Development of a new culture medium and efficient protocol for in vitro micropropagation of Ceratonia siliqua L.

A new basal culture medium was developed and tested using a rapid and efficient protocol of in vitro axillary shoot bud proliferation of Ceratonia siliqua L., an important Mediterranean Fabaceae plant species. In a first experiment, the new formulated ‘LA’ mineral composition significantly improved shoot growth and proliferation as compared with Murashige and Skoog medium (MS, 1962) in both solid and liquid culture media. However, the liquid culture system proved to be the most suitable for shoot induction, shoot length (about fourfold higher), and multiplication rate (about two-fold higher), the difference being significant. The measured growth and proliferation parameters were further improved when LA mineral composition was optimized, in a second experiment. The highest multiplication rate (6.3) was achieved during the second subculture using the optimized ‘LAC’ medium. Noticeably, hyperhydricity and shoot-tip necrosis symptoms were absent in both formulated LA and LAC compositions when using the liquid culture system. In vitro rooting in solid medium showed 41.7 to 46.3% response on a solid medium which was more suitable than the liquid culture system, the difference being significant. In contrast, pretreated microcuttings with 3 μM IBA (indole-3-butyric acid) were successfully rooted ex vitro, showing significantly higher response (91.7%), average root number (8.3), and root length (31.5 mm). The plantlets were successfully acclimatized showing more than 90% survivability and normal morphology. The present study is a first cost-effective protocol for carob micropropagation combining the use of the newly formulated LAC basal medium, a liquid culture system, and ex vitro rooting.

     

Angiogenic Activity of Breast Cancer Patients’ Monocytes Reverted by Combined Use of Systems Modeling and Experimental Approaches

Angiogenesis plays a key role in tumor growth and cancer progression. TIE-2-expressing monocytes (TEM) have been reported to critically account for tumor vascularization and growth in mouse tumor experimental models, but the molecular basis of their pro-angiogenic activity are largely unknown. Moreover, differences in the pro-angiogenic activity between blood circulating and tumor infiltrated TEM in human patients has not been established to date, hindering the identification of specific targets for therapeutic intervention. In this work, we investigated these differences and the phenotypic reversal of breast tumor pro-angiogenic TEM to a weak pro-angiogenic phenotype by combining Boolean modelling and experimental approaches. Firstly, we show that in breast cancer patients the pro-angiogenic activity of TEM increased drastically from blood to tumor, suggesting that the tumor microenvironment shapes the highly pro-angiogenic phenotype of TEM. Secondly, we predicted in silico all minimal perturbations transitioning the highly pro-angiogenic phenotype of tumor TEM to the weak pro-angiogenic phenotype of blood TEM and vice versa. In silico predicted perturbations were validated experimentally using patient TEM. In addition, gene expression profiling of TEM transitioned to a weak pro-angiogenic phenotype confirmed that TEM are plastic cells and can be reverted to immunological potent monocytes. Finally, the relapse-free survival analysis showed a statistically significant difference between patients with tumors with high and low expression values for genes encoding transitioning proteins detected in silico and validated on patient TEM. In conclusion, the inferred TEM regulatory network accurately captured experimental TEM behavior and highlighted crosstalk between specific angiogenic and inflammatory signaling pathways of outstanding importance to control their pro-angiogenic activity. Results showed the successful in vitro reversion of such an activity by perturbation of in silico predicted target genes in tumor derived TEM, and indicated that targeting tumor TEM plasticity may constitute a novel valid therapeutic strategy in breast cancer.     

Core protein machinery for mammalian phosphatidylinositol 3,5-bisphosphate synthesis and turnover that regulates the progression of endosomal transport. Novel Sac phosphatase joins the ArPIKfyve-PIKfyve complex

Perturbations in phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2)-synthesizing enzymes result in enlarged endocytic organelles from yeast to humans, indicating evolutionarily conserved function of PtdIns(3,5)P2 in endosome-related events. This is reinforced by the structural and functional homology of yeast Vac14 and human Vac14 (ArPIKfyve), which activate yeast and mammalian PtdIns(3,5)P2-producing enzymes, Fab1 and PIKfyve, respectively. In yeast, PtdIns(3,5)P2-specific phosphatase, Fig4, in association with Vac14, turns over PtdIns(3,5)P2, but whether such a mechanism operates in mammalian cells and what the identity of mammalian Fig4 may be are unknown. Here we have identified and characterized Sac3, a Sac domain phosphatase, as the Fig4 mammalian counterpart. Endogenous Sac3, a widespread 97-kDa protein, formed a stable ternary complex with ArPIKfyve and PIKfyve. Concordantly, Sac3 cofractionated and colocalized with ArPIKfyve and PIKfyve. The intrinsic Sac3(WT) phosphatase activity preferably hydrolyzed PtdIns(3,5)P2 in vitro, although the other D5-phosphorylated polyphosphoinositides were also substrates. Ablation of endogenous Sac3 by short interfering RNAs elevated PtdIns(3,5)P2 in (32)P-labeled HEK293 cells. Ectopically expressed Sac3(WT) in COS cells colocalized with and dilated EEA1-positive endosomes, consistent with the PtdIns(3,5)P2 requirement in early endosome dynamics. In vitro reconstitution of carrier vesicle formation from donor early endosomes revealed a gain of function upon Sac3 loss, whereas PIKfyve or ArPIKfyve protein depletion produced a loss of function. These data demonstrate a coupling between the machinery for PtdIns(3,5)P2 synthesis and turnover achieved through a physical assembly of PIKfyve, ArPIKfyve, and Sac3. We suggest that the tight regulation in PtdIns(3,5)P2 homeostasis is mechanistically linked to early endosome dynamics in the course of cargo transport.     

Kinesin Adapter JLP Links PIKfyve to Microtubule-based Endosome-to-Trans-Golgi Network Traffic of Furin

