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Late-stage colorectal cancer (CRC) is still a clinically challenging problem. The activity of the tumor suppressor p53 is regulated via post-translational modifications (PTMs). While the relevance of p53 C-terminal acetylation for transcriptional regulation is well defined, it is unknown whether this PTM controls mitochondrially mediated apoptosis directly. We used wild-type p53 or p53-negative human CRC cells, cells with acetylation-defective p53, transformation assays, CRC organoids, and xenograft mouse models to assess how p53 acetylation determines cellular stress responses. The topoisomerase-1 inhibitor irinotecan induces acetylation of several lysine residues within p53. Inhibition of histone deacetylases (HDACs) with the class I HDAC inhibitor entinostat synergistically triggers mitochondrial damage and apoptosis in irinotecan-treated p53-positive CRC cells. This specifically relies on the C-terminal acetylation of p53 by CREB-binding protein/p300 and the presence of C-terminally acetylated p53 in complex with the proapoptotic BCL2 antagonist/killer protein. This control of C-terminal acetylation by HDACs can mechanistically explain why combinations of irinotecan and entinostat represent clinically tractable agents for the therapy of p53-proficient CRC.

Abstract Background The Bonfils intubation fibrescope is a promising alternative device for securing the airway. We examined the success rate of intubation and the ease of use in standardized simulated difficult airway scenarios by physicians. We compared the Bonfils to a classical laryngoscope with Macintosh blade. Methods 30 physicians untrained in the use of rigid fibrescopes but experienced in airway management performed endotracheal intubation in an airway manikin (SimMan, Laerdal, Kent, UK) with three different airway conditions. We evaluated the success rate using the Bonfils (Karl Storz, Tuttlingen, Germany) or the Macintosh laryngoscope, the time needed for securing the airway, and subjective rating of both techniques. Results In normal airway all intubations were successful using laryngoscope (100%) vs. 82% using the Bonfils (p < 0.05). In the scenario "tongue oedema" success rate using the Macintosh laryngoscope was 67% and 83% using the Bonfils. In the scenario "decreased cervical range of motion with jaw trismus", success rate using the Macintosh laryngoscope was 84% vs. 76%. In difficult airway scenarios time until airway was secured did not differ between the two devices. Use of Bonfils was rated "easier" in both difficult airway scenarios. Conclusion The Bonfils can be successfully used by physicians unfamiliar with this technique in an airway manikin. The airway could be secured with at least the same success rate as using a Macintosh laryngoscope in difficult airway scenarios. Use of the Bonfils did not delay intubation in the presence of a difficult airway. These results indicate that intensive special training is advised to use the Bonfils effectively in airway management.

Aim: The purpose of this study was to investigate whether a clinical effect of Lactobacillus reuteri in supportive periodontal therapy can already be detected with a minimum dose of one tablet a day and a minimum observation and intake period of 3 months. Materials and Methods: 28 patients with stage III and IV periodontitis undergoing periodontal follow-up were randomly divided into two groups receiving a lozenge containing L. reuteri or a placebo preparation daily for 90 days. After 0, 4, 8, and 12 weeks, the parameters bleeding on probing (BoP), plaque control record (PCR), periodontal probing depth (PPD), and clinical attachment level (CAL) were recorded in the test and control groups. Results: The results sed a different effect of L. reuteri on the respective patients. In certain patients, clinical parameters worsened or remained largely unchanged. However, in other patients, there were positive effects on the clinical parameters. In the overall analysis, BoP was the only clinical parameter that was statistically significantly reduced. Conclusions: The oral administration of one lozenge per day for 3 months with L. reuteri in supportive periodontal therapy might have a positive influence on clinical parameters in supportive periodontal therapy, depending on the individual.

Late‐stage colorectal cancer (CRC) is still a clinically challenging problem. The activity of the tumor suppressor p53 is regulated via post‐translational modifications (PTMs). While the relevance of p53 C‐terminal acetylation for transcriptional regulation is well defined, it is unknown whether this PTM controls mitochondrially mediated apoptosis directly. We used wild‐type p53 or p53‐negative human CRC cells, cells with acetylation‐defective p53, transformation assays, CRC organoids, and xenograft mouse models to assess how p53 acetylation determines cellular stress responses. The topoisomerase‐1 inhibitor irinotecan induces acetylation of several lysine residues within p53. Inhibition of histone deacetylases (HDACs) with the class I HDAC inhibitor entinostat synergistically triggers mitochondrial damage and apoptosis in irinotecan‐treated p53‐positive CRC cells. This specifically relies on the C‐terminal acetylation of p53 by CREB‐binding protein/p300 and the presence of C‐terminally acetylated p53 in complex with the proapoptotic BCL2 antagonist/killer protein. This control of C‐terminal acetylation by HDACs can mechanistically explain why combinations of irinotecan and entinostat represent clinically tractable agents for the therapy of p53‐proficient CRC.

Abstract Background Despite considerable medical proceedings, cancer is still a leading cause of death. Major problems for tumor therapy are chemoresistance as well as toxic side effects. In recent years, the additional treatment with the antidiabetic drug metformin during chemotherapy showed promising results in some cases. The aim of this study was to develop an in vitro tumor therapy model in order to further investigate the potential of a combined chemotherapy with metformin. Methods Cytotoxic effects of a combined treatment on BALB/c fibroblasts were proven by the resazurin assay. Based on the BALB/c cell transformation assay, the BALB/c tumor therapy model was established successfully with four different and widely used chemotherapeutics from different categories. Namely, Doxorubicin as a type-II isomerase inhibitor, Docetaxel as a spindle toxin, Mitomycin C as an alkylating agent and 5-Fluorouracil as an antimetabolite. Moreover, glucose consumption in the medium supernatant was measured and protein expressions were determined by Western Blotting. Results Initial tests for the combined treatment with metformin indicated unexpected results as metformin could partly mitigate the cytotoxic effects of the chemotherapeutic agents. These results were further confirmed as metformin induced resistance to some of the drugs when applied simultaneously in the tumor therapy model. Mechanistically, an increased glucose consumption was observed in non-transformed cells as well as in the mixed population of malignant transformed cell foci and non-transformed monolayer cells, suggesting that metformin could also increase glucose consumption in transformed cells. Conclusion In conclusion, this study suggests a cautious use of metformin during chemotherapy. Moreover, the BALB/c tumor therapy model offers a potent tool for further mechanistic studies of drug-drug interactions during cancer therapy. Graphical abstract

Abstract Introduction Ewing's sarcoma is an aggressive childhood malignancy whose outcome has not substantially improved over the last two decades. In this study, combination treatments of the HSP90 inhibitor AUY922 with either the ATR inhibitor VE821 or the ATM inhibitor KU55933 were investigated for their effectiveness in Ewing's sarcoma cells. Methods Effects were determined in p53 wild-type and p53 null Ewing's sarcoma cell lines by flow cytometric analyses of cell death, mitochondrial depolarization and cell-cycle distribution as well as fluorescence and transmission electron microscopy. They were molecularly characterized by gene and protein expression profiling, and by quantitative whole proteome analysis. Results AUY922 alone induced DNA damage, apoptosis and ER stress, while reducing the abundance of DNA repair proteins. The combination of AUY922 with VE821 led to strong apoptosis induction independent of the cellular p53 status, yet based on different molecular mechanisms. p53 wild-type cells activated pro-apoptotic gene transcription and underwent mitochondria-mediated apoptosis, while p53 null cells accumulated higher levels of DNA damage, ER stress and autophagy, eventually leading to apoptosis. Impaired PI3K/AKT/mTOR signaling further contributed to the antineoplastic combination effects of AUY922 and VE821. In contrast, the combination of AUY922 with KU55933 did not produce a cooperative effect. Conclusion Our study reveals that HSP90 and ATR inhibitor combination treatment may be an effective therapeutic approach for Ewing's sarcoma irrespective of the p53 status.

Legume grains such as field peas and field beans can be produced on a local level, and may be reliable sources of dietary protein and energy apart from common soybean and rapeseed meals. In ruminants, protein, starch, and carbohydrates from peas and field beans are fermented in large part before reaching the small intestine. The objective of this study was to evaluate the effects of a combination of ensiling and hydro-thermic treatment (i.e., toasting at 160 °C for 30 min) of grains of peas and field beans on the concentrations of post-ruminal crude protein (PRCP) and rumen-undegraded protein (RUP). Moreover, 24-h gas production and methane production were measured. For this, an in vitro batch culture system with ruminal fluid from sheep was used. Rumen-undegraded protein was determined using the Streptomyces griseus protease test. Scanning electron micrographs were used to visualize morphological changes of starch granules and their joint matrices in peas and field beans after ensiling, toasting, or a combination of both. Native pea grains contained crude protein (CP) at 199 g/kg DM, PRCP at 155 g/kg DM at a ruminal passage rate of 0.08/h (Kp8), RUP at 33 g/kg DM at Kp8, and starch at 530 g/kg DM. Native field beans contained CP at 296 g/kg DM, PRCP at 212 g/kg DM at Kp8, RUP at 54 g of/kg DM at Kp8, and starch at 450 g/kg DM. The PRCP did not considerably differ among native and treated peas or field beans. Especially in the peas, RUP at Kp8 increased after ensiling by 10 g/kg DM (i.e., 30%; P

