Chemical genetic screens reveal defective lysosomal trafficking as synthetic lethal with NF1 loss.

Neurofibromatosis type 1, a genetic disorder caused by pathogenic germline variations in NF1, predisposes individuals to the development of tumors, including cutaneous and plexiform neurofibromas (CNs and PNs), optic gliomas, astrocytomas, juvenile myelomonocytic leukemia, high-grade gliomas and malignant peripheral nerve sheath tumors (MPNSTs), which are chemotherapy- and radiation-resistant sarcomas with poor survival. Loss of NF1 also occurs in sporadic tumors, such as glioblastoma (GBM), melanoma, breast, ovarian and lung cancers. We performed a high-throughput screen for compounds that were synthetic lethal with NF1 loss, which identified several leads, including the small molecule Y102. Treatment of cells with Y102 perturbed autophagy, mitophagy and lysosome positioning in NF1-deficient cells. A dual proteomics approach identified BLOC-one-related complex (BORC), which is required for lysosome positioning and trafficking, as a potential target of Y102. Knockdown of a BORC subunit using siRNA recapitulated the phenotypes observed with Y102 treatment. Our findings demonstrate that BORC might be a promising therapeutic target for NF1-deficient tumors.

NRF2 Activation Reprograms Defects in Oxidative Metabolism to Restore Macrophage Function in Chronic Obstructive Pulmonary Disease.

Rationale: Chronic obstructive pulmonary disease (COPD) is a disease characterized by persistent airway inflammation and disordered macrophage function. The extent to which alterations in macrophage bioenergetics contribute to impaired antioxidant responses and disease pathogenesis has yet to be fully delineated. Objectives: Through the study of COPD alveolar macrophages (AMs) and peripheral monocyte-derived macrophages (MDMs), we sought to establish if intrinsic defects in core metabolic processes drive macrophage dysfunction and redox imbalance. Methods: AMs and MDMs from donors with COPD and healthy donors underwent functional, metabolic, and transcriptional profiling. Measurements and Main Results: We observed that AMs and MDMs from donors with COPD display a critical depletion in glycolytic- and mitochondrial respiration-derived energy reserves and an overreliance on glycolysis as a source for ATP, resulting in reduced energy status. Defects in oxidative metabolism extend to an impaired redox balance associated with defective expression of the NADPH-generating enzyme, ME1 (malic enzyme 1), a known target of the antioxidant transcription factor NRF2 (nuclear factor erythroid 2-related factor 2). Consequently, selective activation of NRF2 resets the COPD transcriptome, resulting in increased generation of TCA cycle intermediaries, improved energetic status, favorable redox balance, and recovery of macrophage function. Conclusions: In COPD, an inherent loss of metabolic plasticity leads to metabolic exhaustion and reduced redox capacity, which can be rescued by activation of the NRF2 pathway. Targeting these defects, via NRF2 augmentation, may therefore present an attractive therapeutic strategy for the treatment of the aberrant airway inflammation described in COPD.

Non-invasive tests accurately stratify patients with NAFLD based on their risk of liver-related events.

