Multi-center observational study on occurrence and related clinical factors of neurogenic heterotopic ossification in patients with disorders of consciousness.

Aims: to assess occurrence and clinical correlates of neurogenic heterotopic ossifications (NHO) in patients with prolonged disorder of consciousness (DoC).Design: multi-center cross-sectional observational study.Setting: 23 intensive neurorehabilitation units.Subjects: 287 patients with prolonged disorder of consciousness (DoC; 150 in vegetative state, VS, and 128 in minimally conscious state, MCS) of different etiology (vascular = 125, traumatic = 83, anoxic = 56, others = 14).Main Measures: clinical evidence of NHO confirmed by standard radiological and/or sonographic evaluation; Coma Recovery Scale-Revised; Disability Rating Scale (DRS); Early Rehabilitation Barthel Index; presence of ventilator support, spasticity, bone fractures and paroxysmal sympathetic hyperactivity.Results: 31 patients (11.2%) presented NHO. Univariate analyses showed that NHO was associated with VS diagnosis, traumatic etiology, high DRS category and total score, and high occurrence of limb spasticity and bone fractures. A cluster-corrected binary logistic regression model (excluding spasticity available in a subset of patients) showed that only lower DRS total score and presence of bone fractures were independently associated with NHO.Conclusions: NHO are relatively frequent in patients with DoC, and are independently associated with functional disability, bone fractures and spasticity. These findings contribute to identifying patients with DoC prone to develop NHO and requiring special interventions to improve functional recovery.

Multi-center study on overall clinical complexity of patients with prolonged disorders of consciousness of different etiologies.

Aim: to assess overall clinical complexity of patients with acquired disorders of consciousness (DoC) in vegetative state/unresponsive wakefulness syndrome (VS/UWS) vs. minimally conscious state- MCS) and in different etiologies..Design: Multi-center cross-sectional observational study.Setting: 23 intensive neurorehabilitation units.Subjects: 264 patients with DoC in the post-acute phase: VS/UWS = 141, and MCS = 123 due to vascular (n = 125), traumatic (n = 83) or anoxic (n = 56) brain injury.Main Measures: Coma Recovery Scale-Revised, and Disability Rating Scale (DRS); presence of medical devices (e.g., for eating or breathing); occurrence and severity of medical complications.Results: patients in DoC, and particularly those in VS/UWS, showed severe overall clinical complexity. Anoxic patients had higher overall clinical complexity, lower level of responsiveness/consciousness, higher functional disability, and higher needs of medical devices. Vascular patients had worse premorbid clinical comorbidities. The two etiologies showed a comparable rate of MC, higher than that observed in traumatic etiology.Conclusion: overall clinical complexity is significantly higher in VS/UWS than in MCS, and in non-traumatic vs. traumatic etiology. These findings could explain the worse clinical evolution reported in anoxic and vascular etiologies and in VS/UWS patients and contribute to plan patient-tailored care and rehabilitation programmes.

TFEB inhibition induces melanoma shut-down by blocking the cell cycle and rewiring metabolism.

Melanomas are characterised by accelerated cell proliferation and metabolic reprogramming resulting from the contemporary dysregulation of the MAPK pathway, glycolysis and the tricarboxylic acid (TCA) cycle. Here, we suggest that the oncogenic transcription factor EB (TFEB), a key regulator of lysosomal biogenesis and function, controls melanoma tumour growth through a transcriptional programme targeting ERK1/2 activity and glucose, glutamine and cholesterol metabolism. Mechanistically, TFEB binds and negatively regulates the promoter of DUSP-1, which dephosphorylates ERK1/2. In melanoma cells, TFEB silencing correlates with ERK1/2 dephosphorylation at the activation-related p-Thr185 and p-Tyr187 residues. The decreased ERK1/2 activity synergises with TFEB control of CDK4 expression, resulting in cell proliferation blockade. Simultaneously, TFEB rewires metabolism, influencing glycolysis, glucose and glutamine uptake, and cholesterol synthesis. In TFEB-silenced melanoma cells, cholesterol synthesis is impaired, and the uptake of glucose and glutamine is inhibited, leading to a reduction in glycolysis, glutaminolysis and oxidative phosphorylation. Moreover, the reduction in TFEB level induces reverses TCA cycle, leading to fatty acid production. A syngeneic BRAFV600E melanoma model recapitulated the in vitro study results, showing that TFEB silencing sustains the reduction in tumour growth, increase in DUSP-1 level and inhibition of ERK1/2 action, suggesting a pivotal role for TFEB in maintaining proliferative melanoma cell behaviour and the operational metabolic pathways necessary for meeting the high energy demands of melanoma cells.

Incidents related to critical patient safety during in-hospital transfer.

OBJECTIVE: To analyze the incidents related to patient safety (IRSP) and their risk factors during in-hospital transfer (IHT) of critical patients after the application of a protocol, and to evaluate safety during transfer using quality indicators. DESIGN: A prospective, observational and non-intervention cohort study was carried out. SETTING: A 10-bed multipurpose Intensive Care Unit (ICU) of a second level university hospital. PATIENTS: All IHTs of critical patients in the ICU for diagnostic tests and to the operating room between March 2011 and March 2017 were included in the study. MAIN MEASUREMENTS: Demographic variables, patient severity, transfer priority, moment of the day, reason and type of transfer team. Pre-transport checklist items and IRSP were collected. A biannual analysis was made of quality indicators designed for IHT. RESULTS: A total of 805 transfers were registered, mostly of an urgent nature (53.7%) and for diagnostic tests (77%). In turn, 112 transfers (13.9%) presented some type of IRSP; 54% related to the equipment and 30% related to team and organization. Adverse events occurred in 19 (2.4%) transfers. Risk factors identified in the multivariate analysis were mechanical ventilation and the transport team. The evolution of the indicators related to transport was significantly favorable. CONCLUSIONS: After the application of an IHT protocol, IRSP are low. The main risk factor is invasive mechanical ventilation. The experience of the team performing IHT influences the detection of a greater number of incidents.

Compliance with a sepsis bundle and its effect on intensive care unit mortality in surgical septic shock patients.

BACKGROUND: The Surviving Sepsis Campaign was launched in 2002, aiming at a 25% reduction in mortality in sepsis during a 5-year period. We hypothesized that the compliance with an adapted sepsis bundle would improve intensive care unit (ICU) survival in a cohort of surgical septic shock patients. METHODS: A retrospective, observational study was performed in surgical ICUs from two University hospitals. Seven quality indicators were considered to study the compliance with the sepsis bundle in 182 patients: (1) administration of antibiotics within 6 hours from diagnosis of septic shock, (2) initial effective antibiotic treatment, (3) adequate resuscitation within 6 hours after the diagnosis of septic shock, (4) administration of steroids, (5) use of activated protein C, (6) glucose control, and (7) protective ventilation. Univariate and multivariate logistic regression analyses were performed to make a predictive model to study the probability of survival according to the number of therapeutic guidelines fulfilled and to adjust for other predictive factors. RESULTS: Compliance with individual guidelines was considered adequate in more than 60% of the cases, except in the case of glucose control. For all quality indicators, ICU survival was higher in the bundle-compliant patients. Survival (61%) was associated with the fulfilment of increasing number of therapeutic guidelines (odds ratio, 1.64; 95% confidence interval, 1.28-2.1; p < 0.001). CONCLUSIONS: In surgical septic shock patients, the outcome was significantly related to the number of fulfilled therapeutic guidelines included in a sepsis bundle.

Incidents related to critical patient safety during in-hospital transfer. / Incidentes relacionados con la seguridad del paciente crítico durante los traslados intrahospitalarios.

OBJECTIVE: To analyze the incidents related to patient safety (IRSP) and their risk factors during in-hospital transfer (IHT) of critical patients after the application of a protocol, and to evaluate safety during transfer using quality indicators. DESIGN: A prospective, observational and non-intervention cohort study was carried out. SETTING: A 10-bed multipurpose Intensive Care Unit (ICU) of a second level university hospital. PATIENTS: All IHTs of critical patients in the ICU for diagnostic tests and to the operating room between March 2011 and March 2017 were included in the study. MAIN MEASUREMENTS: Demographic variables, patient severity, transfer priority, moment of the day, reason and type of transfer team. Pre-transport checklist items and IRSP were collected. A biannual analysis was made of quality indicators designed for IHT. RESULTS: A total of 805 transfers were registered, mostly of an urgent nature (53.7%) and for diagnostic tests (77%). In turn, 112 transfers (13.9%) presented some type of IRSP; 54% related to the equipment and 30% related to team and organization. Adverse events occurred in 19 (2.4%) transfers. Risk factors identified in the multivariate analysis were mechanical ventilation and the transport team. The evolution of the indicators related to transport was significantly favorable. CONCLUSIONS: After the application of an IHT protocol, IRSP are low. The main risk factor is invasive mechanical ventilation. The experience of the team performing IHT influences the detection of a greater number of incidents.

Frequency of Mycobacterium bovis as an etiologic agent in extrapulmonary tuberculosis in HIV-positive and -negative Mexican patients.

Mycobacterium bovis can be an important etiological agent for extrapulmonary (EP) manifestations of tuberculosis, especially in HIV-infected persons. From January 2000 to December 2003, M. bovis as a cause of EP tuberculosis was investigated at the Pneumonology Service, Hospital General de Mexico, Mexico City. Eighty HIV-positive (HIV+) patients and 83 HIV-negative (HIV-) with EP involvement (ganglionar, genitourinary, meningeal, cutaneous, peritoneal, and pericardial) were analyzed using clinical, immunological, bacteriological, histopathological, and molecular biology methods. Mycobacterium species were identified by hsp65-RFLP analysis and species of M. tuberculosis complex isolates by spoligotyping. M. bovis was present in 6 HIV- cases (7.2%; 3 with lymphadenitis and 3 genitourinary) vs 11 in HIV+ cases (13.75%; 7 with lymphadenitis, 3 genitourinary, and 1 meningeal). Favorable response to retroviral and specific M. bovis chemotherapy was observed. Spoligotyping showed a unique profile in each isolate, 16 belonging to BOV1 lineage and 1 to BOV2 lineage. M. bovis is an significant re-emerging cause of EPTB in Mexico. Consumption of unpasteurized dairy products is the most likely source of transmission. Successful treatment depends on the adequate and opportune identification of the agent responsible.

[Hemodynamic variability caused by pressure-volume plotting and alveolar recruitment maneuvers in patients with adult respiratory distress syndrome]. / Variaciones hemodinámicas inducidas por la realización de una curva presión-volumen y una maniobra de reclutamiento alveolar en pacientes con síndrome de distrés respiratorio agudo.

OBJECTIVES: The plotting of pressure-volume curves and the performance of alveolar recruitment maneuvers are common practices in the care of patients with adult respiratory distress syndrome (ARDS), even though potentially harmful hemodynamic effects are associated with sustaining a high intrathoracic pressure. Our aim was to analyze hemodynamic and ventilatory changes related to these 2 maneuvers and to assess the short-term effectiveness of recruitment. PATIENTS AND METHODS: The patients had ARDS and were being monitored with a catheter connected to a PiCCO system. All measurements were taken in sinus rhythm and with adequate vascular filling. Values recorded during plotting of the quasistatic pressure-volume curve and the recruitment maneuver (sustained airway pressure of 40 cm H2O) were the cardiac index, mean arterial pressure, heart rate, systolic volume index, and oxygen saturation (SpO2). Blood gas measurements were recorded before the maneuvers and 15 minutes afterwards. RESULTS: All parameters decreased significantly in the 14 patients studied. The mean (SD) maximum decreases, from which all patients recovered within 2 minutes, were as follows: cardiac index, 26% (16%); mean arterial pressure, 6% (6%); heart rate, 4% (5%), systolic volume index, 21% (15%); and SpO2, 3% (3%). Significant increases in PaO2 (7% [6%]) and the ratio of PaO2 to the fraction of inspired oxygen were recorded after the recruitment maneuver (P=.016 and P=.014, respectively), but the changes were not clinically significant. CONCLUSIONS: The hemodynamic disturbances associated with the alveolar recruitment maneuver based on sustaining a high end-expiratory pressure and the minor improvement in oxygenation achieved as a result suggest that the routine use of that maneuver in ARDS patients is of questionable value.

Targeting hedgehog signaling reduces self-renewal in embryonal rhabdomyosarcoma.

Current treatment regimens for rhabdomyosarcoma (RMS), the most common pediatric soft tissue cancer, rely on conventional chemotherapy, and although they show clinical benefit, there is a significant risk of adverse side effects and secondary tumors later in life. Therefore, identifying and targeting sub-populations with higher tumorigenic potential and self-renewing capacity would offer improved patient management strategies. Hedgehog signaling has been linked to the development of embryonal RMS (ERMS) through mouse genetics and rare human syndromes. However, activating mutations in this pathway in sporadic RMS are rare and therefore the contribution of hedgehog signaling to oncogenesis remains unclear. Here, we show by genetic loss- and gain-of-function experiments and the use of clinically relevant small molecule modulators that hedgehog signaling is important for controlling self-renewal of a subpopulation of RMS cells in vitro and tumor initiation in vivo. In addition, hedgehog activity altered chemoresistance, motility and differentiation status. The core stem cell gene NANOG was determined to be important for ERMS self-renewal, possibly acting downstream of hedgehog signaling. Crucially, evaluating the presence of a subpopulation of tumor-propagating cells in patient biopsies identified by GLI1 and NANOG expression had prognostic significance. Hence, this work identifies novel functional aspects of hedgehog signaling in ERMS, redefines the rationale for its targeting as means to control ERMS self-renewal and underscores the importance of studying functional tumor heterogeneity in pediatric cancers.

Active tuberculosis patients have high levels of IgA anti-alpha-crystallin and isocitrate lyase proteins.

SETTING: Mexico City, Mexico. OBJECTIVE: To identify proteins synthetised by Mycobacterium tuberculosis in hypoxic culture, which resemble more closely a granuloma environment than aerobic culture, and to determine if they are recognised by antibodies from patients with active pulmonary tuberculosis (PTB). DESIGN: Soluble extracts from M. tuberculosis H37Rv cultured under aerobic or hypoxic conditions were analysed using two-dimensional polyacrylamide gel electrophoresis, and proteins over-expressed under hypoxia were identified by mass spectrometry. The presence of immunoglobulin (Ig) G, IgA and IgM antibodies against these proteins was determined in the serum of 42 patients with active PTB and 42 healthy controls. RESULTS: We selected three M. tuberculosis H37Rv proteins (alpha-crystallin protein [Acr, Rv2031c], universal stress protein Rv2623 and isocitrate lyase [ICL, RV0467]) that were over-expressed under hypoxia. Titres of anti-Acr and anti-ICL IgA antibodies were higher in patients than in healthy controls, with an area under the receiver operating characteristic curve of 0.71 for anti-ICL IgA antibodies. CONCLUSION: ICL could be used in combination with other M. tuberculosis antigens to improve the sensitivity and specificity of current serological TB diagnostic methods.

Cloning of chicken and mouse alpha 1b adrenergic receptor.

A partial cDNA encoding most of the third intracellular loop of the chicken alpha 1b adrenergic receptor subtype, obtained by reverse transcription-polymerase chain reaction (RT-PCR) techniques using degenerate primers derived from mammalian sequences, was used to isolate an alpha 1b adrenergic receptor cDNA from brain. The cDNA encodes a potential protein of 507 amino acids and Northern hybridization of poly(A)+ RNA from chicken brain of different developmental stages detected a single 3.5 kb transcript. Analysis of receptor expression indicated that the alpha 1b adrenergic receptor is widely distributed in chicken tissues, specially kidney and liver. cDNA and genomic clones encoding sequences of the mouse alpha 1b adrenergic receptor were also isolated.

Inducible site-specific recombination in the brain.

The Cre/loxP recombination system allows the generation of tissue-specific somatic mutations in mice. Additional temporal control of somatic mutagenesis is highly desirable, as this would permit a more precise analysis of gene function in complex systems such as the central nervous system. Extending our previous studies, we compared several ligand-regulated recombinases, in which the ligand-binding domain (LBD) of the progesterone receptor or the estrogen receptor was fused to the Cre recombinase. A fusion protein between the Cre recombinase and a truncated LBD of the progesterone receptor was chosen to obtain inducible recombination in the brain. This fusion protein can be activated by the synthetic steroid RU486, but not by the physiological hormone progesterone. Its expression was targeted to the brain using regulatory sequences of the calcium-calmodulin-dependent kinase IIalpha or the Thy-1 gene. Application of RU486 to the mice induced Cre-mediated recombination of a lacZ reporter transgene in the cortex and hippocampus, showing that spatially and temporally controlled gene targeting can be mediated in the brain.

Chronic consumption of dietary proanthocyanidins modulates peripheral clocks in healthy and obese rats.

Circadian rhythm plays an important role in maintaining homeostasis, and its disruption increases the risk of developing metabolic syndrome. Circadian rhythm is maintained by a central clock in the hypothalamus that is entrained by light, but circadian clocks are also present in peripheral tissues. These peripheral clocks are trained by other cues, such as diet. The aim of this study was to determine whether proanthocyanidins, the most abundant polyphenols in the human diet, modulate the expression of clock and clock-controlled genes in the liver, gut and mesenteric white adipose tissue (mWAT) in healthy and obese rats. Grape seed proanthocyanidin extracts (GSPEs) were administered for 21 days at 5, 25 or 50 mg GSPE/kg body weight in healthy rats and 25 mg GSPE/kg body weight in rats with diet-induced obesity. In healthy animals, GSPE administration led to the overexpression of core clock genes in a positive dose-dependent manner. Moreover, the acetylated BMAL1 protein ratio increased with the same pattern in the liver and mWAT. With regards to clock-controlled genes, Per2 was also overexpressed, whereas Rev-erbα and RORα were repressed in a negative dose-dependent manner. Diet-induced obesity always resulted in the overexpression of some core clock and clock-related genes, although the particular gene affected was tissue specific. GSPE administration counteracted disturbances in the clock genes in the liver and gut but was less effective in normalizing the clock gene disruption in WAT. In conclusion, proanthocyanidins have the capacity to modulate peripheral molecular clocks in both healthy and obese states.

Identification of four splice variants of the mouse stress-activated protein kinase JNK/SAPK alpha-isoform.

Four splice variants (alpha I-IV) of the stress-activated protein kinase JNK/SAPK alpha-isoform have been identified in the mouse. One of them (alpha I) contains an open reading frame of 1269 bp encoding a potential protein of 423 amino acids, whereas the second variant (alpha II) differs in a region encoding 31 amino acids located in subdomain IX. alpha III lacks this region and also differs in the terminal portion of the 3' untranslated region (3' UTR). A fourth variant (alpha IV) which lacks a region of 41 amino acids located in subdomain IX has also been identified. These splice variants are differentially expressed in mouse tissues: alpha I is the most abundant in brain areas, whereas alpha II is mainly expressed in extracerebral tissues, such as liver; alpha III and alpha IV are present in brain and other tissues although in lower amounts.

Analysis of splicing of four mouse JNK/SAPKalpha variants.

The JNK/SAPK (c-Jun NH2-terminal kinase/stress-activated protein kinase) cascade is activated by a variety of stress stimuli and by the inflammatory cytokines interleukin-I (IL-I) and tumor necrosis factor alpha (TNFalpha). Four splice variants of the mouse JNK/SAPKalpha isoform, which differ in a region located in subdomains IX-X of the protein, were previously identified. Analysis of the sequence of the central region of the mouse JNK/SAPKalpha gene indicates that splice variants I and II are generated by a typical alternative splicing mechanism, while splice variants III and IV are generated by a less common mechanism, where alternative 3' splice sites located inside an exon (cryptic sites) are selected. The major splice variants alphaI and all have a wide and similar distribution in hippocampus, cerebral cortex, caudate-putamen, amygdala and the granule cell layer of cerebellum, although their expression is specifically regulated in certain cell types.

Immunodetection of serotonin transporter from mouse brain.

A DNA fragment encoding amino acid sequences from the amino terminal region of the mouse serotonin transporter was isolated and sequenced. This transporter is widely distributed throughout the mouse brain, as deduced by heminested reverse transcription-polymerase chain reaction (RT-PCR) assays. To identify the serotonin transporter protein, we have developed specific antibodies against a fusion protein containing its amino terminal region, a domain which shows a low degree of homology between the different neurotransmitter transporters. Western blot analysis of mouse brain membranes detected the serotonin transporter as a 71 kDa polypeptide.

Identification of a long huntingtin mRNA transcript in mouse brain.

Two mRNA species of the Huntington disease (HD) gene that share identical protein coding sequences but differ in their 3' untranslated region have been identified in human. Although a similar situation has been suggested to occur in mouse, only one cDNA has been isolated to date. We report the isolation of a novel partial cDNA of the mouse HD gene that is identical in its protein coding sequence to the previously reported cDNA, although it differs in the distal portion of the 3' untranslated region. Northern blotting assays indicate that this mRNA transcript is preferentially expressed in brain, with highest levels in cerebellum, cerebral cortex and striatum.

Differential effect of chronic ethanol treatment on barbiturate and steroid modulation of muscimol-binding to rat brain cortex.

We report the differential alterations produced by chronic ethanol treatment on the modulation, by the barbiturate thiopental and the steroid 5 beta-pregnan-3 alpha-ol-20-one, of the binding of [3H]muscimol to membrane preparations from rat brain cortex. We found a clear barbiturate- and steroid-promoted enhancement of muscimol-binding to membranes in both control and ethanol-treated animals. However, the enhancements were higher in control animals, using the barbiturate, and in ethanol-treated rats, using the steroid, Bmax and Kd values were also differentially affected in control and ethanol-treated animals by the presence of the barbiturate or the steroid.

Identification of a cyclic adenosine 3',5'-monophosphate-dependent protein kinase phosphorylation site in the carboxy terminal tail of human D1 dopamine receptor.

Each of the dopamine receptor subtypes contains several consensus sites for phosphorylation in their intracellular domains. We have used fusion proteins of the carboxy terminal tail of D1 and D5 dopamine receptors to study the phosphorylation of these proteins by cyclic adenosine 3',5' monophosphate (cAMP)-dependent protein kinase (PKA) and protein kinase C (PKC). The fusion protein of D1 dopamine receptor was efficiently phosphorylated by PKA, but not by PKC. Site-directed mutagenesis of serine 380 to an alanine residue precluded the phosphorylation by the kinase. No phosphorylation of the D5 dopamine receptor fusion protein was observed with either PKA or PKC, which indicates that these receptor subtypes might differ in their mechanisms of regulation.

Phosphorylation of the third intracellular loop of the mouse alpha1b-adrenergic receptor by cAMP-dependent protein kinase.

The third intracellular loop of adrenergic receptors has been implicated in their interaction with guanine nucleotide-binding proteins (G proteins). One of the mechanisms involved in the modulation of receptor function is the phosphorylation of specific residues by intracellular kinases. alpha1b-Adrenergic receptor is phosphorylated in vitro by cAMP-dependent protein kinase (PKA), although its physiological effect remains to be determined. We have produced fusion proteins formed by glutathione S-transferase and sequences of the third intracellular loop of mouse alpha1a-, alpha1b-, and alpha1d-adrenergic receptor subtypes, and used them as substrates for PKA. Only the fusion protein containing the alpha1b sequence was phosphorylated in vitro by this kinase. Site-directed mutagenesis of a serine (homologue to serine 278 of the rat sequence, RSS) to an alanine residue precluded phosphorylation by PKA.