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Engineering 3D tissue-like constructs for applications such as regenerative medicine remains a major challenge in biomedical research. Recently, self-healing, viscoplastic fluids have been introduced as suspension media to allow lower viscosity, water-rich bioinks to be printed within them for the fabrication of more biomimetic structures. Here, we present gellan gum granular gels produced through the application of shear during gelation, as a candidate suspension medium. We demonstrate that these granular gels exhibit viscoplasticity over a wide range of temperatures, permitting their use for 3D bioprinting of filaments and droplets at low (4°C) as well as physiological temperatures. These granular gels exhibit very low yield stresses (down to 0.4 Pa) which facilitated printing at print speeds up to 60 mm.s-1. Furthermore, we demonstrate the printing of cell-laden droplets maintained over 7 days to show the potential for multiple days of cell culture, as well as the fabrication of hydrogel features within a crosslinkable version of the suspension medium containing granular gellan gum and gelatine-methacryloyl. The combination of ease of preparation, high printing speed, wide temperature tolerance, and crosslinkability makes this gellan gum sheared through cooling-induced gelation an attractive candidate for suspended biofabrication., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 McCormack et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

PTEN is a phosphoinositide lipid phosphatase and an important tumour suppressor protein. PTEN function is reduced or lost in around a third of all human cancers through diverse mechanisms, from gene deletion to changes in the function of proteins which regulate PTEN through direct protein binding. Here we present data from SILAC (Stable Isotope Labelling by Amino acids in Cell culture) proteomic screens to identify proteins which bind to PTEN. These experiments using untransformed epithelial cells and glioma cells identified several novel candidate proteins in addition to many previously identified PTEN binding partners and many proteins which are recognised as common false positives using these methods. From subsequent co-expression pull-down experiments we provide further evidence supporting the physical interaction of PTEN with MMP1, Myosin 18A and SHROOM3. We also performed yeast two-hybrid screens which identify the previously recognised PTEN binding partner MSP58 in addition to the nuclear import export receptor TNPO3. These experiments identify several novel candidate binding partners of PTEN and provide further data addressing the set of proteins that interact with this important tumour suppressor., Competing Interests: Declaration of competing interest None, (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Stress-linked disorders are more prevalent in women than in men and differ in their clinical presentation. Thus, investigating sex differences in factors that promote susceptibility or resilience to stress outcomes, and the circuit elements that mediate their effects, is important. In male rats, instrumental control over stressors engages a corticostriatal system involving the prelimbic cortex (PL) and dorsomedial striatum (DMS) that prevent many of the sequelae of stress exposure. Interestingly, control does not buffer against stress outcomes in females, and here, we provide evidence that the instrumental controlling response in females is supported instead by the dorsolateral striatum (DLS). Additionally, we used in vivo microdialysis, fluorescent in situ hybridization, and receptor subtype pharmacology to examine the contribution of prefrontal dopamine (DA) to the differential impact of behavioral control. Although both sexes preferentially expressed D1 receptor mRNA in PL GABAergic neurons, there were robust sex differences in the dynamic properties of prefrontal DA during controllable stress. Behavioral control potently attenuated stress-induced DA efflux in males, but not females, who showed a sustained DA increase throughout the entire stress session. Importantly, PL D1 receptor blockade (SCH 23390) shifted the proportion of striatal activity from the DLS to the DMS in females and produced the protective effects of behavioral control. These findings suggest a sex-selective mechanism in which elevated DA in the PL biases instrumental responding towards prefrontal-independent striatal circuitry, thereby eliminating the protective impact of coping with stress., (© 2022. The Author(s), under exclusive licence to American College of Neuropsychopharmacology.)

The emergence of circulating DNA analysis in blood during the past decade has responded to the need for noninvasive alternatives to classical tissue biopsies. This has coincided with the development of techniques that allow the detection of low-frequency allele variants in clinical samples that typically carry very low amounts of fragmented DNA, such as plasma or FFPE samples. Enrichment of rare variants by nuclease-assisted mutant allele enrichment with overlapping probes (NaME-PrO) enables a more sensitive detection of mutations in tissue biopsy samples alongside standard qPCR detection assays. Such sensitivity is normally achieved by other more complex PCR methods, such as TaqMan qPCR and digital droplet PCR (ddPCR). Here we describe a workflow of mutation-specific nuclease-based enrichment combined with a SYBR Green real-time quantitative PCR detection method that provides comparable results to ddPCR. Using a PIK3CA mutation as an example, this combined workflow enables detection and accurate prediction of initial variant allele fraction in samples with a low mutant allele frequency (<1%) and could be applied flexibly to detect other mutations of interest., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cell-free DNA has many applications in clinical medicine, in particular in cancer diagnosis and cancer treatment monitoring. Microfluidic-based solutions could provide solutions for rapid, cheaper, decentralized detection of cell-free tumoral DNA from a simple blood draw, or liquid biopsies, replacing invasive procedures or expensive scans. In this method, we present a simple microfluidic system for the extraction of cell-free DNA from low volume of plasma samples (≤500 μL). The technique is suitable for either static or continuous flow systems and can be used as a stand-alone module or integrated within a lab-on-chip system. The system relies on a simple yet highly versatile bubble-based micromixer module whose custom components can be fabricated with a combination of low-cost rapid prototyping techniques or ordered via widely available 3D-printing services. This system is capable of performing cell-free DNA extractions from small volumes of blood plasma with up to a tenfold increase in capture efficiency when compared to control methods., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Loss PTEN function is one of the most common events driving aggressive prostate cancers and biochemically, PTEN is a lipid phosphatase which opposes the activation of the oncogenic PI3K-AKT signalling network. However, PTEN also has additional potential mechanisms of action, including protein phosphatase activity. Using a mutant enzyme, PTEN Y138L, which selectively lacks protein phosphatase activity, we characterised genetically modified mice lacking either the full function of PTEN in the prostate gland or only lacking protein phosphatase activity. The phenotypes of mice carrying a single allele of either wild-type Pten or Pten Y138L in the prostate were similar, with common prostatic intraepithelial neoplasia (PIN) and similar gene expression profiles. However, the latter group, lacking PTEN protein phosphatase activity additionally showed lymphocyte infiltration around PIN and an increased immune cell gene expression signature. Prostate adenocarcinoma, elevated proliferation and AKT activation were only frequently observed when PTEN was fully deleted. We also identify a common gene expression signature of PTEN loss conserved in other studies (including Nkx3.1, Tnf and Cd44 ). We provide further insight into tumour development in the prostate driven by loss of PTEN function and show that PTEN protein phosphatase activity is not required for tumour suppression.

Drug-induced liver injury (DILI) results in over 100 000 hospital attendances per year in the UK alone and is a leading cause for the post-marketing withdrawal of new drugs, leading to significant financial losses. MicroRNA-122 (miR-122) has been proposed as a sensitive DILI marker although no commercial applications are available yet. Extracellular blood microRNAs (miRNAs) are promising clinical biomarkers but their measurement at point of care remains time-consuming, technically challenging, and expensive. For circulating miRNA to have an impact on healthcare, a key challenge to overcome is the development of rapid and reliable low-cost sample preparation. There is an acknowledged issue with miRNA stability in the presence of hemolysis and platelet activation, and no solution has been demonstrated for fast and robust extraction at the site of blood draw. Here, we report a novel microfluidic platform for the extraction of circulating miR-122 from blood enabled by a vertical approach and gravity-based bubble mixing. The performance of this disposable cartridge was verified by standard quantitative polymerase chain reaction analysis on extracted miR-122. The cartridge performed equivalently or better than standard bench extraction kits. The extraction cartridge was combined with electrochemical impedance spectroscopy to detect miR-122 as an initial proof-of-concept toward an application in point-of-care detection. This platform enables the standardization of sample preparation and the detection of miRNAs at the point of blood draw and in resource limited settings and could aid the introduction of miRNA-based assays into routine clinical practice., (© 2022 Author(s).)

PIK3CA is one of the two most frequently mutated genes in breast cancers, occurring in 30-40% of cases. Four frequent 'hotspot' PIK3CA mutations (E542K, E545K, H1047R and H1047L) account for 80-90% of all PIK3CA mutations in human malignancies and represent predictive biomarkers. Here we describe a PIK3CA mutation specific nuclease-based enrichment assay, which combined with a low-cost real-time qPCR detection method, enhances assay detection sensitivity from 5% for E542K and 10% for E545K to 0.6%, and from 5% for H1047R to 0.3%. Moreover, we present a novel flexible prediction method to calculate initial mutant allele frequency in tissue biopsy and blood samples with low mutant fraction. These advancements demonstrated a quick, accurate and simple detection and quantitation of PIK3CA mutations in two breast cancer cohorts (first cohort n = 22, second cohort n = 25). Hence this simple, versatile and informative workflow could be applicable for routine diagnostic testing where quantitative results are essential, e.g. disease monitoring subject to validation in a substantial future study.

Extrusion-based 3D printers have been adopted in pursuit of engineering functional tissues through 3D bioprinting. However, we are still a long way from the promise of fabricating constructs approaching the complexity and function of native tissues. A major challenge is presented by the competing requirements of biomimicry and manufacturability. This opinion article discusses 3D printing in suspension baths as a novel strategy capable of disrupting the current bioprinting landscape. Suspension baths provide a semisolid medium to print into, voiding many of the inherent flaws of printing onto a flat surface in air. We review the state-of-the-art of this approach and extrapolate toward future possibilities that this technology might bring, including the fabrication of vascularized tissue constructs., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Three dimensional (3D) bioprinting of multiple cell types within optimised extracellular matrices has the potential to more closely model the 3D environment of human physiology and disease than current alternatives. In this study, we used a multi-nozzle extrusion bioprinter to establish models of glioblastoma made up of cancer and stromal cells printed within matrices comprised of alginate modified with RGDS cell adhesion peptides, hyaluronic acid and collagen-1. Methods were developed using U87MG glioblastoma cells and MM6 monocyte/macrophages, whilst more disease relevant constructs contained glioblastoma stem cells (GSCs), co-printed with glioma associated stromal cells (GASCs) and microglia. Printing parameters were optimised to promote cell-cell interaction, avoiding the 'caging in' of cells due to overly dense cross-linking. Such printing had a negligible effect on cell viability, and cells retained robust metabolic activity and proliferation. Alginate gels allowed the rapid recovery of printed cell protein and RNA, and fluorescent reporters provided analysis of protein kinase activation at the single cell level within printed constructs. GSCs showed more resistance to chemotherapeutic drugs in 3D printed tumour constructs compared to 2D monolayer cultures, reflecting the clinical situation. In summary, a novel 3D bioprinting strategy is developed which allows control over the spatial organisation of tumour constructs for pre-clinical drug sensitivity testing and studies of the tumour microenvironment., Competing Interests: Declaration of competing interest MA and MB are full time employees of Renishaw PLC., (Copyright © 2019. Published by Elsevier Ltd.)

PTEN is a phosphatase which metabolises PIP 3 , the lipid product of PI 3-Kinase, directly opposing the activation of the oncogenic PI3K/AKT/mTOR signalling network. Accordingly, loss of function of the PTEN tumour suppressor is one of the most common events observed in many types of cancer. Although the mechanisms by which PTEN function is disrupted are diverse, the most frequently observed events are deletion of a single gene copy of PTEN and gene silencing, usually observed in tumours with little or no PTEN protein detectable by immunohistochemistry. Accordingly, with the exceptions of glioblastoma and endometrial cancer, mutations of the PTEN coding sequence are uncommon (<10%) in most types of cancer. Here we review the data relating to PTEN loss in seven common tumour types and discuss mechanisms of PTEN regulation, some of which appear to contribute to reduced PTEN protein levels in cancers., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The function of PTEN in the cytoplasm largely depends on its lipid-phosphatase activity, though which it antagonizes the PI3K-AKT oncogenic pathway. However, molecular mechanisms underlying the role of PTEN in the nucleus remain largely elusive. Here, we report that DNA double-strand breaks (DSB) promote PTEN interaction with MDC1 upon ATM-dependent phosphorylation of T/S398-PTEN. Importantly, DNA DSBs enhance NSD2 (MMSET/WHSC1)-mediated dimethylation of PTEN at K349, which is recognized by the tudor domain of 53BP1 to recruit PTEN to DNA-damage sites, governing efficient repair of DSBs partly through dephosphorylation of γH2AX. Of note, inhibiting NSD2-mediated methylation of PTEN, either through expressing methylation-deficient PTEN mutants or through inhibiting NSD2, sensitizes cancer cells to combinatorial treatment with a PI3K inhibitor and DNA-damaging agents in both cell culture and in vivo xenograft models. Therefore, our study provides a novel molecular mechanism for PTEN regulation of DSB repair in a methylation- and protein phosphatase-dependent manner. SIGNIFICANCE: NSD2-mediated dimethylation of PTEN is recognized by the 53BP1 tudor domain to facilitate PTEN recruitment into DNA-damage sites, governing efficient repair of DNA DSBs. Importantly, inhibiting PTEN methylation sensitizes cancer cells to combinatorial treatment with a PI3K inhibitor combined with DNA-damaging agents in both cell culture and in vivo xenograft models. This article is highlighted in the In This Issue feature, p. 1143 ., (©2019 American Association for Cancer Research.)

Rearrangements of the actin cytoskeleton are regulated in part by dynamic localised activation and inactivation of Rho family small GTPases. SWAP70 binds to and activates the small GTPase RAC1 as well as binding to filamentous actin and PIP 3 . We have developed an encoded biosensor, which uses Forster resonance energy transfer to reveal conformational changes in SWAP70 in live cells. SWAP70 adopts a distinct conformation at the plasma membrane, which in migrating glioma cells is enriched at the leading edge but does not always associate with its PIP 3 -dependent translocation to the membrane. This supports a role for SWAP70 in positive feedback activation of RAC1 at sites of filamentous actin, PIP 3 and active RAC1., (© 2019 Federation of European Biochemical Societies.)

PTEN is a tumor suppressor gene inactivated in over 30% of human cancers. It encodes a lipid phosphatase that serves as a gatekeeper of the phosphoinositide 3-kinase signaling pathway. Germline mutation frequently occurs in this gene in patients diagnosed with PTEN Hamartoma Tumor Syndrome (PHTS). PHTS individuals are characterized by macrocephaly, benign growth of multiple tissues and increased tumor risk. In addition, autistic phenotypes are found in 10-20% of individuals carrying the germline PTEN mutation with macrocephaly. In this report, 13 suspected PHTS patients were screened for mutation in the PTEN gene. A missense variant (c. 302T > C) substituting the isoleucine at codon 101 to a threonine, a single nucleotide insertion (c. 327-328insC) causing a frame shift mutation and termination at codon 109, and a nonsense variant (c. 1003C > T) truncated the protein at codon 335 were identified. The I101T mutation significantly reduced PTEN protein expression levels by 2.5- to 4.0-fold. Mechanistically, I101T reduced the protein half-life of PTEN possibly due to enhanced polyubiquitination at Lysine 13. However, the I101T mutant retained almost 30% of the lipid phosphatase activity of the wild-type protein. Finally, the I101T mutant has reduced phosphorylation at a PTEN auto-dephosphorylation site at Threonine 366 and a lowered ratio of nuclear to cytosolic protein level. These partial losses of multiple PTEN biochemical functions may contribute to the tissue overgrowth and autistic features of this PHTS patient. Autism Res 2018, 11: 1098-1109. © 2018 The Authors Autism Research published by International Society for Autism Research and Wiley Periodicals, Inc. LAY SUMMARY: The genetics of autism spectrum disorders is highly complex with individual risk influenced by both genetic and environmental factors. Mutation in the human PTEN gene confers a high risk of developing autistic behavior. This report revealed that PTEN mutations occurred in 23% of a selected group of Hong Kong patients harboring autistic features with gross overgrowth symptoms. Detailed characterization of a PTEN mutation revealed reduced protein stability as one of the underlying mechanisms responsible for reduced PTEN activity., (© 2018 The Authors Autism Research published by International Society for Autism Research and Wiley Periodicals, Inc.)

Mounting evidence indicates that cytokines secreted by innate immune cells in the brain play a central role in regulating neural circuits that subserve mood, cognition, and sickness responses. A major impediment to the study of neuroimmune signaling in healthy and disease states is the absence of tools for in vivo detection of cytokine release in the brain. Here we describe the design and application of a cytokine detection device capable of serial monitoring of local cytokine release in discrete brain regions. The immunocapture device consisted of a modified optical fiber labeled with a capture antibody specific for the pro-inflammatory cytokine interleukin-1 beta (IL-1β). Using a sandwich immunoassay method, in vitro data demonstrate that the sensing interface of the modified optical fiber has a linear detection range of 3.9 pg mL -1 -500 pg mL -1 and spatial resolution on the order of 200-450 μm. Finally, we show that the immunocapture device can be introduced into a perforated guide cannula for repeated analyte measurements in vivo. An increase in fluorescence detection of spatially localized intrahippocampal IL-1β release was observed following a peripheral lipopolysaccharide challenge in Sprague-Dawley rats. This novel immunosensing technology represents an opportunity for unlocking the function of neuroimmune signaling., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The degree of behavioural control that an organism has over a stressor is a potent modulator of the stressor's impact; controllable stressors produce none of the neurochemical and behavioural sequelae that occur if the stressor is uncontrollable. Research demonstrating the importance of control and the neural mechanisms responsible has been conducted almost entirely with male rats. It is unknown if behavioural control is stress blunting in females, and whether or not a similar resilience circuitry is engaged. Female rats were exposed to controllable, yoked uncontrollable or no tailshock. In separate experiments, behavioural (juvenile social exploration, fear and shuttle box escape) and neurochemical (activation of dorsal raphe serotonin and dorsal raphe-projecting prelimbic neurons) outcomes, which are sensitive to the dimension of control in males, were assessed. Despite successful acquisition of the controlling response, behavioural control did not mitigate dorsal raphe serotonergic activation and behavioural outcomes induced by tailshock, as it does in males. Moreover, behavioural control failed to selectively engage prelimbic cells that project to the dorsal raphe as in males. Pharmacological activation of the prelimbic cortex restored the stress-buffering effects of control. Collectively, the data demonstrate stressor controllability phenomena are absent in females and that the protective prelimbic circuitry is present but not engaged. Reduced benefit from coping responses may represent a novel approach for understanding differential sex prevalence in stress-related psychiatric disorders., (© 2018 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

PIK3CA mutations are seemingly the most common driver mutations in breast cancer with H1047R and E545K being the most common of these, accounting together for around 60% of all PIK3CA mutations and have promising therapeutic implications. Given the low sensitivity and the high cost of current genotyping methods we sought to develop fast, simple and inexpensive assays for PIK3CA H1047R and E545K mutation screening in clinical material. The methods we describe are based on a real-time PCR including a mutation specific primer combined with a non-productive oligonucleotide which inhibits wild-type amplification and a parallel internal control reaction. We demonstrate consistent detection of PIK3CA H1047R mutant DNA in genomic DNA extracted from frozen breast cancer biopsies, FFPE material or cancer cell lines with a detection sensitivity of approximately 5% mutant allele fraction and validate these results using both Sanger sequencing and deep next generation sequencing methods. The detection sensitivity for PIK3CA E545K mutation was approximately 10%. We propose these methods as simple, fast and inexpensive diagnostic tools to determine PIK3CA mutation status.

Glycogen Synthase Kinase-3 (GSK3 or GSK-3) is a promiscuous protein kinase and its phosphorylation of its diverse substrates has major influences on many areas of physiology and pathology, including cellular metabolism, lineage commitment and neuroscience. GSK3 was one of the first identified substrates of the heavily studied oncogenic kinase AKT, phosphorylation by which inhibits GSK3 activity via the formation of an autoinhibitory pseudosubstrate sequence. This has led to investigation of the role of GSK3 inhibition as a key component of the cellular responses to growth factors and insulin, which stimulate the class I PI 3-Kinases and in turn AKT activity and GSK3 phosphorylation. GSK3 has been shown to phosphorylate several upstream and downstream components of the PI3K/AKT/mTOR signalling network, including AKT itself, RICTOR, TSC1 and 2, PTEN and IRS1 and 2, with the potential to apply feedback control within the network. However, it has been clear for some time that functionally distinct, insulated pools of GSK3 exist which are regulated independently, so that for some GSK3 substrates such as β-catenin, phosphorylation by GSK3 is not controlled by input from PI3K and AKT. Instead, as almost all GSK3 substrates require a priming phosphorylated residue to be 4 amino acids C-terminal to the Ser/Thr phosphorylated by GSK3, the predominant form of regulation of the activity of GSK3 often appears to be through control over these priming events, specific to individual substrates. Therefore, a major role of GSK3 can be viewed as an amplifier of the electrostatic effects on protein function which are caused by these priming phosphorylation events. Here we discuss these different aspects to GSK3 regulation and function, and the functions of GSK3 as it integrates with signalling through the PI3K-AKT-mTOR signalling axis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In this issue, Chen et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201604025) show that Importin-11 traffics the tumor suppressor PTEN into the nucleus and in so doing protects it from cytoplasmic proteins that cause PTEN degradation. This work helps explain the nuclear accumulation of PTEN observed in many healthy tissues and, because Ipo11 mutant mice develop lung tumors, also implicates Importin-11 as a novel tumor suppressor., (© 2017 Leslie.)

Loss of function of the PTEN tumour suppressor, resulting in dysregulated activation of the phosphoinositide 3-kinase (PI3K) signalling network, is recognized as one of the most common driving events in prostate cancer development. The observed mechanisms of PTEN loss are diverse, but both homozygous and heterozygous genomic deletions including PTEN are frequent, and often accompanied by loss of detectable protein as assessed by immunohistochemistry (IHC). The occurrence of PTEN loss is highest in aggressive metastatic disease and this has driven the development of PTEN as a prognostic biomarker, either alone or in combination with other factors, to distinguish indolent tumours from those likely to progress. Here, we discuss these factors and the consequences of PTEN loss, in the context of its role as a lipid phosphatase, as well as current efforts to use available inhibitors of specific components of the PI3K/PTEN/TOR signalling network in prostate cancer treatment., (© 2017 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.)

Phosphatase and tensin homolog (PTEN) is a phosphoinositide lipid phosphatase and one of the most frequently disrupted tumor suppressors in many forms of cancer, with even small reductions in the expression levels of PTEN promoting cancer development. Although the post-translational ubiquitination of PTEN can control its stability, activity, and localization, a detailed understanding of how PTEN ubiquitination integrates with other cellular regulatory processes and may be dysregulated in cancer has been hampered by a poor understanding of the significance of ubiquitination at individual sites. Here we show that Lys(66) is not required for cellular activity, yet dominates over other PTEN ubiquitination sites in the regulation of protein stability. Notably, combined mutation of other sites (Lys(13), Lys(80), and Lys(289)) has relatively little effect on protein expression, protein stability, or PTEN polyubiquitination. The present work identifies a key role for Lys(66) in the regulation of PTEN expression and provides both an opportunity to improve the stability of PTEN as a protein therapy and a mechanistic basis for efforts to stabilize endogenous PTEN., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

PTEN has been heavily studied due to its role as a tumour suppressor and as a core inhibitory component of the phosphoinositide 3-kinase (PI3K) signalling network. It is a broadly expressed phosphatase which displays complexity and diversity in both its functions and regulation and accordingly, in the laboratory numerous classes of functionally distinct mutations have been generated. Inherited loss of function mutations in the PTEN gene were originally identified in sufferers of Cowden disease, but later shown to associate with more diverse human pathologies, mostly relating to cell and tissue overgrowth, leading to the use of the broader term, PTEN Hamartoma Tumour Syndrome. Recent phenotypic analysis of clinical cohorts of PTEN mutation carriers, combined with laboratory studies of the consequences of these mutations implies that stable catalytically inactive PTEN mutants may lead to the most severe phenotypes, and conversely, that mutants retaining partial function associate more frequently with a milder phenotype, with autism spectrum disorder often being diagnosed. Future work will be needed to confirm and to refine these genotype-phenotype relationships and convert this developing knowledge into improved patient management and potentially treatment with emerging drugs which target the PI3K pathway., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The phosphatase and tensin homologue deleted on chromosome 10 (PTEN) phosphatase dephosphorylates PIP3, the lipid product of the class I PI 3-kinases, and suppresses the growth and proliferation of many cell types. It has been heavily studied, in large part due to its status as a tumour suppressor, the loss of function of which is observed through diverse mechanisms in many tumour types. Here we present a concise review of our understanding of the PTEN protein and highlight recent advances, particularly in our understanding of its localization and regulation by ubiquitination and SUMOylation., (© 2016 Authors; published by Portland Press Limited.)

PTEN is a one of the most frequently mutated tumor suppressors in human cancers. It is essential for regulating diverse biological processes and through its lipid phosphatase activity regulates the PI 3-Kinase signaling pathway. Sensitive phosphatase assays are employed to study the catalytic activity of PTEN against phospholipid substrates. Here we describe protocols to assay PTEN lipid phosphatase activity using either purified enzyme (purified PTEN lipid phosphatase assay) or PTEN immunopurified from tissues or cultured cells (cellular IP PTEN lipid phosphatase assay) against vesicles containing radiolabeled PIP3 substrate.

The lipid and protein tyrosine phosphatase, PTEN, is one of the most frequently mutated tumor suppressors in human cancers and is essential for regulating the oncogenic pro-survival PI3K/AKT signaling pathway. Because of its diverse physiological functions, PTEN has attracted great interest from researchers in multiple research fields. The functional diversity of PTEN demands a collection of delicate regulatory mechanisms, including transcriptional control and posttranslational mechanisms that include ubiquitination. Addition of ubiquitin to PTEN can have several effects on PTEN function, potentially regulating its stability, localization, and activity. In cell and in vitro ubiquitination assays are employed to study the ubiquitination-mediated regulation of PTEN. However, PTEN ubiquitination assays are challenging to perform and the data published from these assays has been of mixed quality. Here we describe protocols to detect PTEN ubiquitination in cultured cells expressing epitope tagged ubiquitin (in cell PTEN ubiquitination assay) and also using purified proteins (in vitro PTEN ubiquitination assay).

Different bioprinting techniques have been used to produce cell-laden alginate hydrogel structures, however these approaches have been limited to 2D or simple three-dimension (3D) structures. In this study, a new extrusion based bioprinting technique was developed to produce more complex alginate hydrogel structures. This was achieved by dividing the alginate hydrogel cross-linking process into three stages: primary calcium ion cross-linking for printability of the gel, secondary calcium cross-linking for rigidity of the alginate hydrogel immediately after printing and tertiary barium ion cross-linking for long-term stability of the alginate hydrogel in culture medium. Simple 3D structures including tubes were first printed to ensure the feasibility of the bioprinting technique and then complex 3D structures such as branched vascular structures were successfully printed. The static stiffness of the alginate hydrogel after printing was 20.18 ± 1.62 KPa which was rigid enough to sustain the integrity of the complex 3D alginate hydrogel structure during the printing. The addition of 60 mM barium chloride was found to significantly extend the stability of the cross-linked alginate hydrogel from 3 d to beyond 11 d without compromising the cellular viability. The results based on cell bioprinting suggested that viability of U87-MG cells was 93 ± 0.9% immediately after bioprinting and cell viability maintained above 88% ± 4.3% in the alginate hydrogel over the period of 11 d.

In many human cell types, the class I phosphoinositide 3-kinases play key roles in the control of diverse cellular processes including growth, proliferation, survival and polarity. This is achieved through their activation by many cell surface receptors, leading to the synthesis of the phosphoinositide lipid signal, PIP3, which in turn influences the function of numerous direct PIP3-binding proteins. Here we review PI3K pathway biology and analyse the evolutionary distribution of its components and their functions. The broad phylogenetic distribution of class I PI3Ks in metazoa, amoebozoa and choannoflagellates, implies that these enzymes evolved in single celled organisms and were later co-opted into metazoan intercellular communication. A similar distribution is evident for the AKT and Cytohesin groups of downstream PIP3-binding proteins, with other effectors and pathway components appearing to evolve later. The genomic and functional phylogeny of regulatory systems such as the PI3K pathway provides a framework to improve our understanding of the mechanisms by which key cellular processes are controlled in humans., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The PTEN phosphoinositide 3-phosphatase is a tumor suppressor commonly targeted by pathologic missense mutations. Subject to multiple complex layers of regulation, its capital role in cancer relies on its counteracting function of class I phosphoinositide 3-kinase (PI3K), a key feature in oncogenic signaling pathways. Precise assessment of the involvement of PTEN mutations described in the clinics in loss of catalytic activity requires either tedious in vitro phosphatase assays or in vivo experiments involving transfection into mammalian cell lines. Taking advantage of the versatility of the model organism Saccharomyces cerevisiae, we have developed different functional assays by reconstitution of the mammalian PI3K-PTEN switch in this lower eukaryote. This methodology is based on the fact that regulated PI3K expression in yeast cells causes conversion of PtdIns(4,5)P2 in PtdIns(3,4,5)P3 and co-expression of PTEN counteracts this effect. This can be traced by monitoring growth, given that PtdIns(4,5)P2 pools are essential for the yeast cell, or by using fluorescent reporters amenable for microscopy or flow cytometry. Here we describe the methodology and review its application to evaluate the functionality of PTEN mutations. We show that the technique is amenable to both directed and systematic structure-function relationship studies, and present an example of its use for the study of the recently discovered PTEN-L variant., (Copyright © 2014 Elsevier Inc. All rights reserved.)

PTEN is a major tumour suppressor protein and a regulator of numerous diverse biological processes. It has an evolutionarily conserved role as a phosphoinositide lipid phosphatase, regulating the PI3K signalling pathway, but also has catalytic phosphatase activity against protein substrates, although the significance of this latter activity is less well understood. Unlike many tumour suppressors, even modest changes in PTEN activity can have strong effects on phenotypes, including tumour formation. Due to this recognised functional significance, several experimental platforms have been developed to assay the catalytic activity of PTEN against different substrates and are being applied to understand this cellular substrate diversity and the regulation of PTEN. Here we present and discuss methods to assay the phosphatase activity of PTEN in vitro., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Germline mutations in the phosphatase PTEN are associated with diverse human pathologies, including tumour susceptibility, developmental abnormalities and autism, but any genotype-phenotype relationships are poorly understood., Methods: We have studied the functional consequences of seven PTEN mutations identified in patients diagnosed with autism and macrocephaly and five mutations from severe tumour bearing sufferers of PTEN hamartoma tumour syndrome (PHTS)., Results: All seven autism-associated PTEN mutants investigated retained the ability to suppress cellular AKT signalling, although five were highly unstable. Observed effects on AKT also correlated with the ability to suppress soma size and the length and density of dendritic spines in primary neurons. Conversely, all five PTEN mutations from severe cases of PHTS appeared to directly and strongly disrupt the ability to inhibit AKT signalling., Conclusions: Our work implies that alleles causing incomplete loss of PTEN function are more commonly linked to autism than to severe PHTS cases., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Small molecule inhibitors of many classes of enzymes, including phosphatases, have widespread use as experimental tools and as therapeutics. Efforts to develop inhibitors against the lipid phosphatase and tumour suppressor, PTEN, was for some time limited by concerns that their use as therapy could result in increased risk of cancer. However, the accumulation of evidence that short term PTEN inhibition may be valuable in conditions such as nerve injury has raised interest. Here we investigate the inhibition of PTEN by four available PTEN inhibitors, bpV(phen), bpV(pic), VO-OHpic and SF1670 and compared this inhibition with that of only 3 other related enzymes, the tyrosine phosphatase SHP1 and the phosphoinositide phosphatases INPP4A and INPP4B. Even with this very small number of comparators, for all compounds, inhibition of multiple enzymes was observed and with all three vanadate compounds, this was similar or more potent than the inhibition of PTEN. In particular, the bisperoxovanadate compounds were found to inhibit PTEN poorly in the presence of reducing agents including the cellular redox buffer glutathione., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The tumor suppressor PTEN is a major brake for cell transformation, mainly due to its phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] phosphatase activity that directly counteracts the oncogenicity of phosphoinositide 3-kinase (PI3K). PTEN mutations are frequent in tumors and in the germ line of patients with tumor predisposition or with neurological or cognitive disorders, which makes the PTEN gene and protein a major focus of interest in current biomedical research. After almost two decades of intense investigation on the 403-residue-long PTEN protein, a previously uncharacterized form of PTEN has been discovered that contains 173 amino-terminal extra amino acids, as a result of an alternate translation initiation site. To facilitate research in the field and to avoid ambiguities in the naming and identification of PTEN amino acids from publications and databases, we propose here a unifying nomenclature and amino acid numbering for this longer form of PTEN., (Copyright © 2014, American Association for the Advancement of Science.)

The PI3K/PDK1/Akt signaling axis is centrally involved in cellular homeostasis and controls cell growth and proliferation. Due to its key function as regulator of cell survival and metabolism, the dysregulation of this pathway is manifested in several human pathologies including cancers and immunological diseases. Thus, current therapeutic strategies target the components of this signaling cascade. In recent years, numerous feedback loops have been identified that attenuate PI3K/PDK1/Akt-dependent signaling. Here, we report the identification of an additional level of feedback regulation that depends on the negative transcriptional control of phosphatidylinositol 3-kinase (PI3K) class IA subunits. Genetic deletion of 3-phosphoinositide-dependent protein kinase 1 (PDK1) or the pharmacological inhibition of its downstream effectors, that is, Akt and mammalian target of rapamycin (mTOR), relieves this suppression and leads to the upregulation of PI3K subunits, resulting in enhanced generation of phosphatidylinositol-3,4,5-trisphosphate (PIP3). Apparently, this transcriptional induction is mediated by the concerted action of different transcription factor families, including the transcription factors cAMP-responsive element-binding protein and forkhead box O. Collectively, we propose that PDK1 functions as a cellular sensor that balances basal PIP3 generation at levels sufficient for survival but below a threshold being harmful to the cell. Our study suggests that the efficiency of therapies targeting the aberrantly activated PI3K/PDK1/Akt pathway might be increased by the parallel blockade of feedback circuits.

Tumor suppressors block the development of cancer and are often lost during tumor development. Papa et al. show that partial loss of normal PTEN tumor suppressor function can be compounded by additional disruption caused by the expression of inactive mutant PTEN protein. This has significant implications for patients with PTEN gene mutations., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The insulin/IGF-1 (insulin-like growth factor 1)-activated protein kinase Akt (also known as protein kinase B) phosphorylates Ser487 in the 'ST loop' (serine/threonine-rich loop) within the C-terminal domain of AMPK-α1 (AMP-activated protein kinase-α1), leading to inhibition of phosphorylation by upstream kinases at the activating site, Thr172. Surprisingly, the equivalent site on AMPK-α2, Ser491, is not an Akt target and is modified instead by autophosphorylation. Stimulation of HEK (human embryonic kidney)-293 cells with IGF-1 caused reduced subsequent Thr172 phosphorylation and activation of AMPK-α1 in response to the activator A769662 and the Ca2+ ionophore A23187, effects we show to be dependent on Akt activation and Ser487 phosphorylation. Consistent with this, in three PTEN (phosphatase and tensin homologue deleted on chromosome 10)-null tumour cell lines (in which the lipid phosphatase PTEN that normally restrains the Akt pathway is absent and Akt is thus hyperactivated), AMPK was resistant to activation by A769662. However, full AMPK activation could be restored by pharmacological inhibition of Akt, or by re-expression of active PTEN. We also show that inhibition of Thr172 phosphorylation is due to interaction of the phosphorylated ST loop with basic side chains within the αC-helix of the kinase domain. Our findings reveal that a previously unrecognized effect of hyperactivation of Akt in tumour cells is to restrain activation of the LKB1 (liver kinase B1)-AMPK pathway, which would otherwise inhibit cell growth and proliferation.

The stimulation of Toll-like receptors (TLRs) on macrophages by pathogen-associated molecular patterns (PAMPs) results in the activation of intracellular signaling pathways that are required for initiating a host immune response. Both phosphatidylinositol 3-kinase (PI3K)-Akt and p38 mitogen-activated protein kinase (MAPK) signaling pathways are activated rapidly in response to TLR activation and are required to coordinate effective host responses to pathogen invasion. In this study, we analyzed the role of the p38-dependent kinases MK2/3 in the activation of Akt and show that lipopolysaccharide (LPS)-induced phosphorylation of Akt on Thr308 and Ser473 requires p38α and MK2/3. In cells treated with p38 inhibitors or an MK2/3 inhibitor, phosphorylation of Akt on Ser473 and Thr308 is reduced and Akt activity is inhibited. Furthermore, BMDMs deficient in MK2/3 display greatly reduced phosphorylation of Ser473 and Thr308 following TLR stimulation. However, MK2/3 do not directly phosphorylate Akt in macrophages but act upstream of PDK1 and mTORC2 to regulate Akt phosphorylation. Akt is recruited to phosphatidylinositol 3,4,5-trisphosphate (PIP3) in the membrane, where it is activated by PDK1 and mTORC2. Analysis of lipid levels in MK2/3-deficient bone marrow-derived macrophages (BMDMs) revealed a role for MK2/3 in regulating Akt activity by affecting availability of PIP3 at the membrane. These data describe a novel role for p38α-MK2/3 in regulating TLR-induced Akt activation in macrophages.

Genetic changes in HER2, PTEN, PIK3CA and AKT1 are all common in breast cancer and lead to the elevated phosphorylation of downstream targets of the PI3K/AKT signalling pathway. Changes in HER2, PTEN, PIK3CA and AKT have all been reported to lead to both enhanced proliferation and failures in hollow lumen formation in three dimensional epithelial culture models, but it is not clear whether these failures in lumen formation are caused by any failure in the spatial coordination of lumen formation (hollowing) or purely a failure in the apoptosis and clearance of luminal cells (cavitation). Here, we use normal murine mammary gland (NMuMG) epithelial cells, which form a hollow lumen without significant apoptosis, to compare the transformation by these four genetic changes. We find that either mutant PIK3CA expression or PTEN loss, but not mutant AKT1 E17K, cause disrupted epithelial architecture, whereas HER2 overexpression drives strong proliferation without affecting lumen formation in these cells. We also show that PTEN requires both lipid and protein phosphatase activity, its extreme C-terminal PDZ binding sequence and probably Myosin 5A to control lumen formation through a mechanism that does not correlate with its ability to control AKT, but which is selectively lost through mutation in some tumours. These findings correlate AKT-independent signalling activated by mutant PIK3CA or PTEN loss, but not strongly by HER2, with disrupted epithelial architecture and tumour formation.

The individuals carrying melanocortin-1 receptor (MC1R) variants, especially those associated with red hair color, fair skin, and poor tanning ability (RHC trait), are more prone to melanoma; however, the underlying mechanism is poorly defined. Here, we report that UVB exposure triggers phosphatase and tensin homolog (PTEN) interaction with wild-type (WT), but not RHC-associated MC1R variants, which protects PTEN from WWP2-mediated degradation, leading to AKT inactivation. Strikingly, the biological consequences of the failure of MC1R variants to suppress PI3K/AKT signaling are highly context dependent. In primary melanocytes, hyperactivation of PI3K/AKT signaling leads to premature senescence; in the presence of BRAF(V600E), MC1R deficiency-induced elevated PI3K/AKT signaling drives oncogenic transformation. These studies establish the MC1R-PTEN axis as a central regulator for melanocytes' response to UVB exposure and reveal the molecular basis underlying the association between MC1R variants and melanomagenesis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Defects in actin dynamics affect activity-dependent modulation of synaptic transmission and neuronal plasticity, and can cause cognitive impairment. A salient candidate actin-binding protein linking synaptic dysfunction to cognitive deficits is Drebrin (DBN). However, the specific mode of how DBN is regulated at the central synapse is largely unknown. In this study we identify and characterize the interaction of the PTEN tumor suppressor with DBN. Our results demonstrate that PTEN binds DBN and that this interaction results in the dephosphorylation of a site present in the DBN C-terminus--serine 647. PTEN and pS647-DBN segregate into distinct and complimentary compartments in neurons, supporting the idea that PTEN negatively regulates DBN phosphorylation at this site. We further demonstrate that neuronal activity increases phosphorylation of DBN at S647 in hippocampal neurons in vitro and in ex vivo hippocampus slices exhibiting seizure activity, potentially by inducing rapid dissociation of the PTEN:DBN complex. Our results identify a novel mechanism by which PTEN is required to maintain DBN phosphorylation at dynamic range and signifies an unusual regulation of an actin-binding protein linked to cognitive decline and degenerative conditions at the CNS synapse.

Muscle atrophy occurs under various catabolic conditions, including insulin deficiency, insulin resistance, or increased levels of glucocorticoids. This results from reduced levels of insulin receptor substrate 1 (IRS-1), leading to decreased phosphatidylinositol 3-kinase activity and thereby activation of FoxO transcription factors. However, the precise mechanism of reduced IRS-1 under a catabolic condition is unknown. Here, we report that C1-Ten is a novel protein tyrosine phosphatase (PTPase) of IRS-1 that acts as a mediator to reduce IRS-1 under a catabolic condition, resulting in muscle atrophy. C1-Ten preferentially dephosphorylated Y612 of IRS-1, which accelerated IRS-1 degradation. These findings suggest a novel type of IRS-1 degradation mechanism which is dependent on C1-Ten and extends our understanding of the molecular mechanism of muscle atrophy under catabolic conditions. C1-Ten expression is increased by catabolic glucocorticoid and decreased by anabolic insulin. Reflecting these hormonal regulations, the muscle C1-Ten is upregulated in atrophy but downregulated in hypertrophy. This reveals a previously unidentified role of C1-Ten as a relevant PTPase contributing to skeletal muscle atrophy.

PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) is a relatively common anionic lipid that regulates cellular functions by multiple mechanisms. Hydrolysis of PtdIns(4,5)P2 by phospholipase C yields inositol trisphosphate and diacylglycerol. Phosphorylation by phosphoinositide 3-kinase yields PtdIns(3,4,5)P3, which is a potent signal for survival and proliferation. Also, PtdIns(4,5)P2 can bind directly to integral and peripheral membrane proteins. As an example of regulation by PtdIns(4,5)P2, we discuss phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in detail. PTEN is an important tumor suppressor and hydrolyzes PtdIns(3,4,5)P3. PtdIns(4,5)P2 enhances PTEN association with the plasma membrane and activates its phosphatase activity. This is a critical regulatory mechanism, but a detailed description of this process from a structural point of view is lacking. The disordered lipid bilayer environment hinders structural determinations of membrane-bound PTEN. A new method to analyze membrane-bound protein measures neutron reflectivity for proteins bound to tethered phospholipid membranes. These methods allow determination of the orientation and shape of membrane-bound proteins. In combination with molecular dynamics simulations, these studies will provide crucial structural information that can serve as a foundation for our understanding of PTEN regulation in normal and pathological processes.

It is generally assumed that cells synthesize their own intracellular enzymes. Therefore, if expression of a specific gene is silenced in a potential cancer cell, it is expected that loss of protein function will follow. A provocative study indicates an unexpected mechanism of intercellular tumor suppression, showing that PTEN (phosphatase and tensin homolog deleted from chromosome 10), a cytosolic enzyme, can be transferred between cells in exosomes to suppress signaling and proliferation in target cells.

Class I phosphoinositide (PI) 3-kinases act through effector proteins whose 3-PI selectivity is mediated by a limited repertoire of structurally defined, lipid recognition domains. We describe here the lipid preferences and crystal structure of a new class of PI binding modules exemplified by select IQGAPs (IQ motif containing GTPase-activating proteins) known to coordinate cellular signaling events and cytoskeletal dynamics. This module is defined by a C-terminal 105-107 amino acid region of which IQGAP1 and -2, but not IQGAP3, binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdInsP(3)). The binding affinity for PtdInsP(3), together with other, secondary target-recognition characteristics, are comparable with those of the pleckstrin homology domain of cytohesin-3 (general receptor for phosphoinositides 1), an established PtdInsP(3) effector protein. Importantly, the IQGAP1 C-terminal domain and the cytohesin-3 pleckstrin homology domain, each tagged with enhanced green fluorescent protein, were both re-localized from the cytosol to the cell periphery following the activation of PI 3-kinase in Swiss 3T3 fibroblasts, consistent with their common, selective recognition of endogenous 3-PI(s). The crystal structure of the C-terminal IQGAP2 PI binding module reveals unexpected topological similarity to an integral fold of C2 domains, including a putative basic binding pocket. We propose that this module integrates select IQGAP proteins with PI 3-kinase signaling and constitutes a novel, atypical phosphoinositide binding domain that may represent the first of a larger group, each perhaps structurally unique but collectively dissimilar from the known PI recognition modules.

The tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has a well-characterized lipid phosphatase activity and a poorly characterized protein phosphatase activity. We show that both activities are required for PTEN to inhibit cellular invasion and to mediate most of its largest effects on gene expression. PTEN appears to dephosphorylate itself at threonine 366, and mutation of this site makes lipid phosphatase activity sufficient for PTEN to inhibit invasion. We propose that the dominant role for PTEN's protein phosphatase activity is autodephosphorylation-mediated regulation of its lipid phosphatase activity. Because PTEN's regulation of invasion and these changes in gene expression required lipid phosphatase activity, but did not correlate with the total cellular abundance of its phosphatidylinositol 3,4,5-trisphosphate (PIP₃) lipid substrate or AKT activity, we propose that localized PIP₃ signaling may play a role in those PTEN-mediated processes that depend on both its protein and lipid phosphatase activities. Finally, we identified a tumor-derived PTEN mutant selectively lacking protein phosphatase activity, indicating that in some circumstances the regulation of invasion and not that of AKT can correlate with PTEN-mediated tumor suppression.

Two classes of lipid phosphatases selectively dephosphorylate the 3 position of the inositol ring of phosphoinositide signaling molecules: the PTEN and the Myotubularin families. PTEN dephosphorylates PtdIns(3,4,5)P(3), acting in direct opposition to the Class I PI3K enzymes in the regulation of cell growth, proliferation and polarity and is an important tumor suppressor. Although there are several PTEN-related proteins encoded by the human genome, none of these appear to fulfill the same functions. In contrast, the Myotubularins dephosphorylate both PtdIns(3)P and PtdIns(3,5)P(2), making them antagonists of the Class II and Class III PI 3-kinases and regulators of membrane traffic. Both phosphatase groups were originally identified through their causal mutation in human disease. Mutations in specific myotubularins result in myotubular myopathy and Charcot-Marie-Tooth peripheral neuropathy; and loss of PTEN function through mutation and other mechanisms is evident in as many as a third of all human tumors. This chapter will discuss these two classes of phosphatases, covering what is known about their biochemistry, their functions at the cellular and whole body level and their influence on human health.