Inhibition of the AURKA/YAP1 axis is a promising therapeutic option for overcoming cetuximab resistance in colorectal cancer stem cells.

BACKGROUND: Primary resistance to anti-EGFR therapies affects 40% of metastatic colorectal cancer patients harbouring wild-type RAS/RAF. YAP1 activation is associated with this resistance, prompting an investigation into AURKA's role in mediating YAP1 phosphorylation at Ser397, as observed in breast cancer. METHODS: We used transcriptomic analysis along with in vitro and in vivo models of RAS/RAF wild-type CRC to study YAP1 Ser397 phosphorylation as a potential biomarker for cetuximab resistance. We assessed cetuximab efficacy using CCK8 proliferation assays and cell cycle analysis. Additionally, we examined the effects of AURKA inhibition with alisertib and created a dominant-negative YAP1 Ser397 mutant to assess its impact on cancer stem cell features. RESULTS: The RAS/RAF wild-type CRC models exhibiting primary resistance to cetuximab prominently displayed elevated YAP1 phosphorylation at Ser397 primarily mediated by AURKA. AURKA-induced YAP1 phosphorylation was identified as a key trigger for cancer stem cell reprogramming. Consequently, we found that AURKA inhibition had the capacity to effectively restore cetuximab sensitivity and concurrently suppress the cancer stem cell phenotype. CONCLUSIONS: AURKA inhibition holds promise as a therapeutic approach to overcome cetuximab resistance in RAS/RAF wild-type colorectal cancer, offering a potential means to counter the development of cancer stem cell phenotypes associated with cetuximab resistance.

Three dimensional MRF obtains highly repeatable and reproducible multi-parametric estimations in the healthy human brain at 1.5T and 3T.

Magnetic resonance fingerprinting (MRF) is highly promising as a quantitative MRI technique due to its accuracy, robustness, and efficiency. Previous studies have found high repeatability and reproducibility of 2D MRF acquisitions in the brain. Here, we have extended our investigations to 3D MRF acquisitions covering the whole brain using spiral projection k-space trajectories. Our travelling head study acquired test/retest data from the brains of 12 healthy volunteers and 8 MRI systems (3 systems at 3 T and 5 at 1.5 T, all from a single vendor), using a study design not requiring all subjects to be scanned at all sites. The pulse sequence and reconstruction algorithm were the same for all acquisitions. After registration of the MRF-derived PD T1 and T2 maps to an anatomical atlas, coefficients of variation (CVs) were computed to assess test/retest repeatability and inter-site reproducibility in each voxel, while a General Linear Model (GLM) was used to determine the voxel-wise variability between all confounders, which included test/retest, subject, field strength and site. Our analysis demonstrated a high repeatability (CVs 0.7-1.3% for T1, 2.0-7.8% for T2, 1.4-2.5% for normalized PD) and reproducibility (CVs of 2.0-5.8% for T1, 7.4-10.2% for T2, 5.2-9.2% for normalized PD) in gray and white matter. Both repeatability and reproducibility improved when compared to similar experiments using 2D acquisitions. Three-dimensional MRF obtains highly repeatable and reproducible estimations of T1 and T2, supporting the translation of MRF-based fast quantitative imaging into clinical applications.

Accelerated 3D whole-brain T1, T2, and proton density mapping: feasibility for clinical glioma MR imaging.

