Heterologous expression of a plastid EF-Tu reduces protein thermal aggregation and enhances CO\u3csub\u3e2\u3c/sub\u3e fixation in wheat (\u3ci\u3eTriticum aestivum\u3c/i\u3e) following heat stress

Heat stress is a major constraint to wheat production and negatively impacts grain quality, causing tremendous economic losses, and may become a more troublesome factor due to global warming. At the cellular level, heat stress causes denaturation and aggregation of proteins and injury to membranes leading to alterations in metabolic fluxes. Protein aggregation is irreversible, and protection of proteins from thermal aggregation is a strategy a cell uses to tolerate heat stress. Here we report on the development of transgenic wheat (Triticum aestivum) events, expressing a maize gene coding for plastidal protein synthesis elongation factor (EF-Tu), which, compared to non-transgenic plants, display reduced thermal aggregation of leaf proteins, reduced heat injury to photosynthetic membranes (thylakoids), and enhanced rate of CO2 fixation after exposure to heat stress. The results support the concept that EF-Tu ameliorates negative effects of heat stress by acting as a molecular chaperone. This is the first demonstration of the introduction of a plastidal EF-Tu in plants that leads to protection against heat injury and enhanced photosynthesis after heat stress. This is also the first demonstration that a gene other than HSP gene can be used for improvement of heat tolerance and that the improvement is possible in a species that has a complex genome, hexaploid wheat. The results strongly suggest that heat tolerance of wheat, and possibly other crop plants, can be improved by modulating expression of plastidal EF-Tu and/or by selection of genotypes with increased endogenous levels of this protein

POLITICHE DI AMMISSIONE E GESTIONE DEI FLUSSI MIGRATORI DA LAVORO IN ITALIA DALLA TURCO-NAPOLITANO AD OGGI.

L'obiettivo di questo elaborato è quello di analizzare gli strumenti di cui si è dotata l'Italia al fine di regolare i flussi immigratori da lavoro. Dall'analisi svolta emerge la difficoltà dei governi italiani ad abbandonare una gestione emergenziale e contingente del fenomeno in favore di una maggiore consapevolezza di fronte all'immigrazione, in generale, e quella economica in particolare, in quanto elemento strutturale del tessuto socio-economico italiano. Le difficoltà oggettive, incontrate nella stesura dell'elaborato, sono frutto della complessità del fenomeno di per sé di natura transnazionale e che coinvolge una moltitudine di soggetti, oltre a presentare molteplici aspetti, da quelli socio-economici a quelli prettamente politici, fortemente intrecciati tra loro. Data la complessità di questo versante della politica migratoria, ho scelto una chiave di lettura storica, in quanto permette di evidenziare il profondo legame tra la discussione politica, caratterizzata da una forte polarizzazione ideologica, e gli interventi attuati in questa materia. All'interno di questi equilibri s'inserisce, inoltre, il crescente ruolo dell'Unione Europea che chiede, nonostante le resistenze dei singoli Stati, di rinunciare a una porzione di sovranità col fine di creare una politica migratoria comune. Nel primo capitolo vengono analizzati l'origine del fenomeno e la nascita della politica migratoria in Italia. Con l'approvazione della legge Turco-Napolitano nel 1998, e il conseguente raggruppamento delle leggi all'interno del Testo