JIPs (c-Jun N-terminal kinase interacting proteins), which scaffold JNK/p38 MAP kinase signaling modules, also bind conventional kinesins and are implicated in microtubule-based membrane trafficking in neuronal cells. Here we have identified a novel splice variant of the Jip4 gene product JLP_L (JNK-interacting leucine zipper protein) in yeast-two hybrid screens with the phosphoinositide kinase PIKfyve. The interaction was confirmed by pulldown and coimmunoprecipitation assays in native cells. It engages the PIKfyve cpn60_TCP1 consensus sequence and the last 75 residues of the JLP C terminus. Subpopulations of both proteins cofractionated and populated similar structures at the cell perinuclear region. Because PIKfyve is essential in endosome-to-trans-Golgi network (TGN) cargo transport, we tested whether JLP is a PIKfyve functional partner in this trafficking pathway. Short interfering RNA (siRNA)-mediated depletion of endogenous JLP or PIKfyve profoundly delayed the microtubule-based transport of chimeric furin (Tac-furin) from endosomes to the TGN in a CHO cell line, which was rescued upon ectopic expression of siRNA-resistant JLP or PIKfyve constructs. Peptides from the contact sites in PIKfyve and JLP, or a dominant-negative PIKfyve mutant introduced into cells by ectopic expression or microinjection, induced a similar defect. Because Tac-TGN38 delivery from endosomes to the TGN, unlike that of Tac-furin, does not require intact microtubules, we monitored the effect of JLP and PIKfyve depletion or the interacting peptides administration on Tac-TGN38 trafficking. Remarkably, neither maneuver altered the Tac-TGN38 delivery to the TGN. Our data indicate that JLP interacts with PIKfyve and that both proteins and their association are required in microtubule-based, but not in microtubule-independent, endosome-to-TGN cargo transport.In mammalian cells, the endosomal/endocytic system comprises an interconnected and morphologically complex network of membrane organelles that supports fundamental functions such as nutrient entry and delivery for degradation, removal and degradation of plasma membrane or Golgi proteins, regulation and integration of signaling pathways, and protein recycling to the cell surface or the TGN² (1–4). From the plasma membrane, the endocytosed cargo is first delivered to early endosomes/sorting endosomes. Cargoes destined for recycling to the cell surface then enter the endocytic recycling compartment, whereas others, intended for degradation, remain in early endosomes. Early endosomes undergo a series of changes, known as maturation, to give rise to maturing transport intermediates (herein ECV/MVBs; also Ref. 5) and to late endosomes that fuse with lysosomes to deliver cargo for degradation. Recycling or degradation is not the only outcome of the cell surface-originated cargoes. A set of internalized transmembrane proteins, including intracellular sorting receptors, enzymes, and toxins, are retrieved from the endosomal system and transported to the TGN. The endosome-to-TGN trafficking of the acid-hydrolase-sorting receptor, CI-MPR, the endopeptidase furin, and the putative cargo receptor TGN38 are the best studied examples. These cargoes are highly enriched in the TGN at steady state but arrive there from different compartments, utilizing distinct mechanisms. Thus, TGN38 enters the TGN from the endocytic recycling compartment by an iterative removal from the latter compartment, furin reaches the TGN by exiting the early/late endosomal system, and CI-MPR implements features of both pathways (4, 6–9).Whereas the detailed molecular and cellular mechanisms underlying the membrane progression in the course of cargo transport through the endosomal system or retrieval from early/late endosomes to the TGN is still elusive, experimental evidence has been accumulating to implicate PIKfyve, the sole enzyme for PtdIns(3,5)P₂ synthesis (10). Thus, PIKfyve has been found to interact with the late endosome-to-TGN transport factor Rab9 effector p40 (11). Furthermore, disruption of the PtdIns(3,5)P₂ homeostatic mechanism by means of expression of dominant-negative kinase-deficient point mutants of PIKfyve, protein depletion, or pharmacological inhibition of PIKfyve activity was found to impair the exit of a subset of cargoes from early endosomes to the TGN and late endosomes or from the late endosomes (12–16). Phenotypically, these defects are manifested by progressive endosome swelling and cytoplasmic vacuolation, first seen by expression of dominant-negative PIKfyve^K1831E in a number of mammalian cell types (17) and confirmed thereafter by other maneuvers inhibiting PIKfyve protein expression or activity (14, 16). In vitro reconstitution assays indicate that PIKfyve enzymatic activity is required in endosome processing in two ways. It triggers the formation/fission (or maturation) of ECV/MVBs from early endosomes and arrests the rate of fusion events in the endosomal system (18, 19). It is thus conceivable that impaired PIKfyve and PtdIns(3,5)P₂ functioning in the fission and fusion events mechanistically underlies the constraints in the trafficking pathways traversing endosomes.Microtubules aided by the microtubule-associated motor protein families of kinesin and dynein are required for proper performance of the endosomal/endocytic membrane system. Although their role is rather complex and not completely understood, in vivo and in vitro studies implicate microtubule-based dynamics in multiple aspects of the endocytic trafficking, including sorting of endocytic contents, fission/fusion events at early or late endosomes, early endosome maturation, and efficient motility of the transport vesicles to their destination (20–27). Accumulating evidence indicates that the binding of motor proteins to organelles or carrier vesicles is regulated by motor protein adapters. Intriguingly, this newly emerging adapter function has been found to be executed by proteins known as scaffolds of stress signaling enzymes. One such adapter for conventional kinesins is the group of JIPs that scaffold the JNK/p38 MAP kinase signaling modules (28–31). A mutation that causes mislocalization of synaptic vesicles and aberrant axonal transport in Drosophila and Caenorhabditis elegans affects the JIP3 homologs Sunday driver (dSYD) and Unc16, respectively (32, 33). In mammalian cells, JIPs are represented by four proteins (JIP1–4) derived from separate genes and several alternatively spliced variants. JIP1, the founding member, is structurally related to JIP2 (34, 35). JIP3 (also known as Unc16/JSAP1/dSYD) is structurally unrelated to JIP1 or JIP2, but as those two, it is abundant in neuronal cells (30, 32, 36). The latest addition to the group is JIP4 that occurs in three splice variants known thus far: JLP and JIP4 in mouse and SPAG9 in humans (31, 37, 38). JIP4, JLP, and SPAG9 (gene symbol, SPAG9) are structurally homologous to JIP3 but display broader distribution (37–39). Remarkably, all four members of the JIP group interact with the kinesin1 light chain, and potential cargoes for microtubule-based vesicle transport have been proposed for JIP1–JIP3 (32, 33, 38, 40–43). The role of JLP/JIP4 in the context of cargo transport or membrane trafficking events, however, has never been investigated. In the present study we report that JLP is a PIKfyve physical and functional partner in microtubule-based endosome-to-TGN trafficking. The interaction is identified by a yeast two-hybrid screen with the PIKfyve cpn60_TCP1 consensus sequence and mapped to the 75-aa peptide fragment of the extreme JLP C terminus. By monitoring divergent routes of cargo delivery to the TGN, differing by the requirement of microtubule-dependent early endosome maturation, we have determined that JLP assists PIKfyve selective functionality in microtubule-based endosome-to-TGN trafficking.     

