Class I PI3K Biology.

This chapter is an introduction to phosphoinositide 3-kinases (PI3K), with class I PI3Ks as the central focus. First, the various PI3K isoforms in class I are presented with emphasis on their overall structure, subunits, subunit constitutive domains, domain-domain interactions, and functional relevance. This structural analysis is followed by a comprehensive history of seminal investigations into PI3K activity. Next, we highlight the divergent roles of the isoforms: PI3Kα, PI3Kß, PI3Kδ, and PI3Kγ. This section details signaling pathways in which these PI3K isoforms are involved, including the key upstream regulators of PI3K activity and some downstream cellular effects. Nodes of the PI3K pathway are also presented. Inhibitors of some isoforms are discussed to give an overview of the basis of some immunotherapies that are being used to target cell signaling. Finally, the chapter ends with a discussion of the dysregulation of PI3Ks in diseases including APDS, asthma, arthritis, and oncogenic mutations.

A high-flux P84 polyimide mixed matrix membranes incorporated with cadmium-based metal organic frameworks for enhanced simultaneous dyes removal: Response surface methodology.

The water sources contaminated by toxic dyes would pose a serious problem for public health. In view of this, the development of a simple yet effective method for removing dyes from industrial effluent has attracted interest from researchers. In the present work, flat sheet mixed matrix membranes (MMMs) with different physiochemical properties were fabricated by blending P84 polyimide with different concentrations of cadmium-based metal organic frameworks (MOF-2(Cd)). The resultant membranes were then used for simultaneous removal of eosin y (EY), sunset yellow (SY) and methylene blue (MB) under various process conditions. The findings indicated that the membranes could achieve high water permeability (117.8-171.4 L/m2.h.bar) and promising rejection for simultaneous dyes removal, recording value of 99.9%, 81.2% and 68.4% for MB, EY and SY, respectively. When 0.2 wt% MOF-2(Cd) was incorporated into the membrane matrix, the membrane separation efficiency was improved by 110.2% and 213.3% for EY and SY removal, respectively when compared with the pristine membrane. In addition, the optimization and modeling of membrane permeate flux and dye rejection was explored using response surface methodology. The actual and model results are in good agreement with R2 of at least 0.9983 for dye rejection and permeate flux. The high flux of the developed MMMs coupled with effective separation of dyes suggests a promising prospect of using P84 polyimide MMMs incorporated with MOF-2(Cd) for water purification.

Thermally Stable Cross-linked P84 with Superior Membrane H2/CO2 Separation Properties at 100 °C.

Polymers with a strong size-sieving ability and superior H2/CO2 selectivity are of great interests for pre-combustion CO capture at 100 °C or above. Polyimides (such as Matrimid® and 6FDA-durene) have been cross-linked using diamines and show superior H2/CO2 selectivity. However, these cross-linked polymers cannot be used for the pre-combustion CO2 capture because of the lack of thermal stability at 100 C. Herein we demonstrate that commercial P84™ can be chemically cross-linked using 1,4-butanediamine (BuDA) to achieve robust H2/CO2 separation properties at 100 °C to 150 °C. The cross-linked P84 were thoroughly evaluated using Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). The effects of the cross-linking time on the physical properties and H2/CO2 separation properties at various temperatures were determined and interpreted using a free volume model. An exemplary sample based on P84 crosslinked by BuDA for 6 h exhibits a H2 permeability of 47 Barrers (1 Barrer = 3.35 × 10-16 mol m/m2·s·Pa) and H2/CO2 selectivity of 14 at 100 °C, which is on the Robeson's upper bound, indicating their potential for practical applications.

Molecular basis for differential activation of p101 and p84 complexes of PI3Kγ by Ras and GPCRs.