Unico, l'Italia cerca di dotarsi di una legge organica in materia migratoria. Il sistema è incentrato sulla chiamata nominativa del lavoratore ancora all'estero, che avviene secondo i limiti numerici definiti dalla programmazione dei flussi ed attuati attraverso il cosiddetto decreto-flussi. Questo meccanismo dimostra, fin dalla nascita, notevoli inadeguatezze e carenze, per cui si deve ricorrere sistematicamente a strumenti di regolarizzazione ex post di una presenza irregolare che tende a riprodursi, anche a causa del lavoro sommerso che funge da potente fattore d'attrazione dei flussi, e tutto questo perché, sia i datori di lavoro che i lavoratori immigrati, non trovano conveniente utilizzare i canali d'ingresso regolari. Nel secondo capitolo il punto di partenza è rappresentato dalla riforma attuata dalla legge Bossi-Fini nel 2001. Tale legge è frutto della nuova maggioranza di centro-destra che fa dell'immigrazione un tema cruciale per raccogliere consensi, associandola a situazioni problematiche di sicurezza e ordine pubblico. La legge interviene in maniera sostanziale sui meccanismi di ammissione, inasprendo le condizioni di ingresso con l'introduzione del “contratto di soggiorno” (che subordina la presenza alla disponibilità di un'occupazione lavorativa) e precarizzando il soggiorno dei regolari, dimezzando la durata dei permessi. Ai fattori interni si aggiunge l'influenza dell'Unione Europea. L'allargamento verso i paesi dell'Est rappresenta l'apice delle carenze del meccanismo di ammissione: una larga parte degli ingressi si sottrae al potere regolatorio del sistema delle quote, dal momento che buona parte dei flussi si compone di soggetti che non necessitano più di alcun permesso di soggiorno. La medesima incapacità emerge anche con il presentarsi della crisi economica alla fine del 2008. Infatti, nonostante la drastica riduzione delle quote, i flussi, prevalentemente diretti al servizio alle famiglie, si mantengono elevati e subiscono una battuta d'arresto solo a partire dal 2011. Nel terzo capitolo vengono presi in considerazione i provvedimenti più recenti: l'Accordo d'Integrazione all'interno del Piano per l'Integrazione e la Carta Blu sono frutto del crescente ruolo dell'Unione Europea e dell'affermarsi del nuovo (e più impegnativo) concetto d'integrazione e, pertanto, rappresentano il tentativo dell'azione politica di adeguarsi ai principi della politica migratoria dell'Unione Europea. Tali provvedimenti, tuttavia, hanno suscitato perplessità e critiche, sia in termini ideologici che in termini di attuabilità concreta, data la mancata predisposizione di risorse aggiuntive necessarie. Il quadro che emerge è che l'Italia ha attratto ingenti flussi senza che a questi corrispondesse una crescita economica, e questo è frutto del potere d'attrazione costituito dai fattori socio-demografici, come la bassa natalità, l'invecchiamento della popolazione, l'evoluzione delle aspettative lavorative, soprattutto dei giovani. In conclusione i problemi rimangono aperti, anche se oggi meno visibili principalmente per due fattori. Il primo consiste nella mancanza di un dibattito politico e mediatico, nonché scientifico, causato dalla poca notiziabilità dell'immigrazione regolare, in favore degli aspetti più eclatanti e conflittuali. Il secondo invece è rappresentato dalla battuta d'arresto dei flussi di arrivo, che fa apparire il problema lontano. E così l'azione politica aspetta, finché non si presenterà la prossima emergenza