PIKfyve-ArPIKfyve-Sac3 Core Complex: CONTACT SITES AND THEIR CONSEQUENCE FOR Sac3 PHOSPHATASE ACTIVITY AND ENDOCYTIC MEMBRANE HOMEOSTASIS*

The phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P₂) metabolizing enzymes, the kinase PIKfyve and the phosphatase Sac3, constitute a single multiprotein complex organized by the PIKfyve regulator ArPIKfyve and its ability to homodimerize. We previously established that PIKfyve is activated within the triple PIKfyve-ArPIKfyve-Sac3 (PAS) core. These data assign an atypical function for the phosphatase in PtdIns(3,5)P₂ biosynthesis, thus raising the question of whether Sac3 retains its PtdIns(3,5)P₂ hydrolyzing activity within the PAS complex. Herein, we address the issue of Sac3 functionality by a combination of biochemical and morphological assays in triple-transfected COS cells using a battery of truncated or point mutants of the three proteins. We identified the Cpn60_TCP1 domain of PIKfyve as a major determinant for associating the ArPIKfyve-Sac3 subcomplex. Neither Sac3 nor PIKfyve enzymatic activities affected the PAS complex formation or stability. Using the well established formation of aberrant cell vacuoles as a sensitive functional measure of localized PtdIns(3,5)P₂ reduction, we observed a mitigated vacuolar phenotype by kinase-deficient PIKfyve^K1831E if its ArPIKfyve-Sac3 binding region was deleted, suggesting reduced Sac3 access to, and turnover of PtdIns(3,5)P₂. In contrast, PIKfyve^K1831E, which displays intact ArPIKfyve-Sac3 binding, triggered a more severe vacuolar phenotype if coexpressed with ArPIKfyve^WT-Sac3^WT but minimal defects when coexpressed with ArPIKfyve^WT and phosphatase-deficient Sac3^D488A. These data indicate that Sac3 assembled in the PAS regulatory core complex is an active PtdIns(3,5)P₂ phosphatase. Based on these and other data, presented herein, we propose a model of domain interactions within the PAS core and their role in regulating the enzymatic activities.     

Oncolytic parvoviruses as cancer therapeutics

The experimental infectivity and excellent tolerance of some rodent autonomous parvoviruses in humans, together with their oncosuppressive effects in preclinical models, speak for the inclusion of these agents in the arsenal of oncolytic viruses under consideration for cancer therapy. In particular, wild-type parvovirus H-1PV can achieve a complete cure of various tumors in animal models and kill tumor cells that resist conventional anticancer treatments. There is growing evidence that H-1PV oncosuppression involves an immune component in addition to the direct viral oncolytic effect. This article summarizes the recent assessment of H-1PV antineoplastic activity in glioma, pancreatic ductal adenocarcinoma, and non-Hodgkin lymphoma models, laying the foundation for the present launch of a first phase I/IIa clinical trial on glioma patients.     

Prediction of physical performance through muscle enzymes activity

Physical performance deteriorates during strenuous exercise as manifested by a decrease in maximal aerobic power and increased activity of serum muscle enzymes. The relationship between these parameters was investigated in 41 trained subjects during 24 h marches and the following recovery period. Peak O2 uptake and serum activity of creatine phosphokinase (CPK) and glutamic oxalacetic transaminase (GOT) were measured. During the marches there was a simultaneous, significant elevation in serum CPK and GOT activity and a significant reduction in peak O2 uptake. During the early recovery period (24 h) no significant changes occurred in muscle enzyme activity and peak O2 uptake; thereafter (up to 72 h after the end of the march), a gradual decline in enzyme activity levels with a concomitant increase in peak O2 uptake was observed, reaching pre-march values. A "mirror image" relationship between muscle enzyme activity and peak O2 uptake was found during three clearly distinguished phases: a) 24 h march, b) early recovery stage and c) late recovery stage. These findings suggest that muscle enzyme leakage from muscle cells is closely related to the decline in muscular function and aerobic power. Thus, muscle enzyme activity might be a practical measure of physical performance capacity during the early and late stages of recovery from prolonged endurance exercise.     

Plasma-cortisol levels in experimental heatstroke in dogs

The effect of external heat-load, exercise and dehydration on dynamic changes in plasma cortisol during the development of heatstroke was investigated. Thirty-three unanesthetized dogs were tested under two sets of climatic conditions: comfort conditions and hot-dry climatic conditions, half of them while exercising. Half of the dogs in each group were rehydrated. None of the dogs that were investigated at room temperature suffered heatstroke. Of the dogs exposed to high ambient temperature, all of the exercising, as well as five out of six non-hydrated dogs and one rehydrated non-exercising dog suffered heatstroke. Significant dehydration (6%–7% of body weight), occurred only under hgh ambient temperature. Plasma cortisol levels of all dogs that suffered heatstroke rose conspicuously for at least 5 h and returned to normal levels 24 h later. Cortisol levels of dogs who did not experience heatstroke remained within the normal range. Cortisol levels correlated with the severity of the stress leading to heatstroke. High and rising levels of cortisol, several hours after body temperature returns to normal, may support the diagnosis of heatstroke.     

ArPIKfyve homomeric and heteromeric interactions scaffold PIKfyve and Sac3 in a complex to promote PIKfyve activity and functionality

PtdIns(3,5)P(2) (with PtdIns indicating phosphatidylinositol) is vital in the differentiation and development of multicellular organisms because knockout of the PtdIns(3,5)P(2)-synthesizing enzyme PIKfyve (phosphoinositide kinase for position 5 containing a FYVE finger domain) or its associated regulator ArPIKfyve is lethal. In previous work with endogenous proteins, we identified that Sac3, a phosphatase that turns over PtdIns(3,5)P(2), associates with the PIKfyve-ArPIKfyve biosynthetic complex. However, whether the three proteins suffice for the organization/maintenance of this complex [referred to as the PAS (PIKfyve-ArPIKfyve-Sac3) complex], how they interact with one another, and what the functional relevance of this ternary association would be remained unresolved. Using co-immunoprecipitation analyses in transfected mammalian cells with increased or decreased levels of the three proteins, singly or in double versus triple combinations, herein we report that the triad is sufficient to form and maintain the PAS complex. ArPIKfyve is the principal organizer interacting with both Sac3 and PIKfyve, whereas Sac3 is permissive for maximal PIKfyve-ArPIKfyve association in the PAS complex. We further identified that ArPIKfyve scaffolds the PAS complex through homomeric interactions, mediated via its conserved C-terminal domain. Introduction of the C-terminal peptide fragment of the ArPIKfyve-ArPIKfyve contact sites effectively disassembled the PAS complex and reduced the in vitro PIKfyve lipid kinase activity. Exploring insulin-regulated GLUT4 translocation in 3T3L1 adipocytes as a functional readout, a process that is positively regulated by PIKfyve activity and ArPIKfyve levels, we determined that ectopic expression of the ArPIKfyve C-terminal peptide inhibits GLUT4 surface accumulation. Our data indicate that the PAS complex is organized to provide optimal PIKfyve functionality and is maintained via ArPIKfyve homomeric and heteromeric interactions.     

Phosphoinositides in insulin action on GLUT4 dynamics: not just PtdIns(3,4,5)P3

Accumulated evidence over the last several years indicates that insulin regulates multiple steps in the overall translocation of GLUT4 vesicles to the fat/muscle cell surface, including formation of an intracellular storage pool of GLUT4 vesicles, its movement to the proximity of the cell surface, and the subsequent docking/fusion with the plasma membrane. Insulin-stimulated formation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3); and in some cases, of its catabolite PtdIns(3,4)P(2)] plays a pivotal role in this process. PtdIns(3,4,5)P(3) is synthesized by the activated wortmannin-sensitive class IA phosphoinositide (PI) 3-kinase and controls the rate-limiting cell surface terminal stages of the GLUT4 journey. However, recent research is consistent with the conclusion that signals by each of the remaining five PIs, i.e., PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,5)P(2), and PtdIns(4,5)P(2), may act in concert with that of PtdIns(3,4,5)P(3) in integrating the insulin receptor-issued signals with GLUT4 surface translocation and glucose transport activation. This review summarizes the experimental evidence supporting the complementary function of these PIs in insulin responsiveness of fat and muscle cells, with particular reference to mechanistic insights and functional significance in the regulation of overall GLUT4 vesicle dynamics.