showed signs of both oxidative stress and regeneration in Dguok-/- liver. The subcutaneous fat layer was undetectable in Dguok-/-, which, in addition to gene expression analysis, indicated an altered lipid metabolism. We conclude that Dguok has a major role for the synthesis of deoxyribonucleotides for mtDNA replication particularly in the liver, similar to the human disorder. Our data also show a catabolic lipid metabolism in liver tissue of Dguok-/-.

The YIN and YANG of lipoproteins in developing and preventing infectious arthritis by Staphylococcus aureus.

Rapid bone destruction often leads to permanent joint dysfunction in patients with septic arthritis, which is mainly caused by Staphylococcus aureus (S. aureus). Staphylococcal cell wall components are known to induce joint inflammation and bone destruction. Here, we show that a single intra-articular injection of S. aureus lipoproteins (Lpps) into mouse knee joints induced chronic destructive macroscopic arthritis through TLR2. Arthritis was characterized by rapid infiltration of neutrophils and monocytes. The arthritogenic effect was mediated mainly by macrophages/monocytes and partially via TNF-α but not by neutrophils. Surprisingly, a S. aureus mutant lacking Lpp diacylglyceryl transferase (lgt) caused more severe joint inflammation, which coincided with higher bacterial loads of the lgt mutant in local joints than those of its parental strain. Coinjection of pathogenic S. aureus LS-1 with staphylococcal Lpps into mouse knee joints caused improved bacterial elimination and diminished bone erosion. The protective effect of the Lpps was mediated by their lipid moiety and was fully dependent on TLR2 and neutrophils. The blocking of CXCR2 on neutrophils resulted in total abrogation of the protective effect of the Lpps. Our data demonstrate that S. aureus Lpps elicit innate immune responses, resulting in a double-edged effect. On the one hand, staphylococcal Lpps boost septic arthritis. On the other hand, Lpps act as adjuvants and activate innate immunity, which could be useful for combating infections with multiple drug-resistant strains.

Intracellular Neutrophil Oxidants: From Laboratory Curiosity to Clinical Reality.

The phagocyte NADPH oxidase is responsible for the neutrophil's great capacity to produce reactive oxygen species (ROS). The NADPH oxidase can be assembled in the plasma membrane, as well as in membranes of intracellular vesicles, giving neutrophils the ability to direct ROS production to distinct subcellular sites. Neutrophil ROS contribute to microbial killing, trigger formation of neutrophil extracellular traps and appear to partake in inflammation control. Consequently, function-disrupting mutations in the NADPH oxidase lead to chronic granulomatous disease, characterized by severe infections and inflammatory disorders. Recent experimental data and description of a novel chronic granulomatous disease subtype (p40phox-deficiency) imply that ROS generated in intracellular compartments are key for NETosis and for controlling inflammatory signaling. We foresee boosted interest in intracellular ROS production. To fully understand where and how such ROS function, however, limitations of assay systems to measure ROS need to be appreciated, and the development of novel techniques/reagents would be highly useful.

Surface guided frameless positioning for lung stereotactic body radiation therapy.