Kinome rewiring reveals AURKA limits PI3K-pathway inhibitor efficacy in breast cancer.

Dysregulation of the PI3K-AKT-mTOR signaling network is a prominent feature of breast cancers. However, clinical responses to drugs targeting this pathway have been modest, possibly because of dynamic changes in cellular signaling that drive resistance and limit drug efficacy. Using a quantitative chemoproteomics approach, we mapped kinome dynamics in response to inhibitors of this pathway and identified signaling changes that correlate with drug sensitivity. Maintenance of AURKA after drug treatment was associated with resistance in breast cancer models. Incomplete inhibition of AURKA was a common source of therapy failure, and combinations of PI3K, AKT or mTOR inhibitors with the AURKA inhibitor MLN8237 were highly synergistic and durably suppressed mTOR signaling, resulting in apoptosis and tumor regression in vivo. This signaling map identifies survival factors whose presence limits the efficacy of targeted therapies and reveals new drug combinations that may unlock the full potential of PI3K-AKT-mTOR pathway inhibitors in breast cancer

Ca2+/Calmodulin-Dependent Protein Kinase Kinase Is Not Involved in Hypothalamic AMP-Activated Protein Kinase Activation by Neuroglucopenia

Hypoglycemia and neuroglucopenia stimulate AMP-activated protein kinase (AMPK) activity in the hypothalamus and this plays an important role in the counterregulatory responses, i.e. increased food intake and secretion of glucagon, corticosterone and catecholamines. Several upstream kinases that activate AMPK have been identified including Ca2+/Calmodulin-dependent protein kinase kinase (CaMKK), which is highly expressed in neurons. However, the involvement of CaMKK in neuroglucopenia-induced activation of AMPK in the hypothalamus has not been tested. To determine whether neuroglucopenia-induced AMPK activation is mediated by CaMKK, we tested whether STO-609 (STO), a CaMKK inhibitor, would block the effects of 2-deoxy-D-glucose (2DG)-induced neuroglucopenia both ex vivo on brain sections and in vivo. Preincubation of rat brain sections with STO blocked KCl-induced α1 and α2-AMPK activation but did not affect AMPK activation by 2DG in the medio-basal hypothalamus. To confirm these findings in vivo, STO was pre-administrated intracerebroventricularly (ICV) in rats 30 min before 2DG ICV injection (40 µmol) to induce neuroglucopenia. 2DG-induced neuroglucopenia lead to a significant increase in glycemia and food intake compared to saline-injected control rats. ICV pre-administration of STO (5, 20 or 50 nmol) did not affect 2DG-induced hyperglycemia and food intake. Importantly, activation of hypothalamic α1 and α2-AMPK by 2DG was not affected by ICV pre-administration of STO. In conclusion, activation of hypothalamic AMPK by 2DG-induced neuroglucopenia is not mediated by CaMKK

Repair at Single Targeted DNA Double-Strand Breaks in Pluripotent and Differentiated Human Cells

Differences in ex vivo cell culture conditions can drastically affect stem cell physiology. We sought to establish an assay for measuring the effects of chemical, environmental, and genetic manipulations on the precision of repair at a single DNA double-strand break (DSB) in pluripotent and somatic human cells. DSBs in mammalian cells are primarily repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). For the most part, previous studies of DSB repair in human cells have utilized nonspecific clastogens like ionizing radiation, which are highly nonphysiologic, or assayed repair at randomly integrated reporters. Measuring repair after random integration is potentially confounded by locus-specific effects on the efficiency and precision of repair. We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs) based on the site of the DSB within the genome. To overcome locus-specific effects on DSB repair, we used zinc finger nucleases to efficiently target a DSB repair reporter to a safe-harbor locus in hESCs and a panel of somatic human cell lines. We demonstrate that repair at a targeted DSB is highly precise in hESCs, compared to either the somatic human cells or murine embryonic stem cells. Differentiation of hESCs harboring the targeted reporter into astrocytes reduces both the efficiency and precision of repair. Thus, the phenotype of repair at a single DSB can differ based on either the site of damage within the genome or the stage of cellular differentiation. Our approach to single DSB analysis has broad utility for defining the effects of genetic and environmental modifications on repair precision in pluripotent cells and their differentiated progeny

Radiation-Induced Bystander Effects in Cultured Human Stem Cells

The radiation-induced "bystander effect" (RIBE) was shown to occur in a number of experimental systems both in vitro and in vivo as a result of exposure to ionizing radiation (IR). RIBE manifests itself by intercellular communication from irradiated cells to non-irradiated cells which may cause DNA damage and eventual death in these bystander cells. It is known that human stem cells (hSC) are ultimately involved in numerous crucial biological processes such as embryologic development; maintenance of normal homeostasis; aging; and aging-related pathologies such as cancerogenesis and other diseases. However, very little is known about radiation-induced bystander effect in hSC. To mechanistically interrogate RIBE responses and to gain novel insights into RIBE specifically in hSC compartment, both medium transfer and cell co-culture bystander protocols were employed.Human bone-marrow mesenchymal stem cells (hMSC) and embryonic stem cells (hESC) were irradiated with doses 0.2 Gy, 2 Gy and 10 Gy of X-rays, allowed to recover either for 1 hr or 24 hr. Then conditioned medium was collected and transferred to non-irradiated hSC for time course studies. In addition, irradiated hMSC were labeled with a vital CMRA dye and co-cultured with non-irradiated bystander hMSC. The medium transfer data showed no evidence for RIBE either in hMSC and hESC by the criteria of induction of DNA damage and for apoptotic cell death compared to non-irradiated cells (p>0.05). A lack of robust RIBE was also demonstrated in hMSC co-cultured with irradiated cells (p>0.05).These data indicate that hSC might not be susceptible to damaging effects of RIBE signaling compared to differentiated adult human somatic cells as shown previously. This finding could have profound implications in a field of radiation biology/oncology, in evaluating radiation risk of IR exposures, and for the safety and efficacy of hSC regenerative-based therapies