PURPOSE: Advanced MRI-based biomarkers offer comprehensive and quantitative information for the evaluation and characterization of brain tumors. In this study, we report initial clinical experience in routine glioma imaging with a novel, fully 3D multiparametric quantitative transient-state imaging (QTI) method for tissue characterization based on T1 and T2 values. METHODS: To demonstrate the viability of the proposed 3D QTI technique, nine glioma patients (grade II-IV), with a variety of disease states and treatment histories, were included in this study. First, we investigated the feasibility of 3D QTI (6:25 min scan time) for its use in clinical routine imaging, focusing on image reconstruction, parameter estimation, and contrast-weighted image synthesis. Second, for an initial assessment of 3D QTI-based quantitative MR biomarkers, we performed a ROI-based analysis to characterize T1 and T2 components in tumor and peritumoral tissue. RESULTS: The 3D acquisition combined with a compressed sensing reconstruction and neural network-based parameter inference produced parametric maps with high isotropic resolution (1.125 × 1.125 × 1.125 mm3 voxel size) and whole-brain coverage (22.5 × 22.5 × 22.5 cm3 FOV), enabling the synthesis of clinically relevant T1-weighted, T2-weighted, and FLAIR contrasts without any extra scan time. Our study revealed increased T1 and T2 values in tumor and peritumoral regions compared to contralateral white matter, good agreement with healthy volunteer data, and high inter-subject consistency. CONCLUSION: 3D QTI demonstrated comprehensive tissue assessment of tumor substructures captured in T1 and T2 parameters. Aiming for fast acquisition of quantitative MR biomarkers, 3D QTI has potential to improve disease characterization in brain tumor patients under tight clinical time-constraints.

Mammalian polymerase θ promotes alternative NHEJ and suppresses recombination.

The alternative non-homologous end-joining (NHEJ) machinery facilitates several genomic rearrangements, some of which can lead to cellular transformation. This error-prone repair pathway is triggered upon telomere de-protection to promote the formation of deleterious chromosome end-to-end fusions. Using next-generation sequencing technology, here we show that repair by alternative NHEJ yields non-TTAGGG nucleotide insertions at fusion breakpoints of dysfunctional telomeres. Investigating the enzymatic activity responsible for the random insertions enabled us to identify polymerase theta (Polθ; encoded by Polq in mice) as a crucial alternative NHEJ factor in mammalian cells. Polq inhibition suppresses alternative NHEJ at dysfunctional telomeres, and hinders chromosomal translocations at non-telomeric loci. In addition, we found that loss of Polq in mice results in increased rates of homology-directed repair, evident by recombination of dysfunctional telomeres and accumulation of RAD51 at double-stranded breaks. Lastly, we show that depletion of Polθ has a synergistic effect on cell survival in the absence of BRCA genes, suggesting that the inhibition of this mutagenic polymerase represents a valid therapeutic avenue for tumours carrying mutations in homology-directed repair genes.

Retrospective rigid motion correction of three-dimensional magnetic resonance fingerprinting of the human brain.

PURPOSE: To obtain three-dimensional (3D), quantitative and motion-robust imaging with magnetic resonance fingerprinting (MRF). METHODS: Our acquisition is based on a 3D spiral projection k-space scheme. We compared different orderings of trajectory interleaves in terms of rigid motion-correction robustness. In all tested orderings, we considered the whole dataset as a sum of 56 segments of 7-s duration, acquired sequentially with the same flip angle schedule. We performed a separate image reconstruction for each segment, producing whole-brain navigators that were aligned to the first segment using normalized correlation. The estimated rigid motion was used to correct the k-space data, and the aligned data were matched with the dictionary to obtain motion-corrected maps. RESULTS: A significant improvement on the motion-affected maps after motion correction is evident with the suppression of motion artifacts. Correlation with the motionless baseline improved by 20% on average for both T1 and T2 estimations after motion correction. In addition, the average motion-induced quantification bias of 70 ms for T1 and 18 ms for T2 values was reduced to 12 ms and 6 ms, respectively, improving the reliability of quantitative estimations. CONCLUSION: We established a method that allows correcting 3D rigid motion on a 7-s timescale during the reconstruction of MRF data using self-navigators, improving the image quality and the quantification robustness.

Dysregulated lipid metabolism in hepatocellular carcinoma cancer stem cells.

