Search

Fomitiporia mediterranea M. Fisch. (Fmed) is a basidiomycete first described in 2002, and was considered up to then as part of Fomitiporia punctata (P. Karst) Murrill. This fungus can degrade lignocellulosic biomass, causing white rot and leaving bleached fibrous host residues. In Europe Fmed is considered the main grapevine wood rot (Esca) agent within the Esca disease complex, which includes some of the most economically important Grapevine Trunk Diseases (GTDs). This review summarises and evaluates published research on Fmed, on white rot elimination by curettage or management by treatments with specific products applied to diseased grapevines, and on the relationship between wood symptoms and Grapevine Leaf Stripe Disease (GLSD) in the Esca disease complex. Information is also reviewed on the fungus biology, mechanisms of pathogenicity, and their possible relationships with external foliar symptoms of the Esca disease complex. Information on Fmed control strategies is also reviewed.

Downy mildew caused by Plasmopara viticola is probably the most serious disease affecting grapevine (Vitis vinifera), and it is capable of causing consistent yield losses. In organic viticulture, the only acceptable and effective means to control the disease is by applications of copper-based fungicides. However, the use of copper in agriculture is expected to be further restricted by European countries because of its critical ecotoxicological and phytotoxicological profile. Research on ways to reduce the effective amounts of copper by developing innovative formulations as well as optimization of the distribution and persistence of copper-based pesticides for downy mildew control seems to be a promising approach. This research investigated the delivery properties of biomimetic synthetic hydroxyapatite (HA) to enhance the biological activity of Cu(II) ions. To this aim, four Cu(II) compounds were formulated with the innovative HA component and applied in an in vitro antifungal assay against Botrytis cinerea, a common grapevine pathogen suitable for in vitro activity tests, and finally, in in planta efficacy assays against P. viticola under greenhouse conditions. The in vitro results highlighted a different inhibition activity for each Cu(II) compound and indicated a different interaction between the cupric compounds and HA, potentially related to different delivery mechanisms of Cu(II) from HA. Under greenhouse conditions, additional findings on the biological activity of the applied formulations were gained, especially on the efficacy of various concentrations of HA in the formulations, the influence of dose variation of the formulation and the treatment efficiency, and the persistence under rain-washing effect. This study revealed promising findings on the formulation based on the HA particles and the soluble Cu(II) compound, which resulted in reduced disease severity and incidence in all of the experimental conditions, including the lower Cu(II) dosage and the rain-washing effect. This suggests that coformulation of the three insoluble Cu(II) compounds with HA might significantly enhance the adsorption and release of Cu(II) ions by HA particles.

Retinal neurodegeneration can impair visual perception at different levels, involving not only photoreceptors, which are the most metabolically active cells, but also the inner retina. Compensatory mechanisms may hide the first signs of these impairments and reduce the likelihood of receiving timely treatments. Therefore, it is essential to characterize the early critical steps in the neurodegenerative progression to design adequate therapies. This paper describes and correlates early morphological and biochemical changes in the degenerating retina with in vivo functional analysis of retinal activity and investigates the progression of neurodegenerative stages for up to 7 months. For these purposes, Sprague–Dawley rats were exposed to 1000 lux light either for different durations (12 h to 24 h) and examined seven days afterward (7d) or for a fixed duration (24 h) and monitored at various time points following the exposure (up to 210d). Flash electroretinogram (fERG) recordings were correlated with morphological and histological analyses to evaluate outer and inner retinal disruptions, gliosis, trophic factor release, and microglial activation. Twelve hours or fifteen hours of exposure to constant light led to a severe retinal dysfunction with only minor morphological changes. Therefore, early pathological signs might be hidden by compensatory mechanisms that silence retinal dysfunction, accounting for the discrepancy between photoreceptor loss and retinal functional output. The long-term analysis showed a transient functional recovery, maximum at 45 days, despite a progressive loss of photoreceptors and coincident increases in glial fibrillary acidic protein (GFAP) and basic fibroblast growth factor-2 (bFGF-2) expression. Interestingly, the progression of the disease presented different patterns in the dorsal and ventral retina. The information acquired gives us the potential to develop a specific diagnostic tool to monitor the disease’s progression and treatment efficacy.