Class IB phosphoinositide 3-kinase (PI3Kγ) is activated in immune cells and can form two distinct complexes (p110γ-p84 and p110γ-p101), which are differentially activated by G protein-coupled receptors (GPCRs) and Ras. Using a combination of X-ray crystallography, hydrogen deuterium exchange mass spectrometry (HDX-MS), electron microscopy, molecular modeling, single-molecule imaging, and activity assays, we identify molecular differences between p110γ-p84 and p110γ-p101 that explain their differential membrane recruitment and activation by Ras and GPCRs. The p110γ-p84 complex is dynamic compared with p110γ-p101. While p110γ-p101 is robustly recruited by Gßγ subunits, p110γ-p84 is weakly recruited to membranes by Gßγ subunits alone and requires recruitment by Ras to allow for Gßγ activation. We mapped two distinct Gßγ interfaces on p101 and the p110γ helical domain, with differences in the C-terminal domain of p84 and p101 conferring sensitivity of p110γ-p101 to Gßγ activation. Overall, our work provides key insight into the molecular basis for how PI3Kγ complexes are activated.

Low-Voltage Bacterial and Viral Killing Using Laser-Induced Graphene-Coated Non-woven Air Filters.

Laser-induced graphene (LIG) is uniquely positioned to advance applications in which electrically conductive carbon coatings are required. Recently, the antifouling, antiviral, and antibacterial properties of LIG have been proven in both air and water filtration applications. For example, an unsupported LIG based filter (pore size: ∼0.3 µm) demonstrated exceptional air filtration properties, while its joule heating effects successfully sterilized and removed unwanted biological components in air despite persisting challenges such as pressure drop, energy consumption, and lack of mechanical robustness. Here, we developed a polyimide (PI) non-woven supported LIG air filter with negligible pressure drop changes compared to the non-woven support material and showed that low electrical current density inactivates aerosolized bacteria. A current density of 4.5 mA/cm2 did not cause significant joule heating, and 97.2% bacterial removal was obtained. The low-voltage antibacterial mechanism was elucidated using bacterial inhibition experiments on a titanium surface and on an LIG surface fabricated on dense PI films. Complete sterilization was obtained using current densities of ∼8 mA/cm2 applied for 2 min or ∼ 6 mA/cm2 for 10 min upon the dense PI-LIG surface. Lastly, >98% bacterial removal was observed using a low-resistance LIG-coated non-woven polyimide air filter at 5 V. However, only very low voltages (∼0.3 V) were needed to remove ∼99% Pseudomonas aeruginosa bacteria and 100% of T4 virus when the LIG-coated filters were hybridized with a stainless steel mesh. Our results show that low current density levels at very low voltages are sufficient for substantial bacterial and viral inactivation, and that these principles might be effectively used in a wide number of air filtration applications such as air conditioners or other ventilation systems, which might limit the spread of infectious particles in hospitals, homes, workplaces, and the transportation industry.

The utilization of micro-mesoporous carbon-based filler in the P84 hollow fibre membrane for gas separation.

This research involved carrying out a unique micro-mesoporous carbon particle incorporation into P84 co-polyimide membrane for improved gas separation performance. The carbon filler was prepared using a hard template method from zeolite and known as zeolite-templated carbon (ZTC). This research aims to study the loading amount of ZTC into P84 co-polyimide toward the gas separation performance. The ZTC was prepared using simple impregnation method of sucrose into hard template of zeolite Y. The SEM result showing a dispersed ZTC particle on the membrane surface and cross-section. The pore size distribution (PSD) of ZTC revealed that the particle consists of two characteristics of micro and mesoporous region. It was noted that with only 0.5 wt% of ZTC addition, the permeability was boosted up from 4.68 to 7.06 and from 8.95 to 13.15 barrer, for CO2 and H2 respectively when compared with the neat membrane. On the other hand, the optimum loading was at 1 wt%, where the membrane received thermal stability boost of 10% along with the 62.4 and 35% of selectivity boost of CO2/CH4 and H2/CH4, respectively. It was noted that the position of the filler on the membrane surface was significantly affecting the gas transport mechanism of the membrane. Overall, the results demonstrated that the addition of ZTC with proper filler position is a potential candidate to be applicable in the gas separation involving CO2 and H2.

P84/ZCC Hollow Fiber Mixed Matrix Membrane with PDMS Coating to Enhance Air Separation Performance.

This research introduces zeolite carbon composite (ZCC) as a new filler on polymeric membranes based on the BTDA-TDI/MDI (P84) co-polyimide for the air separation process. The separation performance was further improved by a polydimethylsiloxane (PDMS) coating to cover up the surface defect. The incorporation of 1 wt% ZCC into P84 co-polyimide matrix enhanced the O2 permeability from 7.12 to 18.90 Barrer (2.65 times) and the O2/N2 selectivity from 4.11 to 4.92 Barrer (19.71% improvement). The PDMS coating on the membrane further improved the O2/N2 selectivity by up to 60%. The results showed that the incorporation of ZCC and PDMS coating onto the P84 co-polyimide membrane was able to increase the overall air separation performance.

PI3Kγ Regulatory Protein p84 Determines Mast Cell Sensitivity to Ras Inhibition-Moving Towards Cell Specific PI3K Targeting?

Mast cells are the major effector cells in immunoglobulin E (IgE)-mediated allergy. The high affinity IgE receptor FcεRI, as well as G protein-coupled receptors (GPCRs) on the mast cell surface signals to phosphoinositide 3-kinase γ (PI3Kγ) to initiate degranulation, cytokine release, and chemotaxis. PI3Kγ is therefore considered as a target for treatment of allergic disorders. However, leukocyte PI3Kγ is key to many functions in innate and adaptive immunity, and attenuation of host defense mechanisms is an expected adverse effect that complicates treatment of chronic illnesses. PI3Kγ operates as a p110γ/p84 or p110γ/p101 complex, where p110γ/p84 requires Ras activation. Here we investigated if modulation of Ras-isoprenylation could target PI3Kγ activity to attenuate PI3Kγ-dependent mast cell responses without impairment of macrophage functions. In murine bone marrow-derived mast cells, GPCR stimulation triggers activation of N-Ras and H-Ras isoforms, which is followed by the phosphorylation of protein kinase B (PKB/Akt) relayed through PI3Kγ. Although K-Ras is normally not activated in Ras wild-type cells, it is able to compensate for genetically deleted N- and H-Ras isoforms. Inhibition of Ras isoprenylation with farnesyltransferase inhibitor FTI-277 leads to a significant reduction of mast cell degranulation, cytokine production, and migration. Complementation experiments expressing PI3Kγ adaptor proteins p84 or p101 demonstrated a differential sensitivity towards Ras-inhibition depending on PI3Kγ complex composition. Mast cell responses are exclusively p84-dependent and were effectively controlled by FTI-277. Similar results were obtained when GTP-Ras was inactivated by overexpression of the GAP-domain of Neurofibromin-1 (NF-1). Unlike mast cells, macrophages express p84 and p101 but are p101-dominated and thus remain functional under treatment with FTI-277. Our work demonstrates that p101 and p84 have distinct physiological roles, and that Ras dependence of PI3Kγ signaling differs between cell types. FTI-277 reduces GPCR-activated PI3Kγ  responses in p84-expressing but not p101-containing bone marrow derived cells. However, prenylation inhibitors have pleiotropic effects beyond Ras and non-tolerable side-effects that disfavor further clinical validation. Statins are, however, clinically well-established drugs that have previously been proposed to block mast cell degranulation by interference with protein prenylation. We show here that Simvastatin inhibits mast cell degranulation, but that this does not occur via Ras-PI3Kγ pathway alterations.

Function, Regulation and Biological Roles of PI3Kγ Variants.

Phosphatidylinositide 3-kinase (PI3K) γ is the only class IB PI3K member playing significant roles in the G-protein-dependent regulation of cell signaling in health and disease. Originally found in the immune system, increasing evidence suggest a wide array of functions in the whole organism. PI3Kγ occur as two different heterodimeric variants: PI3Kγ (p87) and PI3Kγ (p101), which share the same p110γ catalytic subunit but differ in their associated non-catalytic subunit. Here we concentrate on specific PI3Kγ features including its regulation and biological functions. In particular, the roles of its non-catalytic subunits serving as the main regulators determining specificity of class IB PI3Kγ enzymes are highlighted.

Investigation of imatinib loaded surface decorated biodegradable nanocarriers against glioblastoma cell lines: Intracellular uptake and cytotoxicity studies.

Overexpression of P-glycoprotein (P-gp) efflux transporter in glioma cells thwarts the build-up of therapeutic concentration of drugs usually resulting into poor therapeutic outcome. To surmount aforesaid challenge, Imatinib (IMM) loaded Poly-lactide-co-glycolic acid nanoparticles (IMM-PLGA-NPs) were developed and optimized by Box Behnken Design as a new treatment stratagem in malignant glioma. Optimized NPs were functionalized with Pluronic(®) P84, P-gp inhibitor (IMM-PLGA-P84-NPs) which showed size, PDI, zeta potential, drug loading, 182.63±13.56nm, 0.196±0.021, -15.2±1.49mV, 40.63±2.04µg/mg, respectively. Intracellular uptake study conducted on A172, U251MG and C6 glioma cells demonstrated significantly high uptake of IMM through NPs when compared with IMM solution (IMM-S), p<0.001. IMM-PLGA-P84-NPs showed better uptake in P-gp expressing cell line (U251MG and C6) while uncoated NPs showed higher uptake in non-P-gp expressing cell line (A-172). Cytotoxicity studies demonstrated significantly low IC50 for both IMM-PLGA-NPs and IMM-PLGA-P84-NPs when compared with IC50 of IMM-S. IMM-PLGA-P84-NPs showed a significantly low IC50 against P-gp overexpressing cell lines when compared with IC50 of IMM-PLGA-NPs. In contrary, IMM-PLGA-NPs showed lower IC50 against non P-gp expressing cell line. This study demonstrated the feasibility of targeting surface decorated NPs to multidrug resistant gliomas. However, to address its clinical utility extensive in vivo studies are required.

Fertility reduced by immunization with p84: A human sperm-coating antigen in the mouse.

Background : p84 is an 84-kDa protein with the ABO (H) antigenic epitope, and it is also a member of the family of sperm-coating antigens. In order to investigate whether the antibodies against p84 reduce fertility in mice, p84 and lactotransferrin (Lf), whose N-terminal sequence is similar to that of p84, were injected into female mice. Results : When p84 was used as the immunogen, the fertilization ratio was 37.5% lower than that of female mice immunized with no antigen (control group; P < 0.01). When Lf was used as the immunogen, there was no significant difference in the fertilization ratio between the Lf and control groups. Sera from female mice immunized with these antigens showed no agglutination against human erythrocytes, suggesting that the ABO(H) determinant site on p84 is not the factor reducing fertility. Although the titer of antibody (IgG) against p84 in serum from female mice immunized with p84 was generally higher than that against Lf, the titer was not related to the reduction of fertility. Conclusion : We conclude that an injection of p84 can reduce the fertility of female mice by inducing antibodies against p84. Our results suggest that the antibodies may act to inhibit the sperm-egg interaction, and that the binding site is the carbohydrate chain of p84, excluding the ABO (H) antigenic epitope, rather than the p84 protein itself. (Reprod Med Biol 2002; 1: 35-39).

Chelating polymer modified P84 nanofiltration (NF) hollow fiber membranes for high efficient heavy metal removal.

High performance nanofiltration (NF) membranes for heavy metal removal have been molecularly designed by adsorption of chelating polymers containing negatively charged functional groups such as poly (acrylic acid-co-maleic acid) (PAM), poly (acrylic acid) (PAA) and poly (dimethylamine-co-epichlorohydrin-co-ethylenediamine) (PDMED) on the positively charged polyethyleneimine (PEI) cross-linked P84 hollow fiber substrates. Not only do these chelating polymers change the membrane surface charge and pore size, but also provide an extra mean to remove heavy metal ions through adsorption in addition to traditional steric effect and Donnan exclusion. The adsorbed membranes have comparable water permeability and superior rejections to heavy metals, for instance, Pb(NO3)2, CuSO4, NiCl2, CdCl2, ZnCl2, Na2Cr2O7 and Na2HAsO4, with rejections higher than 98%. The membranes also display excellent rejections to mixed ions with rejections more than 99%. The newly developed membranes show reasonably stability during 60-h tests as well as multiple washes.