According to the stem cell theory for cancer, hepatocellular carcinomas are sustained by a group of cancer stem cells (CSCs) which are responsible for resistance to chemotherapy. In the present study we aimed to examine lipid metabolism in cancer stem cells induced by long-term treatment with sorafenib and its relationship with acquisition of a CSC-like phenotype. Two cell lines (HepG2SF1 and Huh7SF1) were generated by incubation with a step-wise increase of sorafenib concentrations for 10 months. These cell lines displayed stem-like characteristics like increase in the expression of ABCB1A, Nanog and Oct4 as well as an E-cadherin/N-cadherin switch. HepG2SF1 and Huh7SF1 cells showed intracellular accumulation of neutral lipids, assessed by flow cytometry and confocal microscopy. The exam of lipid metabolism revealed that HepG2SF1 and Huh7SF1 cells increased the expression of the enzymes involved in de novo lipid synthesis ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN) and that of the fatty acid transporter CD36. In addition, these CSC-like cells had enhanced expression of the lipogenic transcription factor SREBP1c. Analysis of the key metabolic sensor AMP-activated kinase (AMPK) demonstrated that both AMPK phosphorylation and levels were decreased in the CSC-like cells compared to their parental cells. Interestingly, transfection of HepG2SF1 and Huh7SF1 cells with AMPK, restored the levels of the lipogenic enzymes and SREBP1c and decreased the intracellular lipid accumulation. Furthermore, AMPK transfection decreased the stemness markers and inhibited the E-cadherin/N-cadherin switch. Targeting AMPK and lipid metabolism of hepatocellular cancer stem cells is a promising strategy to face stemness and chemotherapy resistance.

Serum Amyloid A1 as a Potential Intracranial and Extracranial Clinical Severity Biomarker in Traumatic Brain Injury.

Extracranial injury is frequently present in patients with traumatic brain injury (TBI). However, no reliable biomarker exists nowadays to evaluate the magnitude and extension of extracranial injury as well as the identification of patients who are at risk of developing secondary injuries. The purpose of this study was to identify new possible peptide biomarkers by mass spectrometry analysis in patients with TBI and ascertain whether the novel biomarker discovered by peptide mass fingerprinting, serum amyloid A1 (SAA1), is capable of reflecting the condition of the patient and both intracranial and extracranial injury extension. Demographic characteristics, clinical data, and serum samples were prospectively collected from 120 patients with TBI (Glasgow Coma Scale [GCS] score 3-15) on admission. Biomarkers were quantified by enzyme-linked immunosorbent assay. Intracranial lesion volume was measured from the semiautomatic segmentation of hematoma on computed tomography (CT) using Analyze software. Functional outcome was evaluated using the Glasgow Outcome Scale (GOS) at hospital discharge and GOS extended scores at 6 months. The SAA1 levels were significantly associated with intracranial (GCS score at admission, lesion load measured with cranial CT, and pupil responsiveness) and extracranial clinical severity (all Abbreviated Injury Scale regions, Injury Severity Score, major extracranial injury, polytrauma, and orthopedic fractures presence), along with systemic secondary insults and functional outcome. SAA1 was is associated with the volume of traumatic intracranial lesions. The SAA1 levels were correlated with astroglial S100ß and glial fibrillary acidic protein (GFAP), neuronal neuron-specific enolase (NSE), and axonal total tau (T-tau) and phosphorylated neurofilament heavy chain (pNF-H) injury markers. SAA1 predicts unfavorable outcome and mortality at hospital discharge (area under the curve [AUC] = 0.90, 0.82) and 6 months (AUC = 0.89). SAA1 can be established as a marker for the overall patient condition due to its involvement in the neuroendocrine axis of the systemic response to craniocerebral trauma.

Multi-site repeatability and reproducibility of MR fingerprinting of the healthy brain at 1.5 and 3.0 T.