Grapevine trunk diseases (GTDs) are a serious and growing threat to vineyards worldwide. The need for innovative control tools persists since pesticides used against some GTDs have been banned and only methods to prevent infections or to reduce foliar symptoms have been developed so far. In this context, the application of imaging methods, already applied to study plant–microbe interactions, represents an interesting approach to understand the effect of experimental treatments applied to reduce fungal colonization, on GTD-related pathogens activity. To this aim, trials were carried out to evaluate the efficacy of copper-based treatments, formulated with hydroxyapatite (HA) as co-adjuvant with innovative delivery properties, loaded with two different copper(II) compounds (tribasic sulfate and sulfate pentahydrate), and applied to grapevine propagation material to inhibit fungal wood colonization. The treated rootstock (Vitis berlandieri × Vitis riparia cv. K5BB) and scion cuttings (Vitis vinifera L., cv. Chardonnay) had been inoculated with a strain of Phaeoacremonium minimum (Pmi) compared to uninoculated rootstocks. Experimental treatments were applied during the water-soaking process, comparing the copper(II) compounds pure or formulated with HA, to hydrate the cuttings. After callusing, grafted vines were grown under greenhouse conditions in a nursery and inoculated with Pmi::gfp7 or with Pmi wild-type. Fifteen weeks post-inoculation, woody tissues close to the inoculation site were sampled to evaluate the efficiency of the treatments by studying the plant–microbe interaction by confocal laser scanning microscopy (CLSM). Copper and further elements were also quantified in the same tissues immediately after the treatments and on the CLSM samples. Finally, the grapevine defense responses were studied in the leaves of cuttings treated with the same formulations. The present investigation confirmed the relevant interaction of Pmi and the related transformed strain on the vascular tissues of grafted vines. Furthermore, in vitro assay revealed (i) the fungistatic effect of HA and the reduced effect of Cu fungicide when combined with HA. In planta assays showed (ii) the reduction of Pmi infection in propagation material treated with HA-Cu formulations, (iii) the movement of HA-Cu formulations inside the plant tissues and their persistence over time, and (iv) the plant defense reaction following the treatment with pure HA or Cu, or combined.

Age-related macular degeneration (AMD) is one of the leading causes of visual loss in western countries, it has no cure, and its incidence will grow in the future, for the overall population aging. Albino rats with retinal degeneration induced by exposure to high-intensity light (light-damage, LD) have been extensively used as a model of AMD to test neuroprotective agents. Among them, trophic factors (NGF and BDNF) have been shown to play a significant role in photoreceptors&rsquo, survival. Interestingly, cord blood serum (CBS) is an extract full of chemokines and trophic factors, we, therefore, hypothesized that CBS could be an excellent candidate for neuroprotection. Here, we investigate whether CBS-based eye drops might mitigate the effects of light-induced retinal degeneration in albino rats. CBS treatment significantly preserved flash-electroretinogram (f-ERG) response after LD and reduced the &ldquo, hot-spot&rdquo, extension. Besides, CBS-treated animals better preserved the morphology of the outer nuclear layer, together with a reduction in microglia migration and activation. Interestingly, the treatment did not modulate reactive gliosis and activation of the self-protective mechanism (FGF2). In conclusion, our results suggest that CBS-based eye drops might be successfully used to mitigate retinal neurodegenerative processes such as AMD.

Oxidative stress and inflammation determine retinal ganglion cell degeneration, leading to retinal impairment and vision loss. Muller glial cells regulate retinal repair under injury, through gliosis. Meanwhile, reactive gliosis can turn in pathological effects, contributing to neurodegeneration. In the present study, we tested whether Cord Blood Serum (CBS), rich of growth factors, might improve the viability of Muller cells under in vitro damage. BDNF, NGF, TGF-alpha, GDNF and EGF levels were measured in CBS samples by Human Magnetic Luminex Assay. CBS effects were evaluated on rat (rMC-1) and human (MIO-M1) Muller cells, under H2O2 and IL-1 beta damage. Cells grown with FBS or CBS both at 5% were exposed to stress and analyzed in terms of cell viability, GFAP, IL-6 and TNF-alpha expression. CBS was also administrated after treatment with K252a, inhibitor of the neurotrophin receptor Trk. Cell viability of rMC-1 and MIO-M1 resulted significantly improved when pretreated with CBS and exposed to H2O2 and IL-1 beta, in comparison to the standard culture with FBS. Accordingly, the gliosis marker GFAP resulted down-regulated following CBS priming. In parallel, we observed a lower expression of the inflammatory mediators in rMC-1 (TNF-alpha) and MIO-M1 (IL-6, TNF- alpha), especially in presence of inflammatory damage. Trk inhibition through K252a administration impaired the effects of CBS under stress conditions on MIO-M1 and rMC-1 viability, not significantly different from FBS condition. CBS is enriched with neurotrophins and its administration to rMC-1 and MIO-M1 attenuates the cytotoxic effects of H2O2 and IL-1 beta. Moreover, the decrease of the main markers of gliosis and inflammation suggests a promising use of CBS for neuroprotection aims. This study is a preliminary basis that prompts future investigations to deeply explore and confirm the CBS potential.

The activity of two zeolitites, differing in the presence of copper and their particle size, was assessed in a Trebbiano d&rsquo, Abruzzo white berry cultivar, towards grapevine grey mould and sour rot, a serious problem in almost all grapevine-growing areas. In 2018 and 2019, zeolitites were applied before forecast of rainfalls, which likely induce infection. In 2019, further applications were carried out 10 or 2 days before harvest, regardless of the rainfall, in order to verify the effect of the zeolitite residue on leaf reflectance and composition of grapes and wines. Both zeolitites significantly reduced the infections. Only the vines treated 2 days before harvest, especially with copper zeolitite, showed a decrease in leaf reflectance. However, no differences in the yield composition related to the time of zeolitite application, as well as a decrease of yield quality, were ever recorded. Furthermore, analysis of samples collected from vines treated with zeolitites showed increases in grape soluble solids and in wine alcohol content. Our results suggest the use of zeolitites against grapevine rots as a possible effective alternative to the application of synthetic fungicides in the development of environmentally friendly control strategies.