The mutational impact of culturing human pluripotent and adult stem cells

Genetic changes acquired during in vitro culture pose a risk for the successful application of stem cells in regenerative medicine. To assess the genetic risks induced by culturing, we determined all mutations in individual human stem cells by whole genome sequencing. Individual pluripotent, intestinal, and liver stem cells accumulate 3.5 ± 0.5, 7.2 ± 1.1 and 8.3 ± 3.6 base substitutions per population doubling, respectively. The annual in vitro mutation accumulation rate of adult stem cells is nearly 40-fold higher than the in vivo mutation accumulation rate. Mutational signature analysis reveals that in vitro induced mutations are caused by oxidative stress. Reducing oxygen tension in culture lowers the mutational load. We use the mutation rates, spectra, and genomic distribution to model the accumulation of oncogenic mutations during typical in vitro expansion, manipulation or screening experiments using human stem cells. Our study provides empirically defined parameters to assess the mutational risk of stem cell based therapies

Prostaglandin I2 Signaling Drives Th17 Differentiation and Exacerbates Experimental Autoimmune Encephalomyelitis

BACKGROUND: Prostaglandin I(2) (PGI(2)), a lipid mediator currently used in treatment of human disease, is a critical regulator of adaptive immune responses. Although PGI(2) signaling suppressed Th1 and Th2 immune responses, the role of PGI(2) in Th17 differentiation is not known. METHODOLOGY/PRINCIPAL FINDINGS: In mouse CD4(+)CD62L(+) naïve T cell culture, the PGI(2) analogs iloprost and cicaprost increased IL-17A and IL-22 protein production and Th17 differentiation in vitro. This effect was augmented by IL-23 and was dependent on PGI(2) receptor IP signaling. In mouse bone marrow-derived CD11c(+) dendritic cells (BMDCs), PGI(2) analogs increased the ratio of IL-23/IL-12, which is correlated with increased ability of BMDCs to stimulate naïve T cells for IL-17A production. Moreover, IP knockout mice had delayed onset of a Th17-associated neurological disease, experimental autoimmune encephalomyelitis (EAE), and reduced infiltration of IL-17A-expressing mononuclear cells in the spinal cords compared to wild type mice. These results suggest that PGI(2) promotes in vivo Th17 responses. CONCLUSION: The preferential stimulation of Th17 differentiation by IP signaling may have important clinical implications as PGI(2) and its analogs are commonly used to treat human pulmonary hypertension

A Kinome RNAi Screen Identified AMPK as Promoting Poxvirus Entry through the Control of Actin Dynamics

Poxviruses include medically important human pathogens, yet little is known about the specific cellular factors essential for their replication. To identify genes essential for poxvirus infection, we used high-throughput RNA interference to screen the Drosophila kinome for factors required for vaccinia infection. We identified seven genes including the three subunits of AMPK as promoting vaccinia infection. AMPK not only facilitated infection in insect cells, but also in mammalian cells. Moreover, we found that AMPK is required for macropinocytosis, a major endocytic entry pathway for vaccinia. Furthermore, we show that AMPK contributes to other virus-independent actin-dependent processes including lamellipodia formation and wound healing, independent of the known AMPK activators LKB1 and CaMKK. Therefore, AMPK plays a highly conserved role in poxvirus infection and actin dynamics independent of its role as an energy regulator

AMP-Activated Kinase Restricts Rift Valley Fever Virus Infection by Inhibiting Fatty Acid Synthesis

The cell intrinsic innate immune responses provide a first line of defense against viral infection, and often function by targeting cellular pathways usurped by the virus during infection. In particular, many viruses manipulate cellular lipids to form complex structures required for viral replication, many of which are dependent on de novo fatty acid synthesis. We found that the energy regulator AMPK, which potently inhibits fatty acid synthesis, restricts infection of the Bunyavirus, Rift Valley Fever Virus (RVFV), an important re-emerging arthropod-borne human pathogen for which there are no effective vaccines or therapeutics. We show restriction of RVFV both by AMPK and its upstream activator LKB1, indicating an antiviral role for this signaling pathway. Furthermore, we found that AMPK is activated during RVFV infection, leading to the phosphorylation and inhibition of acetyl-CoA carboxylase, the first rate-limiting enzyme in fatty acid synthesis. Activating AMPK pharmacologically both restricted infection and reduced lipid levels. This restriction could be bypassed by treatment with the fatty acid palmitate, demonstrating that AMPK restricts RVFV infection through its inhibition of fatty acid biosynthesis. Lastly, we found that this pathway plays a broad role in antiviral defense since additional viruses from disparate families were also restricted by AMPK and LKB1. Therefore, AMPK is an important component of the cell intrinsic immune response that restricts infection through a novel mechanism involving the inhibition of fatty acid metabolism