Fully-quantitative MR imaging methods are useful for longitudinal characterization of disease and assessment of treatment efficacy. However, current quantitative MRI protocols have not been widely adopted in the clinic, mostly due to lengthy scan times. Magnetic Resonance Fingerprinting (MRF) is a new technique that can reconstruct multiple parametric maps from a single fast acquisition in the transient state of the MR signal. Due to the relative novelty of this technique, the repeatability and reproducibility of quantitative measurements obtained using MRF has not been extensively studied. Our study acquired test/retest data from the brains of nine healthy volunteers, each scanned on five MRI systems (two at 3.0 T and three at 1.5 T, all from a single vendor) located at two different centers. The pulse sequence and reconstruction algorithm were the same for all acquisitions. After registration of the MRF-derived M0, T1 and T2 maps to an anatomical atlas, coefficients-of-variation (CVs) were computed to assess test/retest repeatability and inter-site reproducibility in each voxel, while a General Linear Model (GLM) was used to determine the voxel-wise variability between all confounders, which included test/retest, subject, field strength and site. Our analysis demonstrated an excellent repeatability (CVs of 2-3% for T1, 5-8% for T2, 3% for normalized-M0) and a good reproducibility (CVs of 3-8% for T1, 8-14% for T2, 5% for normalized-M0) in grey and white matter.

Combination of the natural product capsaicin and docetaxel synergistically kills human prostate cancer cells through the metabolic regulator AMP-activated kinase.

BACKGROUND: Current chemotherapy for castration-resistant prostate cancer is established on taxane-based compounds like docetaxel. However, eventually, the development of toxic side effects and resistance limits the therapeutic benefit being the major concern in the treatment of prostate cancer. Combination therapies in many cases, enhance drug efficacy and delay the appearance of undesired effects, representing an important option for the treatment of castration-resistant prostate cancer. In this study, we tested the efficacy of the combination of docetaxel and capsaicin, the pungent ingredient of hot chili peppers, on prostate cancer cells proliferation. METHODS: Prostate cancer LNCaP and PC3 cell lines were used in this study. Levels of total and phosphorylated forms of Akt, mTOR, S6, LKB1, AMPK and ACC were determined by Western blot. AMPK, LKB1 and Akt knock down was performed by siRNA. PTEN was overexpressed by transient transfection with plasmids. Xenograft prostate tumors were induced in nude mice and treatments (docetaxel and capsaicin) were administered intraperitoneally. Statistical analyses were performed with GraphPad software. Combination index was calculated with Compusyn software. RESULTS: Docetaxel and capsaicin synergistically inhibited the growth of LNCaP and PC3 cells, with a combination index lower than 1 for most of the combinations tested. Co-treatment with docetaxel and capsaicin notably decreased Akt and its downstream targets mTOR and S6 phosphorylation. Overexpression of PTEN phosphatase abrogated the synergistic antiproliferative effect of docetaxel and capsaicin. The combined treatment also increased the phosphorylation of AMP-activated kinase (AMPK) and the phosphorylation of its substrate ACC. In addition, pharmacological inhibition of AMPK with dorsomorphin (compound C) as well as knock down by siRNA of AMPK or its upstream kinase LKB1, abolished the synergy of docetaxel and capsaicin. Mechanistically, we showed that the synergistic anti-proliferative effect may be attributed to two independent effects: Inhibition of the PI3K/Akt/mTOR signaling pathway by one side, and AMPK activation by the other. In vivo experiments confirmed the synergistic effects of docetaxel and capsaicin in reducing the tumor growth of PC3 cells. CONCLUSION: Combination of docetaxel and capsaicin represents a therapeutically relevant approach for the treatment of Prostate Cancer.

Capsaicin Targets Lipogenesis in HepG2 Cells Through AMPK Activation, AKT Inhibition and PPARs Regulation.

Obesity, a major risk factor for chronic diseases such as type 2 diabetes (T2D), represents a serious primary health problem worldwide. Dietary habits are of special interest to prevent and counteract the obesity and its associated metabolic disorders, including lipid steatosis. Capsaicin, a pungent compound of chili peppers, has been found to ameliorate diet-induced obesity in rodents and humans. The purpose of this study was to examine the effect of capsaicin on hepatic lipogenesis and to delineate the underlying signaling pathways involved, using HepG2 cells as an experimental model. Cellular neutral lipids, stained with BODIPY493/503, were quantified by flow cytometry, and the protein expression and activity were determined by immunoblotting. Capsaicin reduced basal neutral lipid content in HepG2 cells, as well that induced by troglitazone or by oleic acid. This effect of capsaicin was prevented by dorsomorphin and GW9662, pharmacological inhibitors of AMPK and PPARγ, respectively. In addition, capsaicin activated AMPK and inhibited the AKT/mTOR pathway, major regulators of hepatic lipogenesis. Furthermore, capsaicin blocked autophagy and increased PGC-1α protein. These results suggest that capsaicin behaves as an anti-lipogenic compound in HepG2 cells.

The added prognostic value of magnetic resonance imaging in traumatic brain injury: The importance of traumatic axonal injury when performing ordinal logistic regression.

BACKGROUND AND PURPOSE: This study was performed to investigate the prognostic value of traumatic axonal injury (TAI) in severe head trauma. METHODS: We attempted to determine whether any MR imaging findings of TAI could be related to prognosis in 264 patients with severe head trauma. We performed an ordinal logistic regression, adjusted for the prognostic factors according to the IMPACT studies, adding each MR feature related to prognosis one at a time. A new prognostic model was described by adding these MR features to the classic prognostic factors. The model was externally validated in a prospective series. Harrel's c-statistic and ordinal c-index (ORC) were calculated to measure its predictive accuracy. RESULTS: We found 178 patients with TAI lesions. Lesions in the basal ganglia/thalamus, corpus callosum (CC) and brain stem were associated with poor outcome (P < 0.01). The highest OR was for TAI lesions in the splenium (OR: 2.6) and brain stem dorsal lesions (OR: 3.1). We only found significant differences in outcome between haemorrhagic and non-haemorrhagic TAI lesions in the subgroup of patients with white matter and basal ganglia/thalamus lesions (P = 0.01). We obtained a superior discriminatory capacity by adding these MR findings to the previous prognostic model (Harrel's c-statistic 0.72 and ORC 0.7) in a prospective series of 93 patients. CONCLUSIONS: The prognostic model including MR findings maintained a superior discriminatory capacity than that obtained for the model with the classic prognostic factors alone.

A diffusion model-free framework with echo time dependence for free-water elimination and brain tissue microstructure characterization.

PURPOSE: The compartmental nature of brain tissue microstructure is typically studied by diffusion MRI, MR relaxometry or their correlation. Diffusion MRI relies on signal representations or biophysical models, while MR relaxometry and correlation studies are based on regularized inverse Laplace transforms (ILTs). Here we introduce a general framework for characterizing microstructure that does not depend on diffusion modeling and replaces ill-posed ILTs with blind source separation (BSS). This framework yields proton density, relaxation times, volume fractions, and signal disentanglement, allowing for separation of the free-water component. THEORY AND METHODS: Diffusion experiments repeated for several different echo times, contain entangled diffusion and relaxation compartmental information. These can be disentangled by BSS using a physically constrained nonnegative matrix factorization. RESULTS: Computer simulations, phantom studies, together with repeatability and reproducibility experiments demonstrated that BSS is capable of estimating proton density, compartmental volume fractions and transversal relaxations. In vivo results proved its potential to correct for free-water contamination and to estimate tissue parameters. CONCLUSION: Formulation of the diffusion-relaxation dependence as a BSS problem introduces a new framework for studying microstructure compartmentalization, and a novel tool for free-water elimination.

Final outcome trends in severe traumatic brain injury: a 25-year analysis of single center data.

BACKGROUND: Evidence from the last 25 years indicates a modest reduction of mortality after severe traumatic head injury (sTBI). This study evaluates the variation over time of the whole Glasgow Outcome Scale (GOS) throughout those years. METHODS: The study is an observational cohort study of adults (≥ 15 years old) with closed sTBI (GCS ≤ 8) who were admitted within 48 h after injury. The final outcome was the 1-year GOS, which was divided as follows: (1) dead/vegetative, (2) severely disabled (dependent patients), and (3) good/moderate recovery (independent patients). Patients were treated uniformly according to international protocols in a dedicated ICU. We considered patient characteristics that were previously identified as important predictors and could be determined easily and reliably. The admission years were divided into three intervals (1987-1995, 1996-2004, and 2005-2012), and the following individual CT characteristics were noted: the presence of traumatic subarachnoid or intraventricular hemorrhage (tSAH, IVH), midline shift, cisternal status, and the volume of mass lesions (A × B × C/2). Ordinal logistic regression was performed to estimate associations between predictors and outcomes. The patients' estimated propensity scores were included as an independent variable in the ordinal logistic regression model (TWANG R package). FINDINGS: The variables associated with the outcome were age, pupils, motor score, deterioration, shock, hypoxia, cistern status, IVH, tSAH, and epidural volume. When adjusting for those variables and the propensity score, we found a reduction in mortality from 55% (1987-1995) to 38% (2005-2012), but we discovered an increase in dependent patients from 10 to 21% and just a modest increase in independent patients of 6%. CONCLUSIONS: This study covers 25 years of management of sTBI in a single neurosurgical center. The prognostic factors are similar to those in the literature. The improvement in mortality does not translate to better quality of life.

Prognostic value of corpus callosum injuries in severe head trauma.

BACKGROUND: This study was performed to investigate the relationship between corpus callosum (CC) injury and prognosis in traumatic axonal injury (TAI). METHOD: We retrospectively reviewed 264 patients with severe head trauma who underwent a conventional MR imaging in the first 60 days after injury. They were selected from a prospectively collected database of 1048 patients with severe head trauma admitted in our hospital. TAI lesions were defined as areas of increased signal intensity on T2 and FLAIR or areas of decreased signal on gradient-echo T2. We attempted to determine whether any MR imaging findings of TAI lesions at CC could be related to prognosis. Neurological impairment was assessed at 1 year after injury by means of GOS-E (good outcome being GOS-E 4/5 and bad outcome being GOS-E <4). We adjusted the multivariable analysis for the prognostic factors according to the IMPACT studies: the Core model (age, motor score at admission, and pupillary reactivity) and the Extended model (including CT information and second insults). RESULTS: We found 97 patients (37 %) with TAI at CC and 167 patients (63 %) without CC lesions at MR. A total of 62 % of the patients with CC lesions had poor outcome, whereas 38 % showed good prognosis. The presence of TAI lesions at the corpus callosum was associated with poor outcome 1 year after brain trauma (p < 0.001, OR 3.8, 95 % CI: 2.04-7.06). The volume of CC lesions measured on T2 and FLAIR sequences was negatively correlated with the GOS-E after adjustment for independent prognostic factors (p = 0.01, OR 2.23, 95 % CI:1.17-4.26). Also the presence of lesions at splenium was statistically related to worse prognosis (p = 0.002, OR 8.1, 95 % CI: 2.2-29.82). We did not find statistical significance in outcome between hemorrhagic and non-hemorrhagic CC lesions. CONCLUSIONS: The presence of CC is associated with a poor outcome. The total volume of the CC lesion is an independent prognostic factor for poor outcome in severe head trauma.

Symptomatic ptosis cerebelli after suboccipital craniectomy in a patient with severe brain trauma.

PRIMARY OBJECTIVE: To report the first case of symptomatic cerebellar ptosis after a large suboccipital craniectomy in a patient with severe brain trauma and a review of the literature. PATIENT AND METHODS: A 36-year-old man suffered severe traumatic brain injury after a four-metre fall. He underwent a large suboccipital craniectomy because his computed tomography scan revealed a posterior fossa subdural haematoma and cerebellar swelling. Four weeks after admission, he developed communicating hydrocephalus, and a ventriculoperitoneal shunt was placed. Although he experienced good recovery, seven months after the trauma he complained of cephalea, dizziness, recurrent vomiting and diplopia. Magnetic resonance imaging (MRI) showed descent of the cerebellum through a wide bone defect. RESULTS: We performed a posterior fossa cranioplasty after other causes of delayed worsening were ruled out, such as shunt malfunction, overdrainage and ischaemic lesions. The patient improved, and a post-operative MRI confirmed the upward migration of the cerebellum. CONCLUSIONS: Cerebellar ptosis must be considered in cases of delayed symptoms after large suboccipital craniectomy regardless of pathology. Posterior fossa cranioplasty to provide structural support to slumped cerebellum can improve or resolve symptoms.

[Trends in computed tomography characteristics, intracranial pressure monitoring and surgical management in severe traumatic brain injury: Analysis of a data base of the past 25 years in a neurosurgery department]. / Evolución temporal en las características de la tomografía computarizada, presión intracraneal y tratamiento quirúrgico en el traumatismo craneal grave: análisis de la base de datos de los últimos 25 años en un servicio de neurocirugía.

OBJECTIVE: To describe the radiological characteristics, surgical indications, procedures, and intracranial pressure monitoring of a representative cohort of severe traumatic brain injury (sTBI) cases collected over the past 25years, and to analyse the changes that have occurred by dividing the period into 3 equal time periods. METHODS: An observational cohort study was conducted on consecutive adult patients (>14years of age) with severe closed TBI (Glasgow Coma Scale score [GCS]≤8) who were admitted during the first 48hours after injury to the Hospital 12 de Octubre from 1987 to 2012. The most relevant radiological findings, surgical procedures, and intracranial monitoring indications reported in the literature were defined and compared in 3 equal time periods (1987-1995, 1996-2004, and 2005-2014). RESULTS: A significant increase was observed in subdural haematomas with lesions over 25cc, and midline shift in the last period of time. The incidence of subarachnoid haemorrhage increased significantly with time. There was a progression to a worse computed tomography (CT) classification from the initial CT scan in 33% of cases. Surgery was performed on 721 (39.4%) patients. Early surgery (<12hours) was performed on 585 (81.1%) patients, with the most frequent being for extra-cerebral mass lesions (subdural and epidural haematomas), whereas delayed surgery (>12hours) was most frequently performed due to an intracerebral haematoma. Surgical treatment, both early and late was significantly lower with respect to the first time period. Decompressive craniectomy with evacuation of the mass lesion was the preferred procedure in the last time period. Intracranial pressure monitoring (ICP) was carried out on 1049 (57.3%) patients, with a significantly higher frequency in the second period of time. There was adherence to Guidelines in 64.4% of cases. Elevated/uncontrolled ICP was more significant in the first time period. CONCLUSIONS: As a result of the epidemiological changes seen in traumatic brain injury, a different pattern of morphological injury is described, as depicted in the CT, leading to a difference in practice during this period of observation.

Long-distance RNA-RNA interactions in the coronavirus genome form high-order structures promoting discontinuous RNA synthesis during transcription.

Coronavirus (CoV) transcription requires a high-frequency recombination process that links newly synthesized minus-strand subgenomic RNA copies to the leader region, which is present only once, at the 5' end of the genome. This discontinuous RNA synthesis step is based on the complementarity between the transcription-regulating sequences (TRSs) at the leader region and those preceding each gene in the nascent minus-strand RNA. Furthermore, the template switch requires the physical proximity of RNA genome domains located between 20,000 and 30,000 nucleotides apart. In this report, it is shown that the efficacy of this recombination step is promoted by novel additional long-distance RNA-RNA interactions between RNA motifs located close to the TRSs controlling the expression of each gene and their complementary sequences mapping close to the 5' end of the genome. These interactions would bring together the motifs involved in the recombination process. This finding indicates that the formation of high-order RNA structures in the CoV genome is necessary to control the expression of at least the viral N gene. The requirement of these long-distance interactions for transcription was shown by the engineering of CoV replicons in which the complementarity between the newly identified sequences was disrupted. Furthermore, disruption of complementarity in mutant viruses led to mutations that restored complementarity, wild-type transcription levels, and viral titers by passage in cell cultures. The relevance of these high-order structures for virus transcription is reinforced by the phylogenetic conservation of the involved RNA motifs in CoVs.

Transmissible gastroenteritis coronavirus genome packaging signal is located at the 5' end of the genome and promotes viral RNA incorporation into virions in a replication-independent process.

Preferential RNA packaging in coronaviruses involves the recognition of viral genomic RNA, a crucial process for viral particle morphogenesis mediated by RNA-specific sequences, known as packaging signals. An essential packaging signal component of transmissible gastroenteritis coronavirus (TGEV) has been further delimited to the first 598 nucleotides (nt) from the 5' end of its RNA genome, by using recombinant viruses transcribing subgenomic mRNA that included potential packaging signals. The integrity of the entire sequence domain was necessary because deletion of any of the five structural motifs defined within this region abrogated specific packaging of this viral RNA. One of these RNA motifs was the stem-loop SL5, a highly conserved motif in coronaviruses located at nucleotide positions 106 to 136. Partial deletion or point mutations within this motif also abrogated packaging. Using TGEV-derived defective minigenomes replicated in trans by a helper virus, we have shown that TGEV RNA packaging is a replication-independent process. Furthermore, the last 494 nt of the genomic 3' end were not essential for packaging, although this region increased packaging efficiency. TGEV RNA sequences identified as necessary for viral genome packaging were not sufficient to direct packaging of a heterologous sequence derived from the green fluorescent protein gene. These results indicated that TGEV genome packaging is a complex process involving many factors in addition to the identified RNA packaging signal. The identification of well-defined RNA motifs within the TGEV RNA genome that are essential for packaging will be useful for designing packaging-deficient biosafe coronavirus-derived vectors and providing new targets for antiviral therapies.

Trends in epidemiological and clinical characteristics in severe traumatic brain injury: Analysis of the past 25 years of a single centre data base.

OBJECTIVE: To describe the demographic and clinical profiles of a cohort of environmentally representative severe traumatic brain injury (TBI) cases collected for the past 25 years and to analyse the changes that occurred by dividing the analysis period into 3 equal time periods. MATERIAL AND METHODS: This was an observational cohort study of consecutive adult patients (>14 years of age) with severe closed TBI (Glasgow Coma Scale score [GCS]≤8) who were admitted during the first 48h after injury to the 12 de Octubre hospital from 1987 to 2012. The most relevant epidemiological and clinical variables reported in the literature were defined and compared in 3 equal time periods (1987-1995, 1996-2004 and 2005-2014). RESULTS: There was a 13% reduction in the frequency of severe TBI from the first to the last time period. An increase in the mean age from 35 to 43 years was observed, whereas the frequency of severe TBI according to sex remained approximately the same during the last decades of life. A distinct change was observed in the injury mechanism; traffic accidents decreased from 76% to 55%, particularly those involving 4-wheeled vehicles. However, falls increased significantly, especially in older women, and contusion and subdural haematoma were the most frequent structural injuries. Motor scores could not be reliably assessed for the last time period because of early intubation and sedative drug use. CONCLUSIONS: TBI epidemiology in Western countries has changed. This trend was also observed in our environment as an increase in mean age, which reflected the increase in falls among elderly patients.