BACKGROUND & AIMS: Previous studies on the prognostic significance of non-invasive liver fibrosis tests in non-alcoholic fatty liver disease (NAFLD) lack direct comparison to liver biopsy. We aimed to evaluate the prognostic accuracy of fibrosis-4 (FIB4) and vibration-controlled transient elastography (VCTE), compared to liver biopsy, for the prediction of liver-related events (LREs) in NAFLD. METHODS: A total of 1,057 patients with NAFLD and baseline FIB4 and VCTE were included in a multicenter cohort. Of these patients, 594 also had a baseline liver biopsy. The main study outcome during follow-up was occurrence of LREs, a composite endpoint combining cirrhosis complications and/or hepatocellular carcinoma. Discriminative ability was evaluated using Harrell's C-index. RESULTS: FIB4 and VCTE showed good accuracy for the prediction of LREs, with Harrell's C-indexes >0.80 (0.817 [0.768-0.866] vs. 0.878 [0.835-0.921], respectively, p = 0.059). In the biopsy subgroup, Harrell's C-indexes of histological fibrosis staging and VCTE were not significantly different (0.932 [0.910-0.955] vs. 0.881 [0.832-0.931], respectively, p = 0.164), while both significantly outperformed FIB4 for the prediction of LREs. FIB4 and VCTE were independent predictors of LREs in the whole study cohort. The stepwise FIB4-VCTE algorithm accurately stratified the risk of LREs: compared to patients with "FIB4 <1.30", those with "FIB4 ≥1.30 then VCTE <8.0 kPa" had similar risk of LREs (adjusted hazard ratio [aHR] 1.3; 95% CI 0.3-6.8), whereas the risk of LREs significantly increased in patients with "FIB4 ≥1.30 then VCTE 8.0-12.0 kPa" (aHR 3.8; 95% CI 1.3-10.9), and even more for those with "FIB4 ≥1.30 then VCTE >12.0 kPa" (aHR 12.4; 95% CI 5.1-30.2). CONCLUSION: VCTE and FIB4 accurately stratify patients with NAFLD based on their risk of LREs. These non-invasive tests are alternatives to liver biopsy for the identification of patients in need of specialized management. LAY SUMMARY: The amount of fibrosis in the liver is closely associated with the risk of liver-related complications in non-alcoholic fatty liver disease (NAFLD). Liver biopsy currently remains the reference standard for the evaluation of fibrosis, but its application is limited by its invasiveness. Therefore, we evaluated the ability of non-invasive liver fibrosis tests to predict liver-related complications in NAFLD. Our results show that the blood test FIB4 and transient elastography stratify the risk of liver-related complications in NAFLD, and that transient elastography has similar prognostic accuracy as liver biopsy. These results support the use of non-invasive liver fibrosis tests instead of liver biopsy for the management of patients with NAFLD.

Prevalence estimation of significant fibrosis because of NASH in Spain combining transient elastography and histology.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) has become a major public health problem, but the prevalence of fibrosis associated with non-alcoholic steatohepatitis (NASH) is largely unknown in the general population. This study aimed to provide an updated estimation of the prevalence of NASH fibrosis in Spain. METHODS: This was an observational, retrospective, cross-sectional, population-based study with merged data from two Spanish datasets: a large (N = 12 246) population-based cohort (ETHON), including transient elastography (TE) data, and a contemporary multi-centric biopsy-proven NASH cohort with paired TE data from tertiary centres (N = 501). Prevalence for each NASH fibrosis stage was estimated by crossing TE data from ETHON dataset with histology data from the biopsy-proven cohort. RESULTS: From the patients with valid TE in ETHON dataset (N = 11 440), 5.61% (95% confidence interval [95% CI]: 2.53-11.97) had a liver stiffness measurement (LSM) ≥ 8 kPa. The proportion attributable to NAFLD (using clinical variables and Controlled Attenuation Parameter) was 57.3% and thus, the estimated prevalence of population with LSM ≥ 8 kPa because of NAFLD was 3.21% (95% CI 1.13-8.75). In the biopsy-proven NASH cohort, 389 patients had LSM ≥ 8 kPa. Among these, 37% did not have significant fibrosis (F2-4). The estimated prevalence of NASH F2-3 and cirrhosis in Spain's adult population were 1.33% (95% CI 0.29-5.98) and 0.70% (95% CI 0.10-4.95) respectively. CONCLUSIONS: These estimations provide an accurate picture of the current prevalence of NASH-related fibrosis in Spain and can serve as reference point for dimensioning the therapeutic efforts that will be required as NASH therapies become available.

Fission Yeast Rho1p-GEFs: From Polarity and Cell Wall Synthesis to Genome Stability.

Rho1p is a membrane-associated protein that belongs to the Rho family of small GTPases. These proteins coordinate processes such as actin remodelling and polarised secretion to maintain the shape and homeostasis of yeast cells. In response to extracellular stimuli, Rho1p undergoes conformational switching between a guanosine triphosphate (GTP)-bound active state and a guanosine diphosphate (GDP)-bound inactive state. Cycling is improved with guanine nucleotide exchange factor (GEF) activity necessary to activate signalling and GTPase activating protein (GAP) activity required for subsequent signal depletion. This review focuses on fission yeast Rho1p GEFs, Rgf1p, Rgf2p, and Rgf3p that belong to the family of DH-PH domain-containing Dbl-related GEFs. They are multi-domain proteins that detect biological signals that induce or inhibit their catalytic activity over Rho1p. Each of them activates Rho1p in different places and times. Rgf1p acts preferentially during polarised growth. Rgf2p is required for sporulation, and Rgf3p plays an essential function in septum synthesis. In addition, we outline the noncanonical roles of Rho1p-GEFs in genomic instability.

Impact of liver injury on the severity of COVID-19: a systematic review with meta-analysis.

BACKGROUND AND AIMS: SARS-CoV-2 is mainly a respiratory virus that has relevant systemic effects. We assessed the impact of baseline liver function (aspartate aminotransferase [AST], alanine aminotransferase [ALT], bilirubin) on COVID-19-related outcomes, including mortality, intensive care unit (ICU) admissions, and non-fatal severe complications. METHODS: after a systematic review of the relevant studies the odds ratio (OR), mean difference, sensitivity, specificity, and both positive and negative likelihood ratios were calculated for the prediction of relevant COVID-19 outcomes by performing a meta-analysis using fixed and random effects models. A Fagan nomogram was used to assess clinical usefulness. Heterogeneity was explored by sensitivity analysis and univariate meta-regression. RESULTS: twenty-six studies were included (22 studies and 5,271 patients for AST, 20 studies and 5,440 subjects for ALT, and nine studies and 3,542 patients for bilirubin). The outcomes assessed by these studies were: survival (n = 8), ICU admission (n = 4), and non-fatal severe complications (n = 16). AST > upper limit of normal (ULN) (OR: 3.10 [95 % CI, 2.61-3.68]), ALT > ULN (OR: 2.15 [95 % CI, 1.43-3.23]), and bilirubin > ULN (OR: 2.78 [95 % CI, 1.88-4.13]) were associated with an increased prevalence of severe complications with a specificity of 78 %, 77 %, and 94 %, respectively. The mean difference between mild and severe COVID-19 was 10.7 U/l (95 % CI, 5.8-15.6) for AST, 8 U/l (95 % CI, 1.0-15) for ALT, and 0.3 mg/dl (95 % CI, 0.16-0.45) for bilirubin. CONCLUSIONS: patients showing liver injury had a significantly higher risk of developing severe COVID-19 as compared to those with normal liver function tests at admission. We should include the assessment of AST, ALT, and total bilirubin (TB) routinely in the workup of patients affected by SARS-CoV-2 in order to predict those at risk of developing COVID-19-related outcomes.

Discovery of a crystalline sulforaphane analog with good solid-state stability and engagement of the Nrf2 pathway in vitro and in vivo.

The antioxidant natural product sulforaphane (SFN) is an oil with poor aqueous and thermal stability. Recent work with SFN has sought to optimize methods of formulation for oral and topical administration. Herein we report the design of new analogs of SFN with the goal of improving stability and drug-like properties. Lead compounds were selected based on potency in a cellular screen and physicochemical properties. Among these, 12 had good aqueous solubility, permeability and long-term solid-state stability at 23 °C. Compound 12 also displayed comparable or better efficacy in cellular assays relative to SFN and had in vivo activity in a mouse cigarette smoke challenge model of acute oxidative stress.

Opsonic Phagocytosis in Chronic Obstructive Pulmonary Disease Is Enhanced by Nrf2 Agonists.

RATIONALE: Previous studies have identified defects in bacterial phagocytosis by alveolar macrophages (AMs) in patients with chronic obstructive pulmonary disease (COPD), but the mechanisms and clinical consequences remain incompletely defined. OBJECTIVES: To examine the effect of COPD on AM phagocytic responses and identify the mechanisms, clinical consequences, and potential for therapeutic manipulation of these defects. METHODS: We isolated AMs and monocyte-derived macrophages (MDMs) from a cohort of patients with COPD and control subjects within the Medical Research Council COPDMAP consortium and measured phagocytosis of bacteria in relation to opsonic conditions and clinical features. MEASUREMENTS AND MAIN RESULTS: COPD AMs and MDMs have impaired phagocytosis of Streptococcus pneumoniae. COPD AMs have a selective defect in uptake of opsonized bacteria, despite the presence of antipneumococcal antibodies in BAL, not observed in MDMs or healthy donor AMs. AM defects in phagocytosis in COPD are significantly associated with exacerbation frequency, isolation of pathogenic bacteria, and health-related quality-of-life scores. Bacterial binding and initial intracellular killing of opsonized bacteria in COPD AMs was not reduced. COPD AMs have reduced transcriptional responses to opsonized bacteria, such as cellular stress responses that include transcriptional modules involving antioxidant defenses and Nrf2 (nuclear factor erythroid 2-related factor 2)-regulated genes. Agonists of the cytoprotective transcription factor Nrf2 (sulforaphane and compound 7) reverse defects in phagocytosis of S. pneumoniae and nontypeable Haemophilus influenzae by COPD AMs. CONCLUSIONS: Patients with COPD have clinically relevant defects in opsonic phagocytosis by AMs, associated with impaired transcriptional responses to cellular stress, which are reversed by therapeutic targeting with Nrf2 agonists.

Rgf1p (Rho1p GEF) is required for double-strand break repair in fission yeast.

Rho GTPases are conserved molecules that control cytoskeletal dynamics. These functions are expedited by Rho GEFs that stimulate the release of GDP to enable GTP binding, thereby allowing Rho proteins to initiate intracellular signaling. How Rho GEFs and Rho GTPases protect cells from DNA damage is unknown. Here, we explore the extreme sensitivity of a deletion mutation in the Rho1p exchange factor Rgf1p to the DNA break/inducing antibiotic phleomycin (Phl). The Rgf1p mutant cells are defective in reentry into the cell cycle following the induction of severe DNA damage. This phenotype correlates with the inability of rgf1Δ cells to efficiently repair fragmented chromosomes after Phl treatment. Consistent with this observation Rad11p (ssDNA binding protein, RPA), Rad52p, Rad54p and Rad51p, which facilitate strand invasion in the process of homology-directed repair (HDR), are permanently stacked in Phl-induced foci in rgf1Δ cells. These phenotypes are phenocopied by genetic inhibition of Rho1p. Our data provide evidence that Rgf1p/Rho1p activity positively controls a repair function that confers resistance against the anti-cancer drug Phl.

Mapping Wildfire Ignition Probability Using Sentinel 2 and LiDAR (Jerte Valley, Cáceres, Spain).

Wildfire is a major threat to the environment, and this threat is aggravated by different climatic and socioeconomic factors. The availability of detailed, reliable mapping and periodic and immediate updates makes wildfire prevention and extinction work more effective. An analyst protocol has been generated that allows the precise updating of high-resolution thematic maps. For this protocol, images obtained through the Sentinel 2A satellite, with a return time of five days, have been merged with Light Detection and Ranging (LiDAR) data with a density of 0.5 points/m² in order to obtain vegetation mapping with an accuracy of 88% (kappa = 0.86), which is then extrapolated to fuel model mapping through a decision tree. This process, which is fast and reliable, serves as a cartographic base for the later calculation of ignition-probability mapping. The generated cartography is a fundamental tool to be used in the decision making involved in the planning of preventive silvicultural treatments, extinguishing media distribution, infrastructure construction, etc.

Expanding the p53 regulatory network: LncRNAs take up the challenge.

Long noncoding RNAs (lncRNAs) are rapidly emerging as important regulators of gene expression in a wide variety of physiological and pathological cellular processes. In particular, a number of studies revealed that some lncRNAs participate in the p53 pathway, the unquestioned protagonist of tumor suppressor response. Indeed, several lncRNAs are not only part of the large pool of genes coordinated by p53 transcription factor, but are also required by p53 to fine-tune its response and to fully accomplish its tumor suppressor program. In this review we will discuss the current and fast growing knowledge about the contribution of lncRNAs to the complexity of the p53 network, the different mechanisms by which they affect gene regulation in this context, and their involvement in cancer. The incipient impact of lncRNAs in the p53 biological response may encourage the development of therapies and diagnostic methods focused on these noncoding molecules. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa.

A machine learning classifier trained on cancer transcriptomes detects NF1 inactivation signal in glioblastoma.

BACKGROUND: We have identified molecules that exhibit synthetic lethality in cells with loss of the neurofibromin 1 (NF1) tumor suppressor gene. However, recognizing tumors that have inactivation of the NF1 tumor suppressor function is challenging because the loss may occur via mechanisms that do not involve mutation of the genomic locus. Degradation of the NF1 protein, independent of NF1 mutation status, phenocopies inactivating mutations to drive tumors in human glioma cell lines. NF1 inactivation may alter the transcriptional landscape of a tumor and allow a machine learning classifier to detect which tumors will benefit from synthetic lethal molecules. RESULTS: We developed a strategy to predict tumors with low NF1 activity and hence tumors that may respond to treatments that target cells lacking NF1. Using RNAseq data from The Cancer Genome Atlas (TCGA), we trained an ensemble of 500 logistic regression classifiers that integrates mutation status with whole transcriptomes to predict NF1 inactivation in glioblastoma (GBM). On TCGA data, the classifier detected NF1 mutated tumors (test set area under the receiver operating characteristic curve (AUROC) mean = 0.77, 95% quantile = 0.53 - 0.95) over 50 random initializations. On RNA-Seq data transformed into the space of gene expression microarrays, this method produced a classifier with similar performance (test set AUROC mean = 0.77, 95% quantile = 0.53 - 0.96). We applied our ensemble classifier trained on the transformed TCGA data to a microarray validation set of 12 samples with matched RNA and NF1 protein-level measurements. The classifier's NF1 score was associated with NF1 protein concentration in these samples. CONCLUSIONS: We demonstrate that TCGA can be used to train accurate predictors of NF1 inactivation in GBM. The ensemble classifier performed well for samples with very high or very low NF1 protein concentrations but had mixed performance in samples with intermediate NF1 concentrations. Nevertheless, high-performing and validated predictors have the potential to be paired with targeted therapies and personalized medicine.

Characterization of the Potent, Selective Nrf2 Activator, 3-(Pyridin-3-Ylsulfonyl)-5-(Trifluoromethyl)-2H-Chromen-2-One, in Cellular and In Vivo Models of Pulmonary Oxidative Stress.

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key regulator of oxidative stress and cellular repair and can be activated through inhibition of its cytoplasmic repressor, Kelch-like ECH-associated protein 1 (Keap1). Several small molecule disrupters of the Nrf2-Keap1 complex have recently been tested and/or approved for human therapeutic use but lack either potency or selectivity. The main goal of our work was to develop a potent, selective activator of NRF2 as protection against oxidative stress. In human bronchial epithelial cells, our Nrf2 activator, 3-(pyridin-3-ylsulfonyl)-5-(trifluoromethyl)-2H-chromen-2-one (PSTC), induced Nrf2 nuclear translocation, Nrf2-regulated gene expression, and downstream signaling events, including induction of NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme activity and heme oxygenase-1 protein expression, in an Nrf2-dependent manner. As a marker of subsequent functional activity, PSTC restored oxidant (tert-butyl hydroperoxide)-induced glutathione depletion. The compound's engagement of the Nrf2 signaling pathway translated to an in vivo setting, with induction of Nrf2-regulated gene expression and NQO1 enzyme activity, as well as restoration of oxidant (ozone)-induced glutathione depletion, occurring in the lungs of PSTC-treated rodents. Under disease conditions, PSTC engaged its target, inducing the expression of Nrf2-regulated genes in human bronchial epithelial cells derived from patients with chronic obstructive pulmonary disease, as well as in the lungs of cigarette smoke-exposed mice. Subsequent to the latter, a dose-dependent inhibition of cigarette smoke-induced pulmonary inflammation was observed. Finally, in contrast with bardoxolone methyl and sulforaphane, PSTC did not inhibit interleukin-1ß-induced nuclear factor-κB translocation or insulin-induced S6 phosphorylation in human cells, emphasizing the on-target activity of this compound. In summary, we characterize a potent, selective Nrf2 activator that offers protection against pulmonary oxidative stress in several cellular and in vivo models.

