Comparison of short-term outcomes between people with and without a pre-morbid mental health diagnosis following surgery for traumatic hand injury: a prospective longitudinal study of a multicultural cohort.

BACKGROUND: Following traumatic hand injury, few studies have compared outcomes between people with and without a pre-morbid mental health diagnosis. This study aimed to compare sub-acute outcomes in a multicultural patient cohort with surgically managed traumatic hand injury with and without a pre-morbid mental health diagnosis. METHODS: A prospective, observational cohort study of people with traumatic hand injury presenting pre- surgically to a high-volume hand injury centre in a region of cultural and language diversity was conducted. Participants were assessed face-to-face (baseline) then via telephone (3-months post-surgery) and categorized according to a pre-morbid medically diagnosed mental health diagnosis. Baseline and follow-up assessments included global mental health, and the EuroQol (EQ) 'Health Today' analogue scale (0-100) and health domains. Return-to-work status, complications/symptomatic complaints, and hand function (QuickDASH) were also collected at follow-up. Adjusted analyses-accounting for covariates including cultural identity-were conducted to determine whether 3-month outcomes were associated with a pre-morbid mental health diagnosis. RESULTS: From 405 eligible patients, 386 were enrolled (76% male, mean age 38.9 (standard deviation 15.6)); 57% self-identified as Australian and 22% had a pre-morbid mental health diagnosis. Common injuries regardless of pre-morbid mental health diagnosis were skin (40%), tendon (17%) and bone (17%) injuries. None were complex mutilating injuries. Seventy-eight per cent of the cohort was followed-up. In adjusted analyses, a pre-morbid mental health diagnosis was associated with lower odds for reporting 'good or better' global mental health (Odds Ratio (OR) 0.23 (95% Confidence Interval (CI) 0.18, 0.47), p < 0.001), 'no' anxiety or depression (OR 0.21 (0.11, 0.40), p < 0.001) and no pain (OR 0.56 (0.31, 0.98), p = 0.04)(EQ domains), and worse EQ 'Health Today' (10 points on average (95%CI -14.9, -5.1, p < 0.001). QuickDASH scores, rates of complications/symptomatic complaints and return-to-work profiles were similar. CONCLUSIONS: Despite reporting worse mental and health-related quality-of-life outcomes post-surgery, people with a pre-morbid mental health diagnosis regardless of cultural identity experienced similar clinical and return-to-work outcomes. Future research assessing the value of screening for pre-morbid mental health conditions on post-surgical outcomes is required and should include people with more complex hand injuries.

Metabolic control by AMPK in white adipose tissue.

White adipose tissue (WAT) plays an important role in the integration of whole-body metabolism by storing fat and mobilizing triacylglycerol when needed. The released free fatty acids can then be oxidized by other tissues to provide ATP. AMP-activated protein kinase (AMPK) is a key regulator of metabolic pathways, and can be targeted by a new generation of direct, small-molecule activators. AMPK activation in WAT inhibits insulin-stimulated lipogenesis and in some situations also inhibits insulin-stimulated glucose uptake, but AMPK-induced inhibition of ß-adrenergic agonist-stimulated lipolysis might need to be re-evaluated in vivo. The lack of dramatic effects of AMPK activation on basal metabolism in WAT could be advantageous when treating type 2 diabetes with pharmacological pan-AMPK activators.

Specific post-translational modifications of soluble tau protein distinguishes Alzheimer's disease and primary tauopathies.

Tau protein aggregates in several neurodegenerative disorders, referred to as tauopathies. The tau isoforms observed in post mortem human brain aggregates is used to classify tauopathies. However, distinguishing tauopathies ante mortem remains challenging, potentially due to differences between insoluble tau in aggregates and soluble tau in body fluids. Here, we demonstrated that tau isoforms differ between tauopathies in insoluble aggregates, but not in soluble brain extracts. We therefore characterized post-translational modifications of both the aggregated and the soluble tau protein obtained from post mortem human brain tissue of patients with Alzheimer's disease, cortico-basal degeneration, Pick's disease, and frontotemporal lobe degeneration. We found specific soluble signatures for each tauopathy and its specific aggregated tau isoforms: including ubiquitination on Lysine 369 for cortico-basal degeneration and acetylation on Lysine 311 for Pick's disease. These findings provide potential targets for future development of fluid-based biomarker assays able to distinguish tauopathies in vivo.

Comparison of Different Ex-Vivo Preservation Strategies on Cardiac Metabolism in an Animal Model of Donation after Circulatory Death.

Transplantation of heart following donation after circulatory death (DCD) was recently introduced into clinical practice. Ex vivo reperfusion following DCD and retrieval is deemed necessary in order to evaluate the recovery of cardiac viability after the period of warm ischemia. We tested the effect of four different temperatures (4 °C-18 °C-25 °C-35 °C) on cardiac metabolism during 3-h ex vivo reperfusion in a porcine model of DCD heart. We observed a steep fall in high-energy phosphate (ATP) concentrations in the myocardial tissue at the end of the warm ischemic time and only limited regeneration during reperfusion. Lactate concentration in the perfusate increased rapidly during the first hour of reperfusion and slowly decreased afterward. However, the temperature of the solution does not seem to have an effect on either ATP or lactate concentration. Furthermore, all cardiac allografts showed a significant weight increase due to cardiac edema, regardless of the temperature.

AMPK inhibits liver gluconeogenesis: fact or fiction?

Is there a role for AMPK in the control of hepatic gluconeogenesis and could targeting AMPK in liver be a viable strategy for treating type 2 diabetes? These are frequently asked questions this review tries to answer. After describing properties of AMPK and different small-molecule AMPK activators, we briefly review the various mechanisms for controlling hepatic glucose production, mainly via gluconeogenesis. The different experimental and genetic models that have been used to draw conclusions about the role of AMPK in the control of liver gluconeogenesis are critically discussed. The effects of several anti-diabetic drugs, particularly metformin, on hepatic gluconeogenesis are also considered. We conclude that the main effect of AMPK activation pertinent to the control of hepatic gluconeogenesis is to antagonize glucagon signalling in the short-term and, in the long-term, to improve insulin sensitivity by reducing hepatic lipid content.

Inhibition of basal and glucagon-induced hepatic glucose production by 991 and other pharmacological AMPK activators.

Pharmacological AMPK activation represents an attractive approach for the treatment of type 2 diabetes (T2D). AMPK activation increases skeletal muscle glucose uptake, but there is controversy as to whether AMPK activation also inhibits hepatic glucose production (HGP) and pharmacological AMPK activators can have off-target effects that contribute to their anti-diabetic properties. The main aim was to investigate the effects of 991 and other direct AMPK activators on HGP and determine whether the observed effects were AMPK-dependent. In incubated hepatocytes, 991 substantially decreased gluconeogenesis from lactate, pyruvate and glycerol, but not from other substrates. Hepatocytes from AMPKß1-/- mice had substantially reduced liver AMPK activity, yet the inhibition of glucose production by 991 persisted. Also, the glucose-lowering effect of 991 was still seen in AMPKß1-/- mice subjected to an intraperitoneal pyruvate tolerance test. The AMPK-independent mechanism by which 991 treatment decreased gluconeogenesis could be explained by inhibition of mitochondrial pyruvate uptake and inhibition of mitochondrial sn-glycerol-3-phosphate dehydrogenase-2. However, 991 and new-generation direct small-molecule AMPK activators antagonized glucagon-induced gluconeogenesis in an AMPK-dependent manner. Our studies support the notion that direct pharmacological activation of hepatic AMPK as well as inhibition of pyruvate uptake could be an option for the treatment of T2D-linked hyperglycemia.

AMPK activation by SC4 inhibits noradrenaline-induced lipolysis and insulin-stimulated lipogenesis in white adipose tissue.

The effects of small-molecule AMP-activated protein kinase (AMPK) activators in rat epididymal adipocytes were compared. SC4 was the most effective and submaximal doses of SC4 and 5-amino-4-imidazolecarboxamide (AICA) riboside were combined to study the effects of AMPK activation in white adipose tissue (WAT). Incubation of rat adipocytes with SC4 + AICA riboside inhibited noradrenaline-induced lipolysis and decreased hormone-sensitive lipase (HSL) Ser563 phosphorylation, without affecting HSL Ser565 phosphorylation. Preincubation of fat pads from wild-type (WT) mice with SC4 + AICA riboside inhibited insulin-stimulated lipogenesis from glucose or acetate and these effects were lost in AMPKα1 knockout (KO) mice, indicating AMPKα1 dependency. Moreover, in fat pads from acetyl-CoA carboxylase (ACC)1/2 S79A/S212A double knockin versus WT mice, the effect of SC4 + AICA riboside to inhibit insulin-stimulated lipogenesis from acetate was lost, pinpointing ACC as the main AMPK target. Treatment with SC4 + AICA riboside decreased insulin-stimulated glucose uptake, an effect that was still observed in fat pads from AMPKα1 KO versus WT mice, suggesting the effect was partly AMPKα1-independent. SC4 + AICA riboside treatment had no effect on the insulin-induced increase in palmitate esterification nor on sn-glycerol-3-phosphate-O-acyltransferase activity. Therefore in WAT, AMPK activation inhibits noradrenaline-induced lipolysis and suppresses insulin-stimulated lipogenesis primarily by inactivating ACC and by inhibiting glucose uptake.

Aberrant Membrane Composition and Biophysical Properties Impair Erythrocyte Morphology and Functionality in Elliptocytosis.

Red blood cell (RBC) deformability is altered in inherited RBC disorders but the mechanism behind this is poorly understood. Here, we explored the molecular, biophysical, morphological, and functional consequences of α-spectrin mutations in a patient with hereditary elliptocytosis (pEl) almost exclusively expressing the Pro260 variant of SPTA1 and her mother (pElm), heterozygous for this mutation. At the molecular level, the pEI RBC proteome was globally preserved but spectrin density at cell edges was increased. Decreased phosphatidylserine vs. increased lysophosphatidylserine species, and enhanced lipid peroxidation, methemoglobin, and plasma acid sphingomyelinase (aSMase) activity were observed. At the biophysical level, although membrane transversal asymmetry was preserved, curvature at RBC edges and rigidity were increased. Lipid domains were altered for membrane:cytoskeleton anchorage, cholesterol content and response to Ca2+ exchange stimulation. At the morphological and functional levels, pEl RBCs exhibited reduced size and circularity, increased fragility and impaired membrane Ca2+ exchanges. The contribution of increased membrane curvature to the pEl phenotype was shown by mechanistic experiments in healthy RBCs upon lysophosphatidylserine membrane insertion. The role of lipid domain defects was proved by cholesterol depletion and aSMase inhibition in pEl. The data indicate that aberrant membrane content and biophysical properties alter pEl RBC morphology and functionality.

Inhibition of AMPK activity in response to insulin in adipocytes: involvement of AMPK pS485, PDEs, and cellular energy levels.

Insulin resistance in obesity and type 2 diabetes has been shown to be associated with decreased de novo fatty acid (FA) synthesis in adipose tissue. It is known that insulin can acutely stimulate FA synthesis in adipocytes; however, the mechanisms underlying this effect are unclear. The rate-limiting step in FA synthesis is catalyzed by acetyl-CoA carboxylase (ACC), known to be regulated through inhibitory phosphorylation at S79 by the AMP-activated protein kinase (AMPK). Previous results from our laboratory showed an inhibition of AMPK activity by insulin, which was accompanied by PKB-dependent phosphorylation of AMPK at S485. However, whether the S485 phosphorylation is required for insulin-induced inhibition of AMPK or other mechanisms underlie the reduced kinase activity is not known. To investigate this, primary rat adipocytes were transduced with a recombinant adenovirus encoding AMPK-WT or a nonphosphorylatable AMPK S485A mutant. AMPK activity measurements by Western blot analysis and in vitro kinase assay revealed that WT and S485A AMPK were inhibited to a similar degree by insulin, indicating that AMPK S485 phosphorylation is not required for insulin-induced AMPK inhibition. Further analysis suggested an involvement of decreased AMP-to-ATP ratios in the insulin-induced inhibition of AMPK activity, whereas a possible contribution of phosphodiesterases was excluded. Furthermore, we show that insulin-induced AMPK S485 phosphorylation also occurs in human adipocytes, suggesting it to be of an importance yet to be revealed. Altogether, this study increases our understanding of how insulin regulates AMPK activity, and with that, FA synthesis, in adipose tissue.

Effects of PKB/Akt inhibitors on insulin-stimulated lipogenesis and phosphorylation state of lipogenic enzymes in white adipose tissue.

We investigated acute effects of two allosteric protein kinase B (PKB) inhibitors, MK-2206 and Akti-1/2, on insulin-stimulated lipogenesis in rat epididymal adipocytes incubated with fructose as carbohydrate substrate. In parallel, the phosphorylation state of lipogenic enzymes in adipocytes and incubated epididymal fat pads was monitored by immunoblotting. Preincubation of rat epididymal adipocytes with PKB inhibitors dose-dependently inhibited the following: insulin-stimulated lipogenesis, increased PKB Ser473 phosphorylation, increased PKB activity and decreased acetyl-CoA carboxylase (ACC) Ser79 phosphorylation. In contrast, the effect of insulin to decrease the phosphorylation of pyruvate dehydrogenase (PDH) at Ser293 and Ser300 was not abolished by PKB inhibition. Insulin treatment also induced ATP-citrate lyase (ACL) Ser454 phosphorylation, but this effect was less sensitive to PKB inhibitors than ACC dephosphorylation by insulin. In incubated rat epididymal fat pads, Akti-1/2 treatment reversed insulin-induced ACC dephosphorylation, while ACL phosphorylation by insulin was maintained. ACL and ACC purified from white adipose tissue were poor substrates for PKBα in vitro. However, effects of wortmannin and torin, along with Akti-1/2 and MK-2206, on recognized PKB target phosphorylation by insulin were similar to their effects on insulin-induced ACL phosphorylation, suggesting that PKB could be the physiological kinase for ACL phosphorylation by insulin. In incubated epididymal fat pads from wild-type versus ACC1/2 S79A/S212A knockin mice, effects of insulin to increase lipogenesis from radioactive fructose or from radioactive acetate were reduced but not abolished. Together, the results support a key role for PKB in mediating insulin-stimulated lipogenesis by decreasing ACC phosphorylation, but not by decreasing PDH phosphorylation.

TLR9 and beclin 1 crosstalk regulates muscle AMPK activation in exercise.

The activation of adenosine monophosphate-activated protein kinase (AMPK) in skeletal muscle coordinates systemic metabolic responses to exercise1. Autophagy-a lysosomal degradation pathway that maintains cellular homeostasis2-is upregulated during exercise, and a core autophagy protein, beclin 1, is required for AMPK activation in skeletal muscle3. Here we describe a role for the innate immune-sensing molecule Toll-like receptor 9 (TLR9)4, and its interaction with beclin 1, in exercise-induced activation of AMPK in skeletal muscle. Mice that lack TLR9 are deficient in both exercise-induced activation of AMPK and plasma membrane localization of the GLUT4 glucose transporter in skeletal muscle, but are not deficient in autophagy. TLR9 binds beclin 1, and this interaction is increased by energy stress (glucose starvation and endurance exercise) and decreased by a BCL2 mutation3,5 that blocks the disruption of BCL2-beclin 1 binding. TLR9 regulates the assembly of the endolysosomal phosphatidylinositol 3-kinase complex (PI3KC3-C2)-which contains beclin 1 and UVRAG-in skeletal muscle during exercise, and knockout of beclin 1 or UVRAG inhibits the cellular AMPK activation induced by glucose starvation. Moreover, TLR9 functions in a muscle-autonomous fashion in ex vivo contraction-induced AMPK activation, glucose uptake and beclin 1-UVRAG complex assembly. These findings reveal a heretofore undescribed role for a Toll-like receptor in skeletal-muscle AMPK activation and glucose metabolism during exercise, as well as unexpected crosstalk between this innate immune sensor and autophagy proteins.

Two isoprenylated flavonoids from Dorstenia psilurus activate AMPK, stimulate glucose uptake, inhibit glucose production and lower glycemia.

Root extracts of a Cameroon medicinal plant, Dorstenia psilurus, were purified by screening for AMP-activated protein kinase (AMPK) activation in incubated mouse embryo fibroblasts (MEFs). Two isoprenylated flavones that activated AMPK were isolated. Compound 1 was identified as artelasticin by high-resolution electrospray ionization mass spectrometry and 2D-NMR while its structural isomer, compound 2, was isolated for the first time and differed only by the position of one double bond on one isoprenyl substituent. Treatment of MEFs with purified compound 1 or compound 2 led to rapid and robust AMPK activation at low micromolar concentrations and increased the intracellular AMP:ATP ratio. In oxygen consumption experiments on isolated rat liver mitochondria, compound 1 and compound 2 inhibited complex II of the electron transport chain and in freeze-thawed mitochondria succinate dehydrogenase was inhibited. In incubated rat skeletal muscles, both compounds activated AMPK and stimulated glucose uptake. Moreover, these effects were lost in muscles pre-incubated with AMPK inhibitor SBI-0206965, suggesting AMPK dependency. Incubation of mouse hepatocytes with compound 1 or compound 2 led to AMPK activation, but glucose production was decreased in hepatocytes from both wild-type and AMPKß1-/- mice, suggesting that this effect was not AMPK-dependent. However, when administered intraperitoneally to high-fat diet-induced insulin-resistant mice, compound 1 and compound 2 had blood glucose-lowering effects. In addition, compound 1 and compound 2 reduced the viability of several human cancer cells in culture. The flavonoids we have identified could be a starting point for the development of new drugs to treat type 2 diabetes.

The synthesis of branched-chain fatty acids is limited by enzymatic decarboxylation of ethyl- and methylmalonyl-CoA.

Most fatty acids (FAs) are straight chains and are synthesized by fatty acid synthase (FASN) using acetyl-CoA and malonyl-CoA units. Yet, FASN is known to be promiscuous as it may use methylmalonyl-CoA instead of malonyl-CoA and thereby introduce methyl-branches. We have recently found that the cytosolic enzyme ECHDC1 degrades ethylmalonyl-CoA and methylmalonyl-CoA, which presumably result from promiscuous reactions catalyzed by acetyl-CoA carboxylase on butyryl- and propionyl-CoA. Here, we tested the hypothesis that ECHDC1 is a metabolite repair enzyme that serves to prevent the formation of methyl- or ethyl-branched FAs by FASN. Using the purified enzyme, we found that FASN can incorporate not only methylmalonyl-CoA but also ethylmalonyl-CoA, producing methyl- or ethyl-branched FAs. Using a combination of gas-chromatography and liquid chromatography coupled to mass spectrometry, we observed that inactivation of ECHDC1 in adipocytes led to an increase in several methyl-branched FAs (present in different lipid classes), while its overexpression reduced them below wild-type levels. In contrast, the formation of ethyl-branched FAs was observed almost exclusively in ECHDC1 knockout cells, indicating that ECHDC1 and the low activity of FASN toward ethylmalonyl-CoA efficiently prevent their formation. We conclude that ECHDC1 performs a typical metabolite repair function by destroying methyl- and ethylmalonyl-CoA. This reduces the formation of methyl-branched FAs and prevents the formation of ethyl-branched FAs by FASN. The identification of ECHDC1 as a key modulator of the abundance of methyl-branched FAs opens the way to investigate their function.

Saturated fatty acids induce NLRP3 activation in human macrophages through K+ efflux resulting from phospholipid saturation and Na, K-ATPase disruption.

NLRP3 inflammasome plays a key role in Western diet-induced systemic inflammation and was recently shown to mediate long-lasting trained immunity in myeloid cells. Saturated fatty acids (SFAs) are sterile triggers able to induce the assembly of the NLRP3 inflammasome in macrophages, leading to IL-1ß secretion while unsaturated ones (UFAs) prevent SFAs-mediated NLRP3 activation. Unlike previous studies using LPS-primed bone marrow derived macrophages, we do not see any ROS or IRE-1α involvement in SFAs-mediated NLRP3 activation in human monocytes-derived macrophages. Rather we show that SFAs need to enter the cells and to be activated into acyl-CoA to lead to NLRP3 activation in human macrophages. However, their ß-oxidation is dispensable. Instead, they are channeled towards phospholipids but redirected towards lipid droplets containing triacylglycerol in the presence of UFAs. Lipidomic analyses and Laurdan fluorescence experiments demonstrate that SFAs induce a dramatic saturation of phosphatidylcholine (PC) correlated with a loss of membrane fluidity, both events inhibited by UFAs. The silencing of CCTα, the key enzyme in PC synthesis, prevents SFA-mediated NLRP3 activation, demonstrating the essential role of the de novo PC synthesis. This SFA-induced membrane remodeling promotes a disruption of the plasma membrane Na, K-ATPase, instigating a K+ efflux essential and sufficient for NLRP3 activation. This work opens novel therapeutic avenues to interfere with Western diet-associated diseases such as those targeting the glycerolipid pathway.

Genetic deletion of soluble 5'-nucleotidase II reduces body weight gain and insulin resistance induced by a high-fat diet.

We previously investigated whether inhibition of AMP-metabolizing enzymes could enhance AMP-activated protein kinase (AMPK) activation in skeletal muscle for the treatment of type 2 diabetes. Soluble 5'-nucleotidase II (NT5C2) hydrolyzes IMP and its inhibition could potentially lead to a rise in AMP to activate AMPK. In the present study, we investigated effects of NT5C2 deletion in mice fed a normal-chow diet (NCD) or a high-fat diet (HFD). On a NCD, NT5C2 deletion did not result in any striking metabolic phenotype. On a HFD however, NT5C2 knockout (NT5C2-/-) mice displayed reduced body/fat weight gain, improved glucose tolerance, reduced plasma insulin, triglyceride and uric acid levels compared with wild-type (WT) mice. There was a tendency towards smaller and fewer adipocytes in epididymal fat from NT5C2-/- mice compared to WT mice, consistent with a reduction in triglyceride content. Differences in fat mass under HFD could not be explained by changes in mRNA expression profiles of epididymal fat from WT versus NT5C2-/- mice. However, rates of lipolysis tended to increase in epididymal fat pads from NT5C2-/- versus WT mice, which might explain reduced fat mass. In incubated skeletal muscles, insulin-stimulated glucose uptake and associated signalling were enhanced in NT5C2-/- versus WT mice on HFD, which might contribute towards improved glycemic control. In summary, NT5C2 deletion in mice protects against HFD-induced weight gain, adiposity, insulin resistance and associated hyperglycemia.

Structural Determinants for Small-Molecule Activation of Skeletal Muscle AMPK α2ß2γ1 by the Glucose Importagog SC4.

The AMP-activated protein kinase (AMPK) αßγ heterotrimer regulates cellular energy homeostasis with tissue-specific isoform distribution. Small-molecule activation of skeletal muscle α2ß2 AMPK complexes may prove a valuable treatment strategy for type 2 diabetes and insulin resistance. Herein, we report the small-molecule SC4 is a potent, direct AMPK activator that preferentially activates α2 complexes and stimulates skeletal muscle glucose uptake. In parallel with the term secretagog, we propose "importagog" to define a substance that induces or augments cellular uptake of another substance. Three-dimensional structures of the glucose importagog SC4 bound to activated α2ß2γ1 and α2ß1γ1 complexes reveal binding determinants, in particular a key interaction between the SC4 imidazopyridine 4'-nitrogen and ß2-Asp111, which provide a design paradigm for ß2-AMPK therapeutics. The α2ß2γ1/SC4 structure reveals an interaction between a ß2 N-terminal α helix and the α2 autoinhibitory domain. Our results provide a structure-function guide to accelerate development of potent, but importantly tissue-specific, ß2-AMPK therapeutics.

The interactome of EBV LMP1 evaluated by proximity-based BioID approach.

Epstein-Barr virus LMP1 is an oncoprotein required for immortalizing B lymphocytes and also plays important roles in transforming non-lymphoid tissue. The discovery of LMP1 protein interactions will likely generate targets to treat EBV-associated cancers. Here, we define the broader LMP1 interactome using the recently developed BioID method. Combined with mass spectrometry, we identified over 1000 proteins across seven independent experiments with direct or indirect relationships to LMP1. Pathway analysis suggests that a significant number of the proteins identified are involved in signal transduction and protein or vesicle trafficking. Interestingly, a large number of proteins thought to be important in the formation of exosomes and protein targeting were recognized as probable LMP1 interacting partners, including CD63, syntenin-1, ALIX, TSG101, HRS, CHMPs, and sorting nexins. Therefore, it is likely that LMP1 modifies protein trafficking and exosome biogenesis pathways. In support of this, knock-down of syntenin-1 and ALIX resulted in reduced exosomal LMP1.

HBP1 phosphorylation by AKT regulates its transcriptional activity and glioblastoma cell proliferation.

The HMG-box protein 1 (HBP1) is a transcriptional regulator and a potential tumor suppressor that controls cell proliferation, differentiation and oncogene-mediated senescence. In a previous study, we showed that AKT activation through the PI3K/AKT/FOXO pathway represses HBP1 expression at the transcriptional level in human fibroblasts as well as in cancer cell lines. In the present study, we investigated whether AKT could also regulate HBP1 directly. First, AKT1 phosphorylated recombinant human HBP1 in vitro on three conserved sites, Ser380, Thr484 and Ser509. In living cells, we confirmed the phosphorylation of HBP1 on residues 380 and 509 using phospho-specific antibodies. HBP1 phosphorylation was induced by growth factors, such as EGF or IGF-1, which activated AKT. Conversely, it was blocked by treatment of cells with an AKT inhibitor (MK-2206) or by AKT knockdown. Next, we observed that HBP1 transcriptional activity was strongly modified by mutating its phosphorylation sites. The regulation of target genes such as DNMT1, P47phox, p16INK4A and cyclin D1 was also affected. HBP1 had previously been shown to limit glioma cell growth. Accordingly, HBP1 silencing by small-hairpin RNA increased human glioblastoma cell proliferation. Conversely, HBP1 overexpression decreased cell growth and foci formation. This effect was amplified by mutations that prevented phosphorylation by AKT, and blunted by mutations that mimicked phosphorylation. In conclusion, our results suggest that HBP1 phosphorylation by AKT blocks its functions as transcriptional regulator and tumor suppressor.

Two Case Reports: Using Simulated Reattachment to Treat Persistent Complex Bereavement Disorder and PTSD.

These case reports focus on a rapid treatment for persistent complex bereavement disorder and posttraumatic stress disorder (PTSD), which appears to activate the mirror neuron network. Simulated reattachment is a technique which has been found to repair phantom limb pain in just a few sessions. The same neuroplasticity that accomplishes phantom pain relief has been found to occur in the treatment of complicated grief and PTSD using similar methods. The simulated reattachment for the client in Case one was found to significantly reduce the symptoms of both complicated grief and obsessive-compulsive disorder (OCD) within one session. In Case two, symptoms of PTSD and depression were significantly reduced in a client with lupus after two sessions of simulated reattachment. In addition, inflammatory markers antinuclear autoantibodies (ANA) and C-reactive protein (CRP) declined from the beginning of treatment to the end.