BACKGROUND AND PURPOSE: When treating lung tumors with stereotactic body radiation therapy (SBRT), patient immobilization is of outmost importance. In this study, the intra-fractional shifts of the patient (based on bony anatomy) and the tumor (based on the visible target volume) are quantified, and the associated impact on the delivered dose is estimated for a frameless immobilization approach in combination with surface guided radiation therapy (SGRT) monitoring. METHODS: Cone beam computed tomographies (CBCT) were collected in free breathing prior and after each treatment for 25 patients with lung tumors, in total 137 fractions. The CBCT collected after each treatment was registered to the CBCT collected before each treatment with focus on bony anatomy to determine the shift of the patient, and with focus on the visible target volume to determine the shift of the tumor. Rigid registrations with 6 degrees of freedom were used. The patients were positioned in frameless immobilizations with their position and respiration continuously monitored by a commercial SGRT system. The patients were breathing freely within a preset gating window during treatment delivery. The beam was automatically interrupted if isocenter shifts >4 mm or breathing amplitudes outside the gating window were detected by the SGRT system. The time between the acquisition of the CBCTs was registered for each fraction to examine correlations between treatment time and patient shift. The impact of the observed shifts on the dose to organs at risk (OAR) and the gross tumor volume (GTV) was assessed. RESULTS: The shift of the patient in the CBCTs was ≤2 mm for 132/137 fractions in the vertical (vrt) and lateral (lat) directions, and 134/137 fractions in the longitudinal (lng) direction and ≤4 mm in 134/137 (vrt) and 137/137 (lat, lng) of the fractions. The shift of the tumor was ≤2 mm in 116/137 (vrt), 123/137 (lat) and 115/137 (lng) fractions and ≤4 mm in 136/137 (vrt), 137/137 (lat), and 135/137 (lng) fractions. The maximal observed shift in the evaluated CBCT data was 4.6 mm for the patient and 7.2 mm for the tumor. Rotations were ≤3.3ᵒ for all fractions and the mean/standard deviation were 0.2/1.0ᵒ (roll), 0.1/0.8ᵒ (yaw), and 0.3/1.0ᵒ (pitch). The SGRT system interrupted the beam due to intra-fractional isocenter shifts >4 mm for 21% of the fractions, but the patients always returned within tolerance without the need of repositioning. The maximal observed isocenter shift by the SGRT system during the beam holds was 8 mm. For the respiration monitoring, the beam was interrupted at least one time for 54% of the fractions. The visual tumor was within the planned internal target volume (ITV) for 136/137 fractions in the evaluated CBCT data collected at the end of each fraction. For the fraction where the tumor was outside the ITV, the D98% for the GTV decreased with 0.4 Gy. For the OARs, the difference between planned and estimated dose from the CBCT data (D2% or Dmean ) was ≤2.6% of the prescribed PTV dose. No correlation was found between treatment time and the magnitude of the patient shift. CONCLUSIONS: Using SGRT for motion management and respiration monitoring in combination with a frameless immobilization is a feasible approach for lung SBRT.

Activation of Mitochondrial 2-Oxoglutarate Dehydrogenase by Cocarboxylase in Human Lung Adenocarcinoma Cells A549 Is p53/p21-Dependent and Impairs Cellular Redox State, Mimicking the Cisplatin Action.

Genetic up-regulation of mitochondrial 2-oxoglutarate dehydrogenase is known to increase reactive oxygen species, being detrimental for cancer cells. Thiamine diphosphate (ThDP, cocarboxylase) is an essential activator of the enzyme and inhibits p53-DNA binding in cancer cells. We hypothesize that the pleiotropic regulator ThDP may be of importance for anticancer therapies. The hypothesis is tested in the present work on lung adenocarcinoma cells A549 possessing the p53-p21 pathway as fully functional or perturbed by p21 knockdown. Molecular mechanisms of ThDP action on cellular viability and their interplay with the cisplatin and p53-p21 pathways are characterized. Despite the well-known antioxidant properties of thiamine, A549 cells exhibit decreases in their reducing power and glutathione level after incubation with 5 mM ThDP, not observed in non-cancer epithelial cells Vero. Moreover, thiamine deficiency elevates glutathione in A549 cells. Viability of the thiamine deficient A549 cells is increased at a low (0.05 mM) ThDP. However, the increase is attenuated by 5 mM ThDP, p21 knockdown, specific inhibitor of the 2-oxoglutarate dehydrogenase complex (OGDHC), or cisplatin. Cellular levels of the catalytically competent ThDP·OGDHC holoenzyme are dysregulated by p21 knockdown and correlate negatively with the A549 viability. The inverse relationship between cellular glutathione and holo-OGDHC is corroborated by their comparison in the A549 and Vero cells. The similarity, non-additivity, and p21 dependence of the dual actions of ThDP and cisplatin on A549 cells manifest a common OGDHC-mediated mechanism of the viability decrease. High ThDP saturation of OGDHC compromises the redox state of A549 cells under the control of p53-p21 axes.