Photoreceptor neurodegenerative processes are induced by a variety of factors: genetic mutations, environmental stresses, aging, and a combination of them. The final outcome is photoreceptors malfunction and eventually death leading to progressive deterioration of visual function. At present, there is no cure for most retinal diseases. Recently, saffron has become an interesting candidate to cope with morphological and functional consequences of retinal neurodegeneration. Data obtained in both animal models and clinical trials in age-related macular degeneration and Stargardt patients provided evidence that saffron treatment reduces photoreceptor death, slows down the progression of neuroinflammatory processes, and eventually improves and maintains visual function. The ways of action are complex and not limited to reducing reactive oxygen species production. In fact, saffron treatment is also able to activate many genes. In addition, the chemical composition of saffron's components (mainly the ratio between different crocins) is a critical factor in defining the efficacy of treatment.

Esca of grapevine causes yield losses correlated with incidence and severity symptom expression. Factors associated with leaf symptom mechanisms are yet to be fully clarified. Therefore, in 2019 and 2020, macro and microelement analyses and leaf reflectance measurements were carried out on leaves at different growth stages in a vineyard located in Abruzzo, central Italy. Surveys were carried out on leaves of both never leaf-symptomatic vines and different categories of diseased vine shoots. Never leaf-symptomatic and diseased vines were also treated with a fertilizer mixture that proved to be able to limit the symptom expression. Results showed that untreated asymptomatic diseased vines had high calcium contents for most of the vegetative season. On the contrary, treated asymptomatic diseased vines showed higher contents of calcium, magnesium, and sodium, at berries pea-sized, before the onset of symptoms. These vines had better physiological efficiency showing higher water index (WI), normalized difference vegetation index (NDVI), and green normalized difference vegetation index (GNDVI) values, compared to untreated asymptomatic vines, at fruit set. Results confirmed the strong response of the plant to symptom expression development and the possibility of limiting this response with calcium and magnesium applications carried out before the symptom onset.

Phaeomoniella chlamydospora (Pc) and Phaeoacremonium minimum (Pm) are among the main grapevine trunk disease pathogens affecting vines in the nursery. These pathogens can cause infections of the propagation material often appearing as dark wood streaking, and are associated with decline occurrence in young vineyards. The infection may occur during the grafting process, particularly during hydration. Promising preliminary results were obtained in the nursery using electrolyzed acid water (EAW) for the hydration of cuttings; EAW is characterized by low values of pH (2.5), high oxidation reduction potential (ORP>1000) and a certain amount of free chlorine. No differences in absorption kinetics were recorded between EAW and tap water, nor among 1103P, K5BB and SO4 rootstocks. In all combinations no significant additional absorption was observed after 7 h of immersion. In order to assess the characteristics of EAW after the contact with the inner plant tissues ORP and pH were measured from the liquid extracted from rootstocks after immersion in EAW. The values of the liquid, named “weakened” EAW (wEAW) (625–650 mV ORP and 3.1–3.3 pH), were significantly different from the values of both EAW and the liquid extracted after immersion in tap water. When the propagation material was hydrated for 7 h in EAW no significant effect on vegetating growth performance and percentage of certifiable plants was observed for SO4 or 1103P, whereas a reduction in plant vegetative growth was observed in K5BB hydrated vines at 12hr hydration. EAW and wEAW gave no significant reduction of in vitro mycelial growth, whereas a consistent decrease in conidial germination was observed in Pc and Pm. The three-year experiments carried out in the nursery on cuttings inoculated with Pc and hydrated in EAW showed a remarkable reduction of the infection level in the treated plants.

In Mediterranean environments the adoption of Conservation Agriculture (CA) would allow growers to achieve environmental, agronomic and economic sustainability. We investigated the effect of different combination of crop establishment treatments and crop sequence (conventional tillage (CT) and durum wheat monocropping (WW), conventional tillage (CT) and durum wheat following faba beans (WF), zero tillage (ZT) and durum wheat monocropping (WW), zero tillage (ZF) and durum wheat following faba beans (WF) on yield, grain quality traits as well as on disease incidence and severity in durum wheat (var. Saragolla). The results of two years of data of a long-term experiment (seven-year experiment, split-plot design) are discussed. The CA approach (ZT + WF), which always induced the highest grain yields (6.1 t ha&minus, 1 and 3.3 t ha&minus, 1 in 2016 and 2017) thanks to an increased number of spikes m&minus, 2 (296 vs. 269 and 303 vs. 287 spikes m&minus, 2 in 2016 and 2017, respectively) as well as a more pronounced ear length, demonstrated significantly positive influences in terms of grain quality. It promoted grain protein accumulation (12.1% for ZT + WF vs. 11.4% for ZT + WW and 12.4% for ZT + WF vs. 10.6% for ZT + WW in 2016 and 2017) and improved the gluten quality (in terms of sodium dodecyl sulphate (SDS) sedