Defining the landscape of ATP-competitive inhibitor resistance residues in protein kinases.

Kinases are involved in disease development and modulation of their activity can be therapeutically beneficial. Drug-resistant mutant kinases are valuable tools in drug discovery efforts, but the prediction of mutants across the kinome is challenging. Here, we generate deep mutational scanning data to identify mutant mammalian kinases that drive resistance to clinically relevant inhibitors. We aggregate these data with subsaturation mutagenesis data and use it to develop, test and validate a framework to prospectively identify residues that mediate kinase activity and drug resistance across the kinome. We validate predicted resistance mutations in CDK4, CDK6, ERK2, EGFR and HER2. Capitalizing on a highly predictable residue, we generate resistance mutations in TBK1, CSNK2A1 and BRAF. Unexpectedly, we uncover a potentially generalizable activation site that mediates drug resistance and confirm its impact in BRAF, EGFR, HER2 and MEK1. We anticipate that the identification of these residues will enable the broad interrogation of the kinome and its inhibitors.

SPARC (secreted protein acidic and rich in cysteine) knockdown protects mice from acute liver injury by reducing vascular endothelial cell damage.

Secreted protein, acidic and rich in cysteine (SPARC) is involved in many biological process including liver fibrogenesis, but its role in acute liver damage is unknown. To examine the role of SPARC in acute liver injury, we used SPARC knock-out (SPARC(-/-)) mice. Two models of acute liver damage were used: concanavalin A (Con A) and the agonistic anti-CD95 antibody Jo2. SPARC expression levels were analyzed in liver samples from patients with acute-on-chronic alcoholic hepatitis (AH). SPARC expression is increased on acute-on-chronic AH patients. Knockdown of SPARC decreased hepatic damage in the two models of liver injury. SPARC(-/-) mice showed a marked reduction in Con A-induced necroinflammation. Infiltration by CD4+ T cells, expression of tumor necrosis factor-α and interleukin-6 and apoptosis were attenuated in SPARC(-/-) mice. Sinusoidal endothelial cell monolayer was preserved and was less activated in Con A-treated SPARC(-/-) mice. SPARC knockdown reduced Con A-induced autophagy of cultured human microvascular endothelial cells (HMEC-1). Hepatic transcriptome analysis revealed several gene networks that may have a role in the attenuated liver damaged found in Con A-treated SPARC(-/-) mice. SPARC has a significant role in the development of Con A-induced severe liver injury. These results suggest that SPARC could represent a therapeutic target in acute liver injury.

Thoracic paravertebral anaesthesia for awake video-assisted thoracoscopic surgery daily.

Thoracic paravertebral blockade has been described as an effective alternative to epidural blockade for the management of postoperative pain after thoracic surgery. Here we present what we believe is the first description of the use of thoracic paravertebral block as the sole anaesthetic for video-assisted thoracoscopy. Two oncology patients with severe respiratory disease presented for video-assisted thoracoscopic surgery. Thoracic paravertebral block provided excellent surgical conditions and postoperative pain relief for these patients and allowed an optimal assessment of the anaesthetic impact on respiratory function.

Management of anesthesia in awake craniotomy.

UNLABELLED: The awake craniotomy technique was originally introduced for the surgical treatment of epilepsy and has subsequently been used in patients undergoing surgical management of supratentorial tumors, arteriovenous malformation, deep brain stimulation, and mycotic aneurysms near critical brain regions. This surgical approach aims to maximize lesion resection while sparing important areas of the brain (motor, somatosensory, and language areas). Awake craniotomy offers great advantages with respect to patient outcome. In this type of procedure, the anesthetist's goal is to make the operation safe and effective and reduce the psychophysical distress of the patient. Many authors have described different anesthetic care protocols for awake craniotomy based on monitored or general anesthesia; however, there is still no consensus as to the best anesthetic technique. The most commonly used drugs for awake craniotomies are propofol and remifentanil, but dexmedetomidine is beginning to be used more commonly outside of Europe. Personal experience, careful planning, and attention to detail are the basis for obtaining good awake craniotomy RESULTS: Additional studies are necessary in order to optimize the procedure, reduce complications, and improve patient tolerance. The aim of this review is to present a thorough report of the literature, with particular attention to neuro-oncology surgery.

Androgen 5-alpha-reductase type 2 is highly expressed and active in rat spinal cord motor neurones.

Spinal cord motoneurones express high levels of androgen receptor. However, in responsive tissue, the effects of testosterone is often mediated by the more potent androgenic derivative 5-alpha-dihydrotestosterone (DHT). This compound is formed in androgen target cells by the enzyme 5-alpha-reductase. Two isoforms of the 5-alpha-reductase, with limited degree of homology, have been cloned, type 1 and type 2. The low affinity-constitutive type 1 isoenzyme is widely distributed in the body; the high affinity-androgen regulated 5-alpha-reductase type 2 is confined to androgen-dependent structures and shows a peculiar pH optimum at acidic values. We have previously shown that high levels of 5-alpha-reductase activity are detectable in rat spinal cord. Here, using reverse transcriptase-polymerase chain reaction, we show that both isoforms are expressed in the whole spinal cord of the rat. The enzymatic pH optimum measured in immortalized spinal cord motoneurones (NSC34) is typical of the type 2 isoenzyme. Using in situ hybridization technique, we found that 5-alpha-reductase type 2 is confined to the motoneuronal cells of the anterior horns of the rat spinal cord, the cells that also are known to express high levels of androgen receptor. Because of the close association of androgen receptor and 5-alpha alpha-reductase type 2, motoneuronal cells should be considered as target cells for androgens.

Metabolic response to exercise.

At the beginning, the survival of humans was strictly related to their physical capacity. There was the need to resist predators and to provide food and water for life. Achieving these goals required a prompt and efficient energy system capable of sustaining either high intensity or maintaining prolonged physical activity. Energy for skeletal muscle contraction is supplied by anaerobic and aerobic metabolic pathways. The former can allow short bursts of intense physical activity (60-90 sec) and utilizes as energetic source the phosphocreatine shuttle and anaerobic glycolysis. The aerobic system is the most efficient ATP source for skeletal muscle. The oxidative phosporylation of carbohydrates, fats and, to a minor extent, proteins, can sustain physical activity for many hours. Carbohydrates are the most efficient fuel for working muscle and their contribution to total fuel oxidation is positively related to the intensity of exercise. The first metabolic pathways of carbohydrate metabolism to be involved are skeletal muscle glycogenolysis and glycolysis. Later circulating glucose, formed through activated gluconeogenesis, becomes an important energetic source. Among glucose metabolites, lactate plays a primary role as either direct or indirect (gluconeogenesis) energy source for contracting skeletal muscle. Fat oxidation plays a primary role during either low-moderate intensity exercise or protracted physical activity (over 90-120 min). Severe muscle glycogen depletion results in increased rates of muscle proteolysis and branched chain amino acid oxidation. Endurance training ameliorates physical performance by improving cardiopulmonary efficiency and optimizing skeletal muscle supply and oxidation of substrates.

Diabetes and exercise.

Physical activity has acute and chronic effects on glucose, lipid and protein metabolism. Long-term effects of regular exercise are particularly advantageous for Type 2 diabetic patients. Regular aerobic exercise reduces visceral fat mass and body weight without decreasing lean body mass, ameliorates insulin sensitivity, glucose and BP control, lipid profile and reduces the cardiovascular risk. For these reasons, regular aerobic physical activity must be considered as an essential component of the cure of Type 2 diabetes mellitus. In this regard, individual behavioral strategies have been documented to be effective in motivating sedentary Type 2 diabetic subjects to the adoption and the maintenance of regular physical activity. In Type 1 diabetic subjects, the lack of the physiological inhibition of insulin secretion during exercise results in a potential risk of hypoglycemia. On the other hand, exercise-induced activation of counter-regulatory hormones might trigger an acute metabolic derangement in severe insulin-deficient subjects. Thus, diabetic patients, before starting exercise sessions, must be carefully educated about the consequences of physical activity on their blood glucose and the appropriate modifications of diet and insulin therapy.

Polyglutamine tract expansion of the androgen receptor in a motoneuronal model of spinal and bulbar muscular atrophy.

Spinobulbar muscular atrophy (SBMA) is a late-onset disorder characterized by progressive muscle loss, degeneration of motoneurons in the spinal cord and brainstem, and partial androgen insensitivity. SBMA is directly correlated with the expansion of CAG repeats encoding a polyglutamine tract (polyQ) of extended length. The identification of polyQ expansion in SBMA led to the discovery of an entire class of neurodegenerative disorders. In fact, at least eight different diseases, including Huntington's disease, share a common molecular mechanism involving an expansion of a polyQ tract within different proteins. The elongated polyQ tract causes a toxic gain of function in the mutant protein and is associated with the formation of intracellular aggregates, whose pathogenetic role has not been fully established yet. Our observations in a motoneuron cell line (NSC34), indicate that the expression of the androgen receptor (AR) carrying the elongated polyQ tract (AR-Q48) has a toxic effect in aggregate-independent manner. In fact, in basal condition, AR-Q48 shows a cytoplasmic diffuse distribution, yet it reduces the viability of transfected NSC34. In contrast, testosterone treatment, while inducing aggregation of the mutant AR, also increases cell viability. Aggregates in NSC34 are localized mainly in the perinuclear region and occasionally in the neuropil, whereas no nuclear aggregate has ever been found. Further observations of the minor subset of cells showing neuropil aggregates, reveal an alteration of the neurite morphology, suggesting a different role of the two types of cytoplasmic aggregates.

5Alpha-reductase type 2 and androgen receptor expression in gonadotropin releasing hormone GT1-1 cells.

Gonadal steroids are potent modulators of gonadotropin releasing hormone (GnRH) secretion, and androgen binding sites and 5alpha-reductase activity have been found in the immortalized GnRH secreting cell line GT1-1, suggesting the existence of a direct androgenic control of GnRH dynamics. Two isoforms of the 5alpha-reductase have been cloned with very different biochemical/functional properties: 5alpha-reductase type 1 (widely distributed in the body) and 5alpha-reductase type 2 (confined in androgen target structures). We have analysed whether, in GT1-1, androgen binding sites are linked to "classical" androgen receptor, and which 5alpha-reductase isoform is active. Reverse transcriptase-polymerase chain reaction analysis showed that the mRNAs coding for androgen receptor and for the two 5alpha-reductase isoforms are all expressed in GT1-1 cells. However, the 5alpha-reductase enzymatic reaction showed a peak of activity at a narrow pH around 5.5, the optimum for the 5alpha-reductase type 2. The affinity for testosterone, of the enzyme present in GT1-1 cells, was very similar to that observed for the recombinant type 2 isozyme expressed in yeasts. The data indicate that GT1-1 cells (i) express a "classical" androgen receptor and (ii) contain the 5alpha-reductase type 2 isoform, a specific marker of androgen-responsiveness.