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Cancer initiation and progression result from genetic and epigenetic alterations caused by interactions between environmental and endogenous factors leading to aberrant cell signalling. Colorectal cancers (CRC) are linked to abnormal activation of the Wnt/β-catenin pathway, whose key feature is the nuclear accumulation of acetylated β-catenin in colon epithelial cells. Nuclear β-catenin acts as a transcriptional co-activator, targeting genes involved in cell proliferation and invasion. 1α,25-Dihydroxyvitamin D 3 (1,25(OH) 2 D 3 or calcitriol), the active form of vitamin D, antagonizes Wnt/β-catenin over-activation by engaging its high affinity receptor, VDR. Here we unveil that 1,25(OH) 2 D 3 -bound VDR activates Silent Information Regulator of Transcription, sirtuin 1 (SIRT1), leading to β-catenin deacetylation and nuclear exclusion, downregulation of its pro-tumourigenic target genes and inhibition of human colon carcinoma cell proliferation. Notably, orthogonal SIRT1 activation mimics nuclear exclusion of β-catenin while SIRT1 inhibition blocks the effects of 1,25(OH) 2 D 3 . Thus, SIRT1 emerges as a crucial mediator in the protective action of vitamin D against CRC. The mutual negative feedback loop unveiled here between Wnt and SIRT1 represents an important surrogate target in CRC. Since nuclear localisation of β-catenin is a critical driver of CRC that requires its acetylation, we provide a mechanistic foundation for the epidemiological evidence linking vitamin D deficiency and increased CRC risk and mortality., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

This study aims to examine the 50/50 polypropylene/polyamide 6 (iPP/PA6) system molded under confined flow conditions, both in its original state and after being modified by two different interfacial agents. This study provides two main insights. Firstly, it focuses on a polymer blend close to phase inversion. Secondly, it investigates the impact of using two different types of interfacial agents (derived from polymer waste) to enhance the compatibility between iPP and PA6. Dynamic Mechanical Analysis (DMA) has been employed to achieve these objectives. It is important to note that the investigation of the 50/50 iPP/PA6 system is a crucial focus predicted in previous studies, where a series of mechanical properties were evaluated using Box-Wilson design of experiments (DOEs) over the whole compositional range on the iPP/PA6 binary system. Thus, two interfacial modifiers, namely succinic anhydride (SA)-grafted atactic polypropylene with terminal, side, and bridge SA grafts (aPP-SASA) and succinyl-fluoresceine (SF) with bridge succinic anhydride grafting atactic polypropylene (aPP-SFSA), were employed. The authors obtained and characterized these agents. The quantity of these agents used in the blend was identified as a critical coordinate in prior studies conducted by the authors. The processing method used, compression molding under confined conditions, was chosen to minimize any orientation effect on the emerging morphology. All characterization procedures were performed on samples processed by contour machining to retain the blend morphologies as they emerged from the processing stage. Results from WAXS and SAXS synchrotron tests concluded there were no changes in the crystal morphology of the iPP or the PA6 in the blends nor any co-crystallization process throughout the compositional range. These findings, and the long period fits on the PP crystalline phase for the fifty/fifty blends we are discussing, will support the present DMA study. Finally, the efficiency of these interfacial modifiers has been concluded, even in this unfavorable scenario.

Background: Chemotherapy, the mainstay treatment for metastatic cancer, presents serious side effects due to off-target exposure. In addition to the negative impact on patients' quality of life, side effects limit the dose that can be administered and thus the efficacy of the drug. Encapsulation of chemotherapeutic drugs in nanocarriers is a promising strategy to mitigate these issues. However, avoiding premature drug release from the nanocarriers and selectively targeting the tumour remains a challenge., Results: In this study, we present a pioneering method for drug integration into nanoparticles known as mesoporous organosilica drugs (MODs), a distinctive variant of periodic mesoporous organosilica nanoparticles (PMOs) in which the drug is an inherent component of the silica nanoparticle structure. This groundbreaking approach involves the chemical modification of drugs to produce bis-organosilane prodrugs, which act as silica precursors for MOD synthesis. Mitoxantrone (MTO), a drug used to treat metastatic breast cancer, was selected for the development of MTO@MOD nanomedicines, which demonstrated a significant reduction in breast cancer cell viability. Several MODs with different amounts of MTO were synthesised and found to be efficient nanoplatforms for the sustained delivery of MTO after biodegradation. In addition, Fe 3 O 4 NPs were incorporated into the MODs to generate magnetic MODs to actively target the tumour and further enhance drug efficacy. Importantly, magnetic MTO@MODs underwent a Fenton reaction, which increased cancer cell death twofold compared to non-magnetic MODs., Conclusions: A new PMO-based material, MOD nanomedicines, was synthesised using the chemotherapeutic drug MTO as a silica precursor. MTO@MOD nanomedicines demonstrated their efficacy in significantly reducing the viability of breast cancer cells. In addition, we incorporated Fe 3 O 4 into MODs to generate magnetic MODs for active tumour targeting and enhanced drug efficacy by ROS generation. These findings pave the way for the designing of silica-based multitherapeutic nanomedicines for cancer treatment with improved drug delivery, reduced side effects and enhanced efficacy., (© 2024. The Author(s).)

We report two novel prodrug Pt(IV) complexes with bis-organosilane ligands in axial positions: cis -dichloro(diamine)- trans -[3-(triethoxysilyl)propylcarbamate]platinum(IV) (Pt(IV)-biSi-1) and cis -dichloro(diisopropylamine)- trans -[3-(triethoxysilyl) propyl carbamate]platinum(IV) (Pt(IV)-biSi-2). Pt(IV)-biSi-2 demonstrated enhanced in vitro cytotoxicity against colon cancer cells (HCT 116 and HT-29) compared with cisplatin and Pt(IV)-biSi-1. Notably, Pt(IV)-biSi-2 exhibited higher cytotoxicity toward cancer cells and lower toxicity on nontumorigenic intestinal cells (HIEC6). In preclinical mouse models of colorectal cancer, Pt(IV)-biSi-2 outperformed cisplatin in reducing tumor growth at lower concentrations, with reduced side effects. Mechanistically, Pt(IV)-biSi-2 induced permanent DNA damage independent of p53 levels. DNA damage such as double-strand breaks marked by histone gH2Ax was permanent after treatment with Pt(IV)-biSi-2, in contrast to cisplatin's transient effects. Pt(IV)-biSi-2's faster reduction to Pt(II) species upon exposure to biological reductants supports its superior biological response. These findings unveil a novel strategy for designing Pt(IV) anticancer prodrugs with enhanced activity and specificity, offering therapeutic opportunities beyond conventional Pt drugs.

Exploitation of extrinsic apoptosis signaling via TRAILR2 activation represents a promising therapeutic concept in cancer treatment. The limited clinical success of previous TRAILR2 agonistic agents, to date, has been ascribed to either poor efficacy or hepatotoxicity. TR2/CDH3 BAB is a human bispecific antibody that relies on binding both CDH3 and TRAILR2 on cell surfaces to achieve TRAILR2 hyperclustering and efficient apoptosis induction by TRAILR2 signaling selectively in CDH3-expressing tumor cells. We demonstrate target-dependent TR2/CDH3 BAB anti-tumor activity in CRISPR/Cas9-engineered TRAILR2 or CDH3 knock-out cells. By utilizing the cell line screening platform PRISM, we found selective TR2/CDH3 BAB efficacy in various cancer types, such as pancreatic, gastric, colorectal, and triple negative breast cancer. The efficacy of TR2/CDH3 BAB correlated with caspase activation in cancer cell lines and in xenograft tumor tissues. In pancreatic ductal adenocarcinoma (PDAC), where patient benefit from current cytotoxic therapy options is unsatisfactory, a close to uniform cell surface expression of CDH3 and TRAILR2 was observed, which will qualify the majority of PDAC patients for TR2/CDH3 BAB-based treatment. TR2/CDH3 BAB demonstrated anti-tumor activity in a panel of PDAC patient-derived xenograft models, including tumor regressions. By combining TR2/CDH3 BAB with chemotherapeutic agents, deeper and more sustained anti-tumor responses were observed when compared to monotherapy. Together with the potential to deliver a favorable safety profile, these data support clinical testing of TR2/CDH3 BAB in patients with PDAC.

The present work deeply studied the mechanical behavior of woven non-woven PLA/OLA/MgO electrospun fibers, efibers, by using Box-Wilson surface response methodology. This work follows up a previous one where both the diameters and the thermal response of such efibers were discussed in terms of both the different amounts of magnesium oxide nanoparticles, MgO, as well as of the oligomer (lactic acid), OLA, used as plasticizer. The results of both works, in term of diameters, degree of crystallinity, and mechanical response, can be strongly correlated to each other, as reported here. In particular, the strain mechanism of PLA/OLA/MgO efibers was studied, showing an orientation of efibers parallel to the applied stress and identifying the mechanically weakest points that yielded the start of the breakage of efibers. Moreover, we identified 1.5 wt% as the critical amount of MgO, above which the plasticizing effect of OLA was weaker as the amount of both components increased. Moreover, the minimum elastic modulus value took place at 15 wt% of OLA, in agreement with the previously reported convergence point in the evolution of the degree of crystallinity. Regarding the yield point, a concentration of OLA between 20 and 30 wt% led to a slight improvement in the yielding capability in terms of tensile strength in comparison with neat PLA efibers. Therefore, the approach presented here permits the design of tailor-made electrospun nanocomposites with specific mechanical requirements.

Posttranslational modifications of epigenetic modifiers provide a flexible and timely mechanism for rapid adaptations to the dynamic environment of cancer cells. SIRT1 is an NAD + -dependent epigenetic modifier whose activity is classically associated with healthy aging and longevity, but its function in cancer is not well understood. Here, we reveal that 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3, calcitriol), the active metabolite of vitamin D (VD), promotes SIRT1 activation through auto-deacetylation in human colon carcinoma cells, and identify lysine 610 as an essential driver of SIRT1 activity. Remarkably, our data show that the post-translational control of SIRT1 activity mediates the antiproliferative action of 1,25(OH) 2 D 3 . This effect is reproduced by the SIRT1 activator SRT1720, suggesting that SIRT1 activators may offer new therapeutic possibilities for colon cancer patients who are VD deficient or unresponsive. Moreover, this might be extrapolated to inflammation and other VD deficiency-associated and highly prevalent diseases in which SIRT1 plays a prominent role., Competing Interests: JG, AC, JM, MF, MF, JC, AD, AB, AM, ML, CG No competing interests declared, (© 2023, García-Martínez et al.)

This work has a two-fold objective. First, it attempts to present the excellent efficiency of a maleated interfacial agent (obtained by the authors by using atactic polypropylene industrial waste) when used as interfacial additive in polypropylene/short carbon fiber composites (iPP/SCF). Second, in this paper, we pay attention to the role played by processing in the final properties of the composite. This work has been performed by considering the emerging crystalline morphologies generated by the different shear forces that the molten material suffers depending on the molding method employed. The interfacial agent analyzed here consists of an atactic polypropylene containing succinic anhydride grafts obtained through a chemical modification process performed in solution. It incorporates different types of succinic grafts, such as succinic bridges between aPP chains and backbone and terminal grafts (aPP-SASA) in its structure, and contains 5.6% (5.6 × 10 -4 g/mol) of grafted polar groups in total. The adhesion of the polyamide SCF sizing and the succinic units is followed by Field Emission Scanning Electronic Microscopy (FESEM) and Synchrotron Infrared Microscopy (SIRM). However, the main objective of this work is the study of the thermal and the dynamic mechanical behavior of the materials of a series of both compression- and injection-molded samples to ascertain the enhanced interfacial interactions in the material and further comparison between the results obtained by both processing operations. Therefore, we detect improvements of 200% in stiffness and 400% in the viscous response of the same SCF content composites caused by aPP-SASA, depending on the processing method used.

In the present work, a statistical study of the morphology and thermal behavior of poly(lactic acid) (PLA)/oligomer(lactic acid) (OLA)/magnesium oxide nanoparticles (MgO), electrospun fibers (efibers) has been carried out. The addition of both, OLA and MgO, is expected to modify the final properties of the electrospun PLA-based nanocomposites for their potential use in biomedical applications. Looking for the compositional optimization of these materials, a Box−Wilson design of experiment was used, taking as dependent variables the average fiber diameter as the representative of the fiber morphologies, as well as the glass transition temperature (Tg) and the degree of crystallinity (Xc) as their thermal response. The results show values of 73.76% (diameter), 88.59% (Tg) and 75.61% (Xc) for each polynomial fit, indicating a good correlation between both OLA and MgO, along with the morphological as well as the thermal behavior of the PLA-based efibers in the experimental space scanned.

Colorectal cancer (CRC) and diabetes are two of the most prevalent chronic diseases worldwide with dysregulated receptor tyrosine kinase signaling and strong co-occurrence correlation. Plasma autoantibodies represent a promising early diagnostic marker for both diseases before symptoms appear. In this study, we explore the value of autoantibodies against receptor-type tyrosine-protein phosphatase-like N (PTPRN; full-length or selected domains) as diagnostic markers using a cohort of individuals with type 2 diabetes (T2D), CRC, or both diseases or healthy individuals. We show that PTPRN autoantibody levels in plasma discriminated between patients with T2D with and without CRC. Consistently, high PTPRN expression correlated with decreased survival of patients with CRC. Mechanistically, PTPRN depletion significantly reduced invasiveness of CRC cells in vitro and liver homing and metastasis in vivo by means of a dysregulation of the epithelial-mesenchymal transition and a decrease of the insulin receptor signaling pathway. Therefore, PTPRN autoantibodies may represent a particularly helpful marker for the stratification of patients with T2D at high risk of developing CRC. Consistent with the critical role played by tyrosine kinases in diabetes and tumor biology, we provide evidence that tyrosine phosphatases such as PTPRN may hold potential as therapeutic targets in patients with CRC., (© 2022 by the American Diabetes Association.)

Chaetomium sp. is a mold, member of the phylum Ascomycota. Clinical disease in humans is rare, particularly in children, for which only five cases have been reported. We report a 7-months-old female patient with a diagnosis of visceral heterotaxy syndrome who was admitted to a private center in Mexico. After two episodes of focal myoclonic seizure, a brain magnetic resonance imaging (MRI) revealed a right porencephalic cyst and a right frontal abscess with ventriculitis. Seventy-two hours after temporal abscesses drainage procedure, the culture showed a rapidly growing pale white fungal colony. Sequencing of internal transcribed spacer (ITS) and D1/D2 led to the identification of Chaetomium strumarium. Although Chaetomium sp. is a rare fungal infection in humans, clinicians should consider it as a plausible etiologic agent that can form brain abscess.

Obesity is the strongest known risk factor to develop type 2 diabetes (T2D) and both share a state of chronic, diffuse and low-grade inflammation, impaired immune responses and alterations in the composition and function of the microbiome. Notably, these hallmarks are shared with colorectal cancer (CRC), which is epidemiologically associated to obesity and T2D. Gut barrier damages in T2D destabilize the microbiome that metabolizes the diet and modulates the host immune response triggering inflammatory and proliferative pathways. In this review, we discuss the pathways altered by defects in the immune response and microbiota that may link T2D to CRC development. Stressed adipocytes, metabolic incongruity in blood and gut barrier failure with dysbiosis cooperate to establish imbalances between immune innate and adaptive cells and cytokines such as interleukin 6 (IL6) or TNFA that define low-grade diffuse inflammation in T2D. Inflammation drives tissue repair through proliferation and migration (critical mechanisms for tumourigenesis) and under physiological conditions feeds anti-inflammatory cytokine production to resolve the process. The disproportion in pro- vs anti-inflammatory cells and cytokines imposed by T2D will impact the tumour micro- and macro-environment, favouring tumour proliferation, angiogenesis and decreased immune responses. Complex bidirectional relationships between the metabolic environment of T2D, gut microbiota, and immune dysfunctions may favour tumour cell demands and will define the outcome. Animal models developed to study the relationships between T2D and CRC in the context of microbiota and immune system are discussed.

The existence of molecular links that facilitate colorectal cancer (CRC) development in the population with type 2 diabetes (T2D) is supported by substantial epidemiological evidence. This review summarizes how the systemic, metabolic and hormonal imbalances from T2D alter CRC cell metabolism, signalling and gene expression as well as their reciprocal meshing, with an overview of CRC molecular subtypes and animal models to study the diabetes-CRC cancer links. Metabolic and growth factor checkpoints ensure a physiological cell proliferation rate compatible with limited nutrient supply. Hyperinsulinaemia and hyperleptinaemia in prediabetes and excess circulating glucose and lipids in T2D overcome formidable barriers for tumour development. Increased nutrient availability favours metabolic reprogramming, alters signalling and generates mutations and epigenetic modifications through increased reactive oxygen species and oncometabolites. The reciprocal control between metabolism and hormone signalling is lost in diabetes. Excess adipose tissue at the origin of T2D unbalances adipokine (leptin/adiponectin) secretion ratios and function and disrupts the insulin/IGF axes. Leptin/adiponectin imbalances in T2D are believed to promote proliferation and invasion of CRC cancer cells and contribute to inflammation, an important component of CRC tumourigenesis. Disruption of the insulin/IGF axes in T2D targets systemic and CRC cell metabolic reprogramming, survival and proliferation. Future research to clarify the molecular diabetes-CRC links will help to prevent CRC and reduce its incidence in the diabetic population and must guide therapeutic decisions.

Activation of TRAILR2 has emerged as an important therapeutic concept in cancer treatment. TRAILR2 agonistic molecules have only had limited clinical success, to date, due either to lack of efficacy or hepatotoxicity. BI 905711 is a novel tetravalent bispecific antibody targeting both TRAILR2 and CDH17 and represents a novel liver-sparing TRAILR2 agonist specifically designed to overcome the disadvantages of previous strategies. Here, we show that BI 905711 effectively triggered apoptosis in a broad panel of CDH17-positive colorectal cancer tumor cells in vitro . Efficient induction of apoptosis was dependent on the presence of CDH17, as exemplified by the greater than 1,000-fold drop in potency in CDH17-negative cells. BI 905711 demonstrated single-agent tumor regressions in CDH17-positive colorectal cancer xenografts, an effect that was further enhanced upon combination with irinotecan. Antitumor efficacy correlated with induction of caspase activation, as measured in both the tumor and plasma. Effective tumor growth inhibition was further demonstrated across a series of different colorectal cancer PDX models. BI 905711 induced apoptosis in both a cis (same cell) as well as trans (adjacent cell) fashion, translating into significant antitumor activity even in xenograft models with heterogeneous CDH17 expression. In summary, we demonstrate that BI 905711 has potent and selective antitumor activity in CDH17-positive colorectal cancer models both in vitro and in vivo . The high prevalence of over 95% CDH17-positive tumors in patients with colorectal cancer, the molecule preclinical efficacy together with its potential for a favorable safety profile, support the ongoing BI 905711 phase I trial in colorectal cancer and additional CDH17-positive cancer types (NCT04137289)., (©2020 American Association for Cancer Research.)

This work deals with the changes of the glass transition temperature (T g ) of the polymer in polypropylene/mica composites due to the combined and synergistic effect of the reinforcement and the interfacial modifier. In our case, we studied the effect on T g of platy mica and an interfacial modifier with p -phenylen-bis-maleamic acid ( p PBMA) grafted groups onto atactic polypropylene (aPP- p PBMA). This one contains 5.0 × 10 -4 g·mol -1 (15% w/w ) grafted p PBMA and was previously obtained by the author's labs by using industrial polymerization wastes (aPP). The objective of the article must be perceived as two-fold. On one hand, the determination of the changes in the glass transition temperature of the isotactic polypropylene phase (iPP) due to both the reinforcement and the agent as determined form the damp factor in DMA analysis. On the other hand, forecasting the variation of this parameter (T g ) as a function of both the interfacial agent and reinforcement content. For such purposes, and by assuming the complex character of the iPP/aPP- p PBMA/Mica system, wherein interaction between the components will define the final behaviour, a Box-Wilson experimental design considering the amount of mica particles and of interface agent as the independent variables, and the T g as the dependent one, has been used. By taking in mind that the glass transition is a design threshold for the ultimate properties of parts based in this type of organic-inorganic hybrid materials, the final purpose of the work is the prediction and interpretation of the effect of both variables on this key parameter.

Coordination of gene expression with nutrient availability supports proliferation and homeostasis and is shaped by protein acetylation. Yet how physiological/pathological signals link acetylation to specific gene expression programs and whether such responses are cell-type-specific is unclear. AMP-activated protein kinase (AMPK) is a key energy sensor, activated by glucose limitation to resolve nutrient supply-demand imbalances, critical for diabetes and cancer. Unexpectedly, we show here that, in gastrointestinal cancer cells, glucose activates AMPK to selectively induce EP300, but not CREB-binding protein (CBP). Consequently, EP300 is redirected away from nuclear receptors that promote differentiation towards β-catenin, a driver of proliferation and colorectal tumorigenesis. Importantly, blocking glycogen synthesis permits reactive oxygen species (ROS) accumulation and AMPK activation in response to glucose in previously nonresponsive cells. Notably, glycogen content and activity of the ROS/AMPK/EP300/β-catenin axis are opposite in healthy versus tumor sections. Glycogen content reduction from healthy to tumor tissue may explain AMPK switching from tumor suppressor to activator during tumor evolution., Competing Interests: The authors have declared that no competing interests exist.

The present article adequately supports a twofold objective. On one hand, the study of the dynamic mechanical behavior of polypropylene/polyamide-6 blends modified by a novel compatibilizer was the objective. This was previously obtained by chemical modification of an atactic polypropylene polymerization waste. On the other hand, the accurate predictions of these properties in the experimental space scanned was the objective. As a novelty, this compatibilizer contains grafts rather than just maleated ones. Therefore, it consists precisely of an atactic polymer containing succinic anhydride (SA) bridges and both backbone and terminal grafted succinyl-fluorescein groups (SFSA) attached to the atactic backbone (aPP-SFSA). Therefore, it contains 6.2% of total grafting (2.5% as SA and 3.7% as SF), which is equivalent to 6.2·× 10 -4 g·mol -1 . This interfacial agent was uniquely designed and obtained by the authors themselves. Essentially, this article focuses on how the beneficial effect of both PA6 and aPP-SFSA varies the elastic ( E ') and the viscous ( E '') behavior of the iPP/aPP-SFSA/PA6 blend at the iPP glass transition. Thus, we accurately measured the Dynamic Mechanical Analysis (DMA) parameters ( E ', E '') at this specific point considering it represents an extremely unfavorable scenario for the interfacial modifier due to mobility restrictions. Hence, this evidences the real interfacial modifications caused by aPP-SFSA to the iPP/PA6 system. Even more, and since each of the necessary components in the blend typically interacts with one another, we employed a Box-Wilson experimental design by its marked resemblance to the "agent-based models". In this manner, we obtained complex algorithms accurately forecasting the dynamic mechanical behavior of the blends for all the composition range of the iPP/aPP-SFSA/PA6 system at the glass transition of iPP.

Background: Sex bias in immune function has been contributed in part to a preponderance of immune system-related genes (ISRG) on the X-chromosome. We verified whether ISRG are more abundant on the X chromosome as compared to autosomal chromosomes and reflected on the impact of our findings., Methods: Consulting freely accessible databases, we performed a comparative study consisting of three complementary strategies. First, among coding X/Y-linked genes, the abundance of ISRG was compared to the abundance of genes dedicated to other systems. Genes were assigned considering three criteria: disease, tissue expression, and function (DEF approach). In addition, we carried out two genome-wide approaches to compare the contribution of sex and autosomal chromosomes to immune genes defined by an elevated expression in lymphatic tissues (LTEEG approach) or annotation to an immune system process, GO:0002376 (GO approach)., Results: The X chromosome had less immune genes than the median of the autosomal chromosomes. Among X-linked genes, ISRG ranked fourth after the reproductive and nervous systems and genes dedicated to development, proliferation and apoptosis. On the Y chromosome, ISRG ranked second, and at the pseudoautosomal region (PAR) first. According to studies on the expression of X-linked genes in a variety of (mostly non-lymphatic) tissues, almost two-thirds of ISRG are expressed without sex bias, and the remaining ISRG presented female and male bias with similar frequency. Various epigenetic controllers, X-linked MSL3 and Y-linked KDM5D and UTY, were preferentially expressed in leukocytes and deserve further attention for a possible role in sex biased expression or its neutralisation., Conclusions: The X chromosome is not enriched for ISRG, though particular X-linked genes may be responsible for sex differences in certain immune responses. So far, there is insufficient information on sex-biased expression of X/Y-linked ISRG in leukocytes to draw general conclusions on the impact of X/Y-linked ISRG in immune function. More research on the regulation of the expression X-linked genes is required with attention to 1) female and male mechanisms that may either augment or diminish sex biased expression and 2) tissue-specific expression studies.

Background: There is increasing evidence of weight regain in patients after bariatric surgery (BS), generally occurring from 12 to 24 months postoperatively. Postoperative exercise has been suggested to ad long-term weight maintenance and to improve physical function in BS patients. However, there are a limited number of intervention studies investigating the possible benefits of exercise in this population. The aim of the current report is to provide a comprehensive CERT (Consensus on Exercise Reporting Template)-based description of the rationale and details of the exercise programme implemented in the EFIBAR Study (Ejercicio FÍsico tras cirugía BARiátrica), a randomised controlled trial investigating the effects of a 16-week supervised concurrent (aerobic and strength) exercise intervention program on weight loss (primary outcome), body composition, cardiometabolic risk, physical fitness, physical activity and quality of life (secondary outcomes) in patients with severe/morbid obesity following bariatric surgery., Methods: A total of 80 BS patients [60-80% expected women, aged 18 to 60 years, body mass index (BMI) ≥ 40 kg/m 2 or ≥ 35 kg/m 2 with comorbid conditions)] will be enrolled in the EFIBAR Randomized Control Trial (RCT). Participants allocated in the exercise group (n = 40) will undertake a 16-week supervised concurrent (strength and aerobic) exercise programme (three sessions/week, 60 min/session), starting 7 to 14 days after surgery. The rationale of the exercise programme will be described following the CERT criteria detailing the 16 key items. The study has been reviewed and approved by the Ethics Committee of the Torrecárdenas University Hospital (Almería, Spain) (ref. N° 76/2016)., Discussion: The present study details the exercise programme of the EFIBAR RCT, which may serve: 1) exercise professionals who would like to implement an evidence-based exercise programme for BS patients, and 2) as an example of the application of the CERT criteria., Trial Registration: The trial was prospectively registered at Clinicaltrials.gov NCT03497546 on April 13, 2018.

This study aimed to analyse the association between socioeconomic status (SES) and fatness and fitness in preschoolers. 2,638 preschoolers (3-5 years old; 47.2% girls) participated. SES was estimated from the parental educational and occupational levels, and the marital status. Fatness was assessed by body mass index (BMI), waist circumference (WC), and waist-to-height ratio (WHtR). Physical fitness components were assessed using the PREFIT battery. Preschoolers whose parents had higher educational levels had lower fatness (P < 0.05). BMI significantly differed across occupational levels of each parent (P < 0.05) and WHtR across paternal levels (P = 0.004). Musculoskeletal fitness was different across any SES factor (P < 0.05), except handgrip across paternal occupational levels (P ≥ 0.05). Preschoolers with high paternal occupation had higher speed/agility (P = 0.005), and those with high or low maternal education had higher VO 2 max (P = 0.046). Odds of being obese and having low musculoskeletal fitness was lower as SES was higher (P < 0.05). Those with married parents had higher cardiorespiratory fitness than single-parent ones (P = 0.010). School-based interventions should be aware of that children with low SES are at a higher risk of obesity and low fitness already in the first years of life.

Background: Gastrointestinal adverse effects occurring during cancer chemotherapy are well known and feared; those persisting once treatment has finished are relatively unknown. We characterized the alterations occurring in the rat small intestine, after repeated treatment with cisplatin., Methods: Male Wistar rats received saline or cisplatin (2 mg kg -1  week -1 , for 5 weeks, ip). Gastric motor function was studied non-invasively throughout treatment (W1-W5) and 1 week after treatment finalization (W6). During W6, upper gastrointestinal motility was also invasively studied and small intestinal samples were collected for histopathological and molecular studies. Structural alterations in the small intestinal wall, mucosa, submucosa, muscle layers, and lymphocytic nodules were histologically studied. Periodic acid-Schiff staining and immunohistochemistry for Ki-67, chromogranin A, and neuronal-specific enolase were used to detect secretory, proliferating, endocrine and neural cells, respectively. The expression of different markers in the tunica muscularis was analyzed by RT/qPCR., Key Results: Repeated cisplatin induced motility alterations during and after treatment. After treatment (W6), the small intestinal wall showed histopathological alterations in most parameters measured, including a reduction in the thickness of circular and longitudinal muscle layers. Expression of c-KIT (for interstitial cells of Cajal), nNOS (for inhibitory motor neurons), pChAT, and cChAT (for excitatory motor neurons) increased significantly (although both ChATs to a lesser extent)., Conclusions & Inferences: Repeated cisplatin induces relatively long-lasting gut dysmotility in rat associated with important histopathological and molecular alterations in the small intestinal wall. In cancer survivors, the possible chemotherapy-induced histopathological, molecular, and functional intestinal sequelae should be evaluated., (© 2017 John Wiley & Sons Ltd.)

The association between diabetes and cancer was hypothesized almost one century ago. Today, a vast number of epidemiological studies support that obese and diabetic populations are more likely to experience tissue-specific cancers, but the underlying molecular mechanisms remain unknown. Obesity, diabetes, and cancer share many hormonal, immune, and metabolic changes that may account for the relationship between diabetes and cancer. In addition, antidiabetic treatments may have an impact on the occurrence and course of some cancers. Moreover, some anticancer treatments may induce diabetes. These observations aroused a great controversy because of the ethical implications and the associated commercial interests. We report an epidemiological update from a mechanistic perspective that suggests the existence of many common and differential individual mechanisms linking obesity and type 1 and 2 diabetes mellitus to certain cancers. The challenge today is to identify the molecular links responsible for this association. Classification of cancers by their molecular signatures may facilitate future mechanistic and epidemiological studies., (Copyright © 2016 SEEN. Publicado por Elsevier España, S.L.U. All rights reserved.)

Bowlby's attachment theory suggested that the attachment experiences of early childhood influence adult approaches to close relationships. As a result of these experiences, the child develops typical mental schemas or internal working models. The aim of this study was to analyze how young people with different attachment styles perceive the benefits and costs involved in spending as much time as possible with their partner, and to determine whether their beliefs reflect the internal working models associated with their attachment style. A sample of 1,539 university students responded to the Relationship Questionnaire (Bartholomew & Horowith, 1991), and to a questionnaire about behavioral beliefs (perceived benefits and costs). Results show that young people with different attachment styles hold different beliefs about the consequences derived from engaging in a specific behavior in romantic relationships. Secure and preoccupied individuals perceived more benefits than costs associated with the behavior, whereas dismissing and fearful individuals perceived more costs than benefits. Furthermore, secure and preoccupied individuals rated those behavioral consequences leading to enhanced intimacy or closeness more positively than avoidant individuals, whereas dismissing individuals rated more negatively those consequences that involved a loss of independence. These results confirm that a congruity exists between the beliefs associated with the behavior studied and the internal working models related to each adult attachment style.

Prolactin (PRL) is a polypeptide hormone/cytokine mainly synthesized by the lactotrophic cells of the adenohypophysis. In addition to the best-known role in mammary gland development and the functional differentiation of its epithelium, PRL is involved in regulation of multiple physiological processes in higher organisms contributing to their homeostasis. PRL has been also associated with pathology, including breast cancer. Therefore, it is relevant to determine the molecular mechanisms by which PRL controls cellular functions. Here, we analyze the role of Src family kinases (SFKs) in the intracellular signaling pathways controlled by PRL in several model systems. The data show that SFKs are essential components in transmitting signals upon PRL receptor stimulation, as they control activation of Jak2/Stat5 and other routes that regulate PRL cellular responses.

Minutes after ingestion of fat or carbohydrates, vesicles stored in enteroendocrine cells release their content of incretin peptide hormones that, together with absorbed glucose, enhance insulin secretion by beta-pancreatic cells. Freshly-made incretins must therefore be packed into new vesicles in anticipation of the next meal with cells adjusting new incretin production to be proportional to the level of previous insulin release and absorbed blood glucose. Here we show that insulin stimulates the expression of the major human incretin, glucose-dependent insulinotropic peptide (GIP) in enteroendocrine cells but requires glucose to do it. Akt-dependent release of FoxO1 and glucose-dependent binding of LEF1/β-catenin mediate induction of Gip expression while insulin-induced phosphorylation of β-catenin does not alter its localization or transcriptional activity in enteroendocrine cells. Our results reveal a glucose-regulated feedback loop at the entero-insular axis, where glucose levels determine basal and insulin-induced Gip expression; GIP stimulation of insulin release, physiologically ensures a fine control of glucose homeostasis. How enteroendocrine cells adjust incretin production to replace incretin stores for future use is a key issue because GIP malfunction is linked to all forms of diabetes., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Extensive epidemiological studies suggest that the diabetic population is at higher risk of site-specific cancers. The diabetes-cancer link has been hypothesized to rely on various hormonal (insulin, IGF1, adipokines), immunological (inflammation), or metabolic (hyperglycemia) characteristics of the disease and even on certain treatments. Inflammation may have an important but incompletely understood role. As a growth factor, insulin directly, or indirectly through IGF1, has been considered the major link between diabetes and cancer, while high glucose has been considered as a subordinate cause. Here we discuss the evidence that supports a role for insulin/IGF1 in general in cancer, and the mechanism by which hyperglycemia may enhance the appearance, growth and survival of diabetes-associated cancers. High glucose triggers several direct and indirect mechanisms that cooperate to promote cancer cell proliferation, migration, invasion and immunological escape. In particular, high glucose enhancement of WNT/β-catenin signaling in cancer cells promotes proliferation, survival and senescence bypass, and represents a previously unrecognized direct mechanism linking diabetes-associated hyperglycemia to cancer. Increased glucose uptake is a hallmark of tumor cells and may ensure enhanced WNT signaling for continuous proliferation. Mechanistically, high glucose unbalances acetylation through increased p300 acetyl transferase and decreased sirtuin 1 deacetylase activity, leading to β-catenin acetylation at lysine K354, a requirement for nuclear accumulation and transcriptional activation of WNT-target genes. The impact of high glucose on β-catenin illustrates the remodeling of cancer-associated signaling pathways by metabolites. Metabolic remodeling of cancer-associated signaling will receive much research attention in the coming years. Future epidemiological studies may be guided and complemented by the identification of these metabolic interplays. Together, these studies should lead to the development of new preventive strategies for diabetes-associated cancers.

Nuclear accumulation of β-catenin, a widely recognized marker of poor cancer prognosis, drives cancer cell proliferation and senescence bypass and regulates incretins, critical regulators of fat and glucose metabolism. Diabetes, characterized by elevated blood glucose levels, is associated with increased cancer risk, partly because of increased insulin growth factor 1 signaling, but whether elevated glucose directly impacts cancer-associated signal-transduction pathways is unknown. Here, we show that high glucose is essential for nuclear localization of β-catenin in response to Wnt signaling. Glucose-dependent β-catenin nuclear retention requires lysine 354 and is mediated by alteration of the balance between p300 and sirtuins that trigger β-catenin acetylation. Consequently β-catenin accumulates in the nucleus and activates target promoters under combined glucose and Wnt stimulation, but not with either stimulus alone. Our results reveal a mechanism by which high glucose enhances signaling through the cancer-associated Wnt/β-catenin pathway and may explain the increased frequency of cancer associated with obesity and diabetes., (Copyright © 2013 Elsevier Inc. All rights reserved.)

G protein-coupled receptor oligomerization is a concept which is changing the understanding of classical pharmacology. Both, oligomerization and functional interaction between adenosine A(2A,) dopamine D(2) and metabotropic glutamate type 5 receptors have been demonstrated in the striatum. However, the transcriptional consequences of receptors co-activation are still unexplored. We aim here to determine the changes in gene expression of striatal primary cultured neurons upon isolated or simultaneous receptor activation. Interestingly, we found that 95 genes of the total analyzed (15,866 transcripts and variants) changed their expression in response to simultaneous stimulation of all three receptors. Among these genes, we focused on the β-synuclein (β-Syn) gene (SCNB). Quantitative PCR verified the magnitude and direction of change in expression of SCNB. Since β-Syn belongs to the homologous synuclein family and may be considered a natural regulator of α-synuclein (α-Syn), it has been proposed that β-Syn might act protectively against α-Syn neuropathology., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Aim: To assess the efficacy of multiple treatment of phosphatidylinositol-3-kinase (PI3K) inhibitor on autochthonous tumours in phosphatase and tensin homologue (Pten)-deficient genetically engineered mouse cancer models using a longitudinal magnetic resonance imaging (MRI) protocol., Materials and Methods: Using 3D MRI, B-cell follicular lymphoma growth was quantified in a Pten(+/-)Lkb1(+/hypo) mouse line, before, during and after repeated treatments with a PI3K inhibitor GDC-0941 (75 mg/kg)., Results: Mean pre-treatment linear tumour growth rate was 16.5±12.8 mm(3)/week. Repeated 28-day GDC-0941 administration, with 21 days 'off-treatment', induced average tumour regression of 41±7%. Upon cessation of the second treatment (which was not permanently cytocidal), tumours re-grew with an average linear growth rate of 40.1±15.5 mm(3)/week. There was no evidence of chemoresistance., Conclusion: This protocol can accommodate complex dosing schedules, as well as combine different cancer therapies. It reduces biological variability problems and resulted in a 10-fold reduction in mouse numbers compared with terminal assessment methods. It is ideal for preclinical efficacy studies and for phenotyping molecularly characterized mouse models when investigating gene function.

Research to date has revealed that the association between gender, attachment and the quality and functioning of intimate relationships is complex. This study examined the relationship between gender and attachment styles in attitudes to communication with one's partner and in the number of past relationships in a sample of 746 Spanish undergraduates. The Relationship Questionnaire was administered to them to determine the adult attachment style. The results revealed the existence of differences according to the adult attachment style and gender with regard to the two measured variables, and a significant effect of the interaction between gender and attachment. Dismissing men reported the highest average scores in the number of past relationships, with significant differences appearing when they were compared with secure and preoccupied men. However, dismissing women did not differ from the rest of the women with other attachment styles. When men and women with the same attachment styles were compared in this variable, the only significant differences were found between dismissing men and women (with the latter reporting fewer partners). In the case of attitudes to expressing feelings to one's partner, dismissing men reported the most negative attitudes, compared with secure and preoccupied men. Dismissing women, unlike the men, did not differ in their attitudes either from preoccupied or fearful women. Moreover, clear differences were shown between dismissing men and women in these attitudes (more negative in the case of men).

Although Candida parapsilosis is the most prevalent among the 3 species of the *psilosis group, studies applying DNA-based diagnostic techniques with isolates previously identified as C. parapsilosis have revealed that both C. orthopsilosis and C. metapsilosis account for 0-10% of all these isolates, depending on the geographical area. Differences in the degrees of antifungal susceptibility and virulence have been found, so a more precise identification is required. In a first approach, we reidentified 38 randomly chosen clinical isolates, previously identified as C. parapsilosis, using the RPO2 (CA2) RAPD marker. Among them, we reclassified 4 as C. metapsilosis and 5 as C. orthopsilosis. We previously developed a method to identify different pathogen yeast species, including C. parapsilosis, based on the amplification of the RPS0 gene intron. In this work, we extend this approach to the new *psilosis species by partially sequencing their RPS0 gene, including the intron sequence. Based on intron sequences, we designed specific primers capable of identifying C. orthopsilosis and C. metapsilosis species, and we reidentified species among the initial isolates. These new primers have allowed a specific and rapid identification of C. orthopsilosis and C. metapsilosis., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Background: The PI3K-mTOR (phosphoinositide 3-kinase-mammalian target of rapamycin kinase) pathway is activated in the majority of tumours, and there is interest in assessing whether inhibitors of PI3K or mTOR kinase have efficacy in treating cancer. Here, we define the effectiveness of specific mTOR (AZD8055) and PI3K (GDC-0941) inhibitors, currently in clinical trials, in treating spontaneous B-cell follicular lymphoma that develops in PTEN(+/-)LKB1(+/hypo) mice., Methods: The PTEN(+/-)LKB1(+/hypo) mice were administered AZD8055 or GDC-0941, and the volumes of B-cell follicular lymphoma were measured by MRI. Tumour samples were analysed by immunohistochemistry, immunoblot and flow cytometry., Results: The AZD8055 or GDC-0941 induced ∼40% reduction in tumour volume within 2 weeks, accompanied by ablation of phosphorylation of AKT, S6K and SGK (serum and glucocorticoid protein kinase) protein kinases. The drugs reduced tumour cell proliferation, promoted apoptosis and suppressed centroblast population. The AZD8055 or GDC-0941 treatment beyond 3 weeks caused a moderate additional decrease in tumour volume, reaching ∼50% of the initial volume after 6 weeks of treatment. Tumours grew back at an increased rate and displayed similar high grade and diffuse morphology as the control untreated tumours upon cessation of drug treatment., Conclusion: These results define the effects that newly designed and specific mTOR and PI3K inhibitors have on a spontaneous tumour model, which may be more representative than xenograft models frequently employed to assess effectiveness of kinase inhibitors. Our data suggest that mTOR and PI3K inhibitors would benefit treatment of cancers in which the PI3K pathway is inappropriately activated; however, when administered alone, may not cause complete regression of such tumours.

Background: The autoimmune lymphoproliferative syndrome (ALPS) is caused by genetic defect in lymphocyte apoptosis. Chronic lymphadenopathy and splenomegaly are the consequence of lymphoproliferation. The diagnosis is based on the assessment of the defective lymphocyte apoptosis and the identification of lymphocyte T subset that are double negative (CD4-CD8-). The susceptibility to lymphoma and autoimmune diseases, mainly blood cytopenias is increased., Methods: We studied a 14 year-old boy with chronic splenomegaly and familial history of splenomegaly and lymphadenopathy. T lymphocyte phenotypes, and molecular defect of TNFRSF6 gene were studied in the child, his sister and his father. Lymphocyte apoptosis was also analysed in the child and his father., Results: The boy and his father showed in vitro apoptosis defects, an increased number of double negative T lymphocytes (18% and 5%, respectively) and the same mutation in the TNFRSF6 gene. His sister had 16% of double negative T lymphocytes and the mutation in the TNFRSF6 gene., Comments: Chronic familial splenomegaly can be the only clinical sign of autoimmune lymphoproliferative syndrome., (2009 Asociación Española de Pediatría. Published by Elsevier Espana. All rights reserved.)

The cytokine prolactin (PRL) plays important roles in the proliferation and differentiation of the mammary gland and it has been implicated in tumorigenesis. The prolactin receptor (PRLR) is devoid of catalytic activity and its mitogenic response is controlled by cytoplasmic tyrosine kinases of the Src (SFK) and Jak families. How PRLR uses these kinases for signaling is not well understood. Previous studies indicated that PRLR-induced Jak2 activation does not require SFK catalytic activity in favor of separate signaling operating on this cellular response. Here we show that, nevertheless, PRLR requires Src-SH2 and -SH3 domains for Jak2 signaling. In W53 lymphoid cells, conditional expression of two c-Src non-catalytic mutants, either SrcK295M/Y527F or SrcK, whose SH3 and SH2 domains are exposed, controls Jak2/Stat5 activation by recruiting Jak2, avoiding its activation by endogenous active SFK. In contrast, the kinase inactive SrcK295M mutant, with inaccessible SH3 and SH2 domains, does not. Furthermore, all three mutants attenuate PRLR-induced Akt and p70S6K activation. Accordingly, PRLR-induced Jak2/Stat5 signaling is inhibited in MCF7 breast cancer cells by Src depletion, expression of SrcK295M/Y527F or active Src harboring an inactive SH2 (SrcR175L) or SH3 domain (SrcW118A). Finally, Jak2/Stat5 pathway is also reduced in Src-/- mice mammary glands. We thus conclude that, in addition to Akt and p70S6K, SFK regulate PRLR-induced Jak2 signaling through a kinase-independent mechanism.

Dysregulation of gene expression is one of the mechanisms involved in the pathophysiology of Huntington's disease (HD). Here, we examined whether mutant huntingtin regulates the levels of PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), a phosphatase that specifically dephosphorylates Akt at Ser473. Our results show decreased PHLPP1 protein levels in knock-in models (Hdh(Q111/Q111) mouse striatum and STHdh(Q111/Q111) cells), in the striatum of N-terminal exon-1 mutant huntingtin transgenic mouse models (R6/1; R6/1 : BDNF + or - , R6/2 and Tet/HD94) and in the putamen of HD patients. Quantitative PCR analysis revealed a reduction in PHLPP1 mRNA levels in the striatum of R6/1 compared with wild-type mice. Coincident with reduced PHLPP1 protein levels, we observed increased phosphorylated Akt (Ser473) levels specifically in the striatum. The analysis of the conditional mouse model Tet/HD94 disclosed that after mutant huntingtin shutdown PHLPP1 levels returned to wild-type levels whereas phospho-Akt levels were partially reduced. In conclusion, our results show that mutant huntingtin downregulates PHLPP1 expression. In the striatum, these reduced levels of PHLPP1 can contribute to maintain high levels of activated Akt that may delay cell death and allow the recovery of neuronal viability after mutant huntingtin silencing.

Calcineurin is a serine/threonine phosphatase involved in the regulation of glutamate receptors signaling. Here, we analyzed whether the regulation of calcineurin protein levels and activity modulates the susceptibility of striatal neurons to excitotoxicity in R6/1 and R6/1:BDNF+/- mouse models of Huntington's disease. We show that calcineurin inhibition in wild-type mice drastically reduced quinolinic acid-induced striatal cell death. Moreover, calcineurin A and B were differentially regulated during disease progression with a specific reduction of calcineurin A protein levels and calcineurin activity at the onset of the disease in R6/1:BDNF+/- mice. Analysis of the conditional mouse model Tet/HD94 showed that mutant huntingtin specifically controls calcineurin A protein levels. Finally, calcineurin activation induced by intrastriatal quinolinic acid injection in R6/1 mouse was lower than in wild-type mice. Therefore, reduction of calcineurin activity by alteration of calcineurin A expression participates in the pathophysiology of Huntington's disease and contributes to the excitotoxic resistance observed in exon-1 mouse models.

Aims/hypothesis: Glucose-dependent insulinotropic peptide (GIP) plays a pivotal role in the regulation of glucose homeostasis. Rates of diet-induced obesity, insulin resistance and type 2 diabetes are decreased when GIP signalling is disturbed in mice, suggesting that GIP plays a role in the onset of type 2 diabetes. WNT signalling is linked to type 2 diabetes and induces synthesis of the other incretin, glucagon-like peptide 1 (GLP-1). GLP-1 analogues improve treatment of type 2 diabetes patients in whom GLP-1 signalling is intact and have captured clinical attention. GIP levels are altered at the onset of type 2 diabetes and later on, while GIP signalling is impaired. Thus, GIP is not a candidate for treatment but might be an important target from a prevention perspective. Hypothesising that hypersecretion of GIP links altered WNT signalling to the onset of type 2 diabetes, we sought to determine whether WNT signalling induces GIP production by entero-endocrine cells., Methods: RT-PCR and chromatin immunoprecipitation (ChIP) were used to study Gip gene induction. Gip promoter elements mediating WNT/lithium induction were identified (electrophoretic mobility shift assay, co-transfection of deletion mutants, ChIP)., Results: Lithium or WNT/beta-catenin signalling enhanced GIP production by entero-endocrine cells through a conserved site in the proximal Gip promoter. Lithium favours lymphoid enhancer factor-1/beta-catenin binding to Gip promoter and diminishes ChIP through T cell factor-4 and histone deacetylase 1., Conclusions/interpretation: Lithium and WNT are incretin inducers in general. This work provides a novel link between WNT signalling, obesity and diabetes.

mTOR (mammalian target of rapamycin) stimulates cell growth by phosphorylating and promoting activation of AGC (protein kinase A/protein kinase G/protein kinase C) family kinases such as Akt (protein kinase B), S6K (p70 ribosomal S6 kinase) and SGK (serum and glucocorticoid protein kinase). mTORC1 (mTOR complex-1) phosphorylates the hydrophobic motif of S6K, whereas mTORC2 phosphorylates the hydrophobic motif of Akt and SGK. In the present paper we describe the small molecule Ku-0063794, which inhibits both mTORC1 and mTORC2 with an IC50 of approximately 10 nM, but does not suppress the activity of 76 other protein kinases or seven lipid kinases, including Class 1 PI3Ks (phosphoinositide 3-kinases) at 1000-fold higher concentrations. Ku-0063794 is cell permeant, suppresses activation and hydrophobic motif phosphorylation of Akt, S6K and SGK, but not RSK (ribosomal S6 kinase), an AGC kinase not regulated by mTOR. Ku-0063794 also inhibited phosphorylation of the T-loop Thr308 residue of Akt phosphorylated by PDK1 (3-phosphoinositide-dependent protein kinase-1). We interpret this as implying phosphorylation of Ser473 promotes phosphorylation of Thr308 and/or induces a conformational change that protects Thr308 from dephosphorylation. In contrast, Ku-0063794 does not affect Thr308 phosphorylation in fibroblasts lacking essential mTORC2 subunits, suggesting that signalling processes have adapted to enable Thr308 phosphorylation to occur in the absence of Ser473 phosphorylation. We found that Ku-0063794 induced a much greater dephosphorylation of the mTORC1 substrate 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) than rapamycin, even in mTORC2-deficient cells, suggesting a form of mTOR distinct from mTORC1, or mTORC2 phosphorylates 4E-BP1. Ku-0063794 also suppressed cell growth and induced a G1-cell-cycle arrest. Our results indicate that Ku-0063794 will be useful in delineating the physiological roles of mTOR and may have utility in treatment of cancers in which this pathway is inappropriately activated.

The mammalian target of rapamycin complex 2 (mTORC2) plays critical roles in regulating cell growth and proliferation. mTORC2 promotes the activation of the serum glucocorticoid-induced protein kinase (SGK). This mTOR complex also promotes the constitutive phosphorylation of proline-directed serine or threonine sites in the turn motif of Akt and protein kinase C isoforms. mTORC2 may control phosphorylation of the turn motif by promoting the activity of a kinase that targets the Ser/Thr-Pro sequence or by inhibiting the activity of a phosphatase.

SGK1 (serum- and glucocorticoid-induced protein kinase 1) is a member of the AGC (protein kinase A/protein kinase G/protein kinase C) family of protein kinases and is activated by agonists including growth factors. SGK1 regulates diverse effects of extracellular agonists by phosphorylating regulatory proteins that control cellular processes such as ion transport and growth. Like other AGC family kinases, activation of SGK1 is triggered by phosphorylation of a threonine residue within the T-loop of the kinase domain and a serine residue lying within the C-terminal hydrophobic motif (Ser(422) in SGK1). PDK1 (phosphoinositide-dependent kinase 1) phosphorylates the T-loop of SGK1. The identity of the hydrophobic motif kinase is unclear. Recent work has established that mTORC1 [mTOR (mammalian target of rapamycin) complex 1] phosphorylates the hydrophobic motif of S6K (S6 kinase), whereas mTORC2 (mTOR complex 2) phosphorylates the hydrophobic motif of Akt (also known as protein kinase B). In the present study we demonstrate that SGK1 hydrophobic motif phosphorylation and activity is ablated in knockout fibroblasts possessing mTORC1 activity, but lacking the mTORC2 subunits rictor (rapamycin-insensitive companion of mTOR), Sin1 (stress-activated-protein-kinase-interacting protein 1) or mLST8 (mammalian lethal with SEC13 protein 8). Furthermore, phosphorylation of NDRG1 (N-myc downstream regulated gene 1), a physiological substrate of SGK1, was also abolished in rictor-, Sin1- or mLST8-deficient fibroblasts. mTORC2 immunoprecipitated from wild-type, but not from mLST8- or rictor-knockout cells, phosphorylated SGK1 at Ser(422). Consistent with mTORC1 not regulating SGK1, immunoprecipitated mTORC1 failed to phosphorylate SGK1 at Ser(422), under conditions which it phosphorylated the hydrophobic motif of S6K. Moreover, rapamycin treatment of HEK (human embryonic kidney)-293, MCF-7 or HeLa cells suppressed phosphorylation of S6K, without affecting SGK1 phosphorylation or activation. The findings of the present study indicate that mTORC2, but not mTORC1, plays a vital role in controlling the hydrophobic motif phosphorylation and activity of SGK1. Our findings may explain why in previous studies phosphorylation of substrates, such as FOXO (forkhead box O), that could be regulated by SGK, are reduced in mTORC2-deficient cells. The results of the present study indicate that NDRG1 phosphorylation represents an excellent biomarker for mTORC2 activity.

Excitotoxicity has been proposed as one of the mechanisms involved in the specific loss of striatal neurons that occurs in Huntington's disease. Here, we studied the role of calcineurin in the vulnerability of striatal neurons expressing mutant huntingtin to excitotoxicity. To this end, we induced excitotoxicity by adding NMDA to a striatal precursor cell line expressing full-length wild-type (STHdh(Q7/Q7)) or mutant (STHdh(Q111/Q111)) huntingtin. We observed that cell death appeared earlier in STHdh(Q111/Q111) cells than in STHdh(Q7/Q7) cells. Interestingly, these former cells expressed higher levels of calcineurin A that resulted in a greater increase of its activity after NMDA receptor stimulation. Moreover, transfection of full-length mutant huntingtin in different striatal-derived cells (STHdh(Q7/Q7), M213 and primary cultures) increased calcineurin A protein levels. To determine whether high levels of calcineurin A might account for the earlier activation of cell death in mutant huntingtin knock-in cells, wild-type cells were transfected with calcineurin A. Calcineurin A-transfected STHdh(Q7/Q7) cells displayed a significant increase in cell death compared with that recorded in green fluorescent protein-transfected cells after NMDA treatment. Notably, addition of the calcineurin inhibitor FK-506 produced a more robust reduction in cell death in mutant huntingtin knock-in cells than it did in wild-type cells. These results suggest that high levels of calcineurin A could account for the increased vulnerability of striatal cells expressing mutant huntingtin to excitotoxicity.

PDK1 activates a group of kinases, including protein kinase B (PKB)/Akt, p70 ribosomal S6 kinase (S6K), and serum and glucocorticoid-induced protein kinase (SGK), that mediate many of the effects of insulin as well as other agonists. PDK1 interacts with phosphoinositides through a pleckstrin homology (PH) domain. To study the role of this interaction, we generated knock-in mice expressing a mutant of PDK1 incapable of binding phosphoinositides. The knock-in mice are significantly small, insulin resistant, and hyperinsulinemic. Activation of PKB is markedly reduced in knock-in mice as a result of lower phosphorylation of PKB at Thr308, the residue phosphorylated by PDK1. This results in the inhibition of the downstream mTOR complex 1 and S6K1 signaling pathways. In contrast, activation of SGK1 or p90 ribosomal S6 kinase or stimulation of S6K1 induced by feeding is unaffected by the PDK1 PH domain mutation. These observations establish the importance of the PDK1-phosphoinositide interaction in enabling PKB to be efficiently activated with an animal model. Our findings reveal how reduced activation of PKB isoforms impinges on downstream signaling pathways, causing diminution of size as well as insulin resistance.