The putative exchange factor Gef3p interacts with Rho3p GTPase and the septin ring during cytokinesis in fission yeast.

The small GTP-binding proteins of the Rho family and its regulatory proteins play a central role in cytokinetic actomyosin ring assembly and cytokinesis. Here we show that the fission yeast guanine nucleotide exchange factor Gef3p interacts with Rho3p at the division site. Gef3p contains a putative DH homology domain and a BAR/IMD-like domain. The protein localized to the division site late in mitosis, where it formed a ring that did not constrict with actomyosin ring (cytokinetic actomyosin ring) invagination; instead, it split into a double ring that resembled the septin ring. Gef3p co-localized with septins and Mid2p and required septins and Mid2p for its localization. Gef3p interacts physically with the GTP-bound form of Rho3p. Although Gef3p is not essential for cell separation, the simultaneous disruption of gef3(+) and Rho3p-interacting proteins, such as Sec8p, an exocyst component, Apm1p, a subunit of the clathrin adaptor complex or For3p, an actin-polymerizing protein, yielded cells with strong defects in septation and polarity respectively. Our results suggest that interactions between septins and Rho-GEFs provide a new targeting mechanism for GTPases in cytokinesis, in this case probably contributing to Rho3p function in vesicle tethering and vesicle trafficking in the later steps of cell separation.

Neuro-otologic manifestations of tuberculosis. "The great imitator".

OBJECTIVE: To demonstrate the different neuro-otologic clinical presentations of tuberculosis. STUDY DESIGN: Retrospective clinical analysis. RESULT: 83.3% of the cases of ear or central nervous system TB were without concomitant lung disease. 2 cases had primary infection in the central nervous system. The neuro-otologic manifestation was as follows: 85.7% sensorineural hearing loss; 42% polyneuropathy. 71.4% had granulation tissue. 2 had normal otoscopy. In 6 patients the histopathology and Ziehl Neelsen were confirmatory. One case was confirmed by the positive response to treatment with antituberculosis drugs. CONCLUSIONS: Tuberculosis has a wide variety of neurotologic manifestations from chronic otitis media cadres to vestibular, audiological and neurological manifestations as well as a large variability in imaging studies.

Analytical approaches for quantification of a Nrf2 pathway activator: overcoming bioanalytical challenges to support a toxicity study.

Activation of the Nrf2 stress pathway is known to play an important role in the defense mechanism against electrophilic and oxidative damage to biological macromolecules (DNA, lipids, and proteins). Chemical inducers of Nrf2 such as sulforaphane, dimethyl fumarate (Tecfidera®), CDDO-Me (bardoxolone-methyl), and 3-(dimethylamino)-4-((3-isothiocyanatopropyl)(methyl)amino)cyclobut-3-ene-1,2-dione (a synthetic sulforaphane analogue; will be referred to as ) have the ability to react with Keap1 cysteine residues, leading to activation of the Antioxidant Response Element (ARE). Due to their electrophilic nature and poor matrix stability, these compounds represent great challenges when developing bioanalytical methods to evaluate in vivo exposure. like SFN reacts rapidly with glutathione (GSH) and nucleophilic groups in proteins to form covalent adducts. In this work, three procedures were developed to estimate the exposure of in a non-GLP 7 day safety study in rats: (1) protein precipitation of blood samples with methanol containing the free thiol trapping reagent 4-fluoro-7-aminosulfonylbenzofurazan (ABD-F) to measure GSH- and N-acetylcysteine conjugated metabolites of ; (2) an Edman degradation procedure to cleave and analyze N-terminal adducts of at the valine moiety; and (3) treatment with ammonium hydroxide to measure circulating free- and all sulfhydryl bound .

Proteins in the nutrient-sensing and DNA damage checkpoint pathways cooperate to restrain mitotic progression following DNA damage.

Checkpoint pathways regulate genomic integrity in part by blocking anaphase until all chromosomes have been completely replicated, repaired, and correctly aligned on the spindle. In Saccharomyces cerevisiae, DNA damage and mono-oriented or unattached kinetochores trigger checkpoint pathways that bifurcate to regulate both the metaphase to anaphase transition and mitotic exit. The sensor-associated kinase, Mec1, phosphorylates two downstream kinases, Chk1 and Rad53. Activation of Chk1 and Rad53 prevents anaphase and causes inhibition of the mitotic exit network. We have previously shown that the PKA pathway plays a role in blocking securin and Clb2 destruction following DNA damage. Here we show that the Mec1 DNA damage checkpoint regulates phosphorylation of the regulatory (R) subunit of PKA following DNA damage and that the phosphorylated R subunit has a role in restraining mitosis following DNA damage. In addition we found that proteins known to regulate PKA in response to nutrients and stress either by phosphorylation of the R subunit or regulating levels of cAMP are required for the role of PKA in the DNA damage checkpoint. Our data indicate that there is cross-talk between the DNA damage checkpoint and the proteins that integrate nutrient and stress signals to regulate PKA.

Cutaneous metastasis of primitive neuroectodermal lung tumor.

Primary sarcomas of the chest are rare. Although primitive neuroectodermal tumor (PNET) usually develops in the chest wall, it has been described as a primary pulmonary tumor. We present an unusual case of PNET arising in the lung of an 89-year-old man.

Cultural adaptation of the iSupport online training and support programme for caregivers of people with dementia in Castilla y León, Spain.

BACKGROUND: E-learning has shown to be an effective intervention in helping informal caregivers of people living with dementia. It has the potential to reach people living in remote areas, increasing service coverage. As a response to the demographic context in Spain associated with a higher percentage of ageing, depopulation, and the complexities of health service delivery in rural areas, this paper describes the cultural adaptation and co-design of the iSupport online training and support programme for Castilla y León, Spain, as a potential e-health intervention to mitigate these constraints. METHODS: The translation and cultural adaptation were performed following the WHO guidelines, with some adaptation due to the cultural context of Spain. Three focus groups were conducted with informal caregivers, health professionals, and a group of experts on cognitive impairment and dementia. The co-design process was performed as a Patient and Public Involvement activity with three groups consisting of people living with dementia, informal caregivers, rural population and experts on technology and dementia. RESULTS: A total of 435 suggestions were proposed for adaptation associated with erroneous terminology, rewording text/writing, grammatical or punctuation marks errors, and repeated information or need for additional content. Several recommendations were exposed during the co-design process: preference for interactive material such as videos or images, a forum to receive feedback from health care professionals and to leave satisfaction comments, availability in multiple platforms (e.g., tablet, laptop, mobile), slide format for information presentation, and availability to edit letter size and background colours. CONCLUSIONS: A culturally adapted version of the iSupport was developed for Castilla y León, Spain. The need for modification of words and expressions, information links to local resources websites, adjustments of characters' names and caregivers' scenarios, and additional content to some sections were recommended. Suggestions for the design should be taken into account for further adapted versions and platform developments.

Evaluation of Change in Radiographic Fractal Dimension around Dental Implants Placed with Low-Speed Drilling and Standard Drilling Protocols.

Osseointegration is a process that depends on a multitude of factors, including the type of drilling, whether biological or conventional. OBJECTIVE: Establish box-counting dimension values for radiological images in patients with implants placed with both drilling methods. MATERIAL AND METHOD: The sample included 129 implants corresponding to 50 patients. A double-blind study of data collection was carried out with the subsequent analysis of the fractal dimension as a comparative value of the state of the trabecular architecture. RESULTS: We found no significant differences (p ≥ 0.05) between the two study groups comparing both drilling techniques. The values for the conventional drilling technique are 0.24 ± 0.07 and for biological drilling: 0.19 ± 0.11 with a p-value of 0.767. CONCLUSIONS: The drilling technique does not influence the success of the procedure and the osseointegration process.