Abstract Pseudomonas aeruginosa is a Gram‐negative bacterium of the proteobacteria class, and one of the most common causes of nosocomial infections. For example, it causes chronic pneumonia in cystic fibrosis patients. Patient sputum contains 2‐heptyl‐4‐hydroxyquinoline N‐oxide [HQNO] and Pseudomonas quorum sensing molecules such as the Pseudomonas quinolone signal [PQS]. It is known that HQNO inhibits the enzyme activity of mitochondrial and bacterial complex III at the Qi (quinone reduction) site, but the target of PQS is not known. In this work we have shown that PQS has a negative effect on mitochondrial respiration in HeLa and A549 cells. It specifically inhibits the complex I of the respiratory chain. In vitro analyses showed a partially competitive inhibition with respect to ubiquinone at the IQ site. In competing studies with Rotenone, PQS suppressed the ROS‐promoting effect of Rotenone, which is typical for a B‐type inhibitor. Prolonged incubation with PQS also had an effect on the activity of complex III.

The precise cell dynamics of early development have not yet been visualized. Here, the authors use custom 4-lens light sheet microscopy to image and analyze the dynamics of all three fluorescently labeled germlayers, yielding a comprehensive, pan-embryo description of early zebrafish gastrulation.

Abstract Background In Germany, a decreasing number of general practitioners (GPs) face a growing number of patients with multimorbidity. Whilst care for patients with multimorbidity involves various healthcare providers, the coordination of this care is one of the many responsibilities of GPs. The aims of this study are to identify the barriers to the successful coordination of multimorbid patient care and these patients’ complex needs, and to explore the support needed by GPs in the care of multimorbid patients. Interviewees were asked for their opinion on concepts which involve the support by additional employees within the practice or, alternatively, external health care professionals, providing patient navigation. Methods Thirty-two semi-structured, qualitative interviews were conducted with 16 GPs and 16 medical practice assistants (MPAs) from 16 different practices in Berlin. A MPA is a qualified non-physician practice employee. He or she undergoes a three years vocational training which qualifies him or her to provide administrative and clinical support. The interviews were digitally recorded, transcribed and analysed using the framework analysis methodology. Results The results of this paper predominantly focus on GPs’ perspectives of coordination within and external to general practice. Coordination in the context of care for multimorbid patients consists of a wide range of different tasks. Organisational and administrative obstacles under the regulatory framework of the German healthcare system, and insufficient communication with other healthcare providers constitute barriers described by the interviewed GPs and MPAs. In order to ensure optimal care for patients with multimorbidity, GPs may have to delegate responsibilities associated with coordinating tasks. GPs consider the deployment of an additional specifically qualified employee inside the general practice to take on coordinative and social and legal duties to be a viable option. Conclusions The cross-sectoral cooperation between all involved key players working within the healthcare system, as well as the coordination of the whole care process, is seemingly challenging for GPs within the complex care system of multimorbid patients. GPs are generally open to the assignment of a person to support them in coordination tasks, preferably situated within the practice team.

Selectively methylated analogues of naturally occurring 2-heptyl-4(1H)-quinolones, which are alkaloids common within the Rutaceae family and moreover are associated with quorum sensing and virulence of the human pathogen Pseudomonas aeruginosa, have been prepared. While the synthesis by direct methylation was successful for 3-unsubstituted 2-heptyl-4(1H)-quinolones, methylated derivatives of the Pseudomonas quinolone signal (PQS) were synthesized from 3-iodinated quinolones by methylation and iodine–metal exchange/oxidation. The two N- and O-methylated derivatives of the PQS showed strong quorum sensing activity comparable to that of PQS itself. Staphylococcus aureus, another pathogenic bacterium often co-occurring with P. aeruginosa especially in the lung of cystic fibrosis patients, was inhibited in planktonic growth and cellular respiration by the 4-O-methylated derivatives of HQNO and HHQ, respectively.

TraCurate is an open-source software tool to curate and manually annotate cell tracking data from time-lapse microscopy. Although many studies of cellular behaviour require high-quality, long-term observations of single cells across several generations, automated tracking of individual cells is often imperfect and typically yields fragmented results that still contain many errors. TraCurate supports the user to efficiently curate and extract complete cell tracks and genealogies from a variety of cell tracking data. Source code and binary packages for TraCurate and all related tools are freely available for Linux, macOS, and Windows at https://tracurate.gitlab.io/.

The risk of the development of tendon disorders or ruptures increases with age, but it is unclear whether intrinsic fitness during lifetime might also affect tendon properties. To investigate this, a contrasting rat model of high-capacity runners (HCR with high intrinsic fitness) and low-capacity runners (LCR with low intrinsic fitness) was employed. Histological and molecular changes in rotator cuff (RC) tendons from 10 weeks old (young; HCR-10 and LCR-10) and 100 weeks old (old; HCR-100 and LCR-100) female rats were investigated. Age-dependent changes of RC tendons observed in HCR and LCR were increase of weight, decrease of tenocytes and RNA content, reduction of the wavy pattern of collagen and elastic fibers, repressed expression of Col1a1, Eln, Postn, Tnmd, Tgfb3 and Egr1 and reduction of the Col1:Col3 and Col1:Eln ratio. The LCR rats showed less physical activity, increased body weight, signs of metabolic disease and a reduced life expectancy. Their RC tendons revealed increased weight (more than age-dependent) and enlargement of the tenocyte nuclei (consistent with degenerative tendons). Low intrinsic fitness led to repressed expression of a further nine genes (Col3a1, Fbn1, Dcn, Tnc, Scx, Mkx, Bmp1, Tgfb1, Esr1) as well as the rise of the Col1:Col3 and Col1:Eln ratios (related to the lesser expression of Col3a1 and Eln). The intrinsic fitness influences the female RC tendons at least as much as age. Lower intrinsic fitness accelerates aging of RC tendons and leads to further impairment; this could result in decreased healing potential and elasticity and increased stiffness.

The BALB/c cell transformation assay (BALB-CTA) considers inter- and intra-tumor heterogeneities and affords the possibility of a direct comparison between untransformed and malignant cells. In the present study, we established monoclonal cell lines that originate from the BALB-CTA and mimic heterogeneous tumor cell populations, in order to investigate phenotype-specific effects of the anti-diabetic drug metformin and the short-chain fatty acid butyrate. Growth inhibitory effects were measured with a ViCell XR cell counter. The BALB/c tumor therapy model (BALB-TTM) was performed, and the extracellular glucose level was measured in the medium supernatant. Using a Seahorse Analyzer, the metabolic phenotypes of four selected clones were characterized, and effects on energy metabolism were investigated. Anti-carcinogenic effects and reduced glucose uptake after butyrate application were observed in the BALB-TTM. Metabolic characterization of the cell clones revealed three different phenotypes. Surprisingly, treatment with metformin or butyrate induced opposite metabolic shifts with similar patterns in all cell clones tested. In conclusion, the BALB-TTM is a relevant model for mechanistic cancer research, and the generation of monoclonal cell lines offers a novel possibility to investigate specific drug effects in a heterogeneous tumor cell population. The results indicate that induced alterations in energy metabolism seem to be independent of the original metabolic phenotype.

Old age, adiposity, and metabolic disorders are known as risk factors for chronic tendinopathy, which is a common problem in both athletes and the general population. However, the importance of these influencing factors has not yet been well understood. This study investigated alterations in gene expression and histology of Achilles tendons of young (10 weeks) and old (100 weeks) rats bred for low (low capacity runners, LCR) and high (high capacity runners, HCR) intrinsic aerobic exercise capacity. In this rat model, LCR displayed a phenotype of reduced exercise capacity, higher body weight, and metabolic dysfunctions compared to HCR. We hypothesized that the risk factors for tendinopathy in old LCR could lead to more pronounced impairments in Achilles tendon tissue. In quantitative real-time PCR (qPCR), age-related downregulation of tenocyte markers e.g., tenomodulin, genes related to matrix modeling and remodeling (e.g., collagens, elastin, biglycan, fibronectin, tenascin C) as well as transforming growth factor beta 3 (Tgfb3) have been detected. Inflammation marker cyclooxygenase 2 (Cox2) was downregulated in old rats, while microsomal prostaglandin E synthase 2 (Ptges2) was upregulated in old HCR and old LCR. In all groups, interleukin 6 (Il6), interleukin 1 beta (Il1b), and tumor necrosis factor alpha (Tnfa) showed no significant alteration. In histological evaluation, tendons of old rats had fewer and more elongated tenocyte nuclei than young rats. Even though a higher content of glycosaminoglycans, a sign of degeneration, was found in old HCR and LCR, no further signs of tendinopathy were detectable in tendons of old rats by histological evaluation. Low intrinsic aerobic exercise capacity and the associated phenotype did not show significant effects on gene expression and tendon histology. These findings indicate that aging seems to play a prominent role in molecular and structural alterations of Achilles tendon tissue and suggests that other risk factors associated with intrinsic aerobic exercise capacity are less influential in this rat model.

Friedreich ataxia is a neurodegenerative disease caused by a GAA triplet repeat expansion in the first intron of the frataxin gene, which results in reduced expression levels of the corresponding protein. Despite numerous animal and cellular models, therapeutic options that mechanistically address impaired frataxin expression are lacking. Here, we have developed a new mammalian cell model employing the Cre/loxP recombination system to induce a homozygous or heterozygous frataxin knockout in mouse embryonic fibroblasts. Induction of Cre-mediated disruption by tamoxifen was successfully tested on RNA and protein levels. After loss of frataxin protein, cell division, aconitase activity and oxygen consumption rates were found to be decreased, while ROS production was increased in the homozygous state. By contrast, in the heterozygous state no such changes were observed. A time-resolved analysis revealed the loss of aconitase activity as an initial event after induction of complete frataxin deficiency, followed by secondarily elevated ROS production and a late increase in iron content. Initial impairments of oxygen consumption and ATP production were found to be compensated in the late state and seemed to play a minor role in Friedreich ataxia pathophysiology. In conclusion and as predicted from its proposed role in iron sulfur cluster (ISC) biosynthesis, disruption of frataxin primarily causes impaired function of ISC-containing enzymes, whereas other consequences, including elevated ROS production and iron accumulation, appear secondary. These parameters and the robustness of the newly established system may additionally be used for a time-resolved study of pharmacological candidates in a HTS manner.

Introduction: Matrix Metalloproteinase (MMP) -8 plays crucial role in pathogenesis of periodontitis and is also a possible biomarker candidate in peri-implantitis. Aim: The aim of the study was to analyse MMP-8 levels in periImplant Sulcus Fluid (PISF) from peri-implantitis affected implants in smoking and non-smoking patients with different periodontal health status of natural teeth before and after peri-implantitis treatment. Settings and Design: Altogether 29 patients with peri-implantitis were recruited and divided into two study groups (11 with healthy periodontium or gingivitis, i.e. no marginal bone loss, and 18 with chronic periodontitis). Materials and Methods: PISF sample from one implant with peri-implantitis from each patient was collected at the baseline and six months after conservative and surgical peri-implantitis treatment, and clinical parameters were registered. Samples were analysed for MMP-8 with dento ELISA method applying a monoclonal antibody. Mucosal cell samples were also analysed for IL-1 gene polymorphism. PISF MMP-8 levels’ differences between periodontal diagnosis groups and between smokers’ and non-smokers’ were analysed. Also, IL-1 polymorphism profiles were compared between study groups. Results: PISF MMP-8 levels were higher at the baseline compared to and after the treatment when all sampled implant sites were analysed together (p = 0.001). MMP-8 levels’ distribution was broader in periodontitis patients’ PISF samples, and only in periodontitis patients’ group levels decreased statistically significantly after the treatment (p = 0.005). Smokers’and nonsmokers’ PISF MMP-8 was at similar level both at the baseline and after the treatment. No difference between distributions of IL-1 genotypes was found between study groups. Conclusion: MMP-8 levels increase in peri-implantitis affected implants both in non-periodontitis and periodontitis patients, but levels still after treatment of the condition reflect intensified host response around implants and indicate challenges of controlling peri-Implantitis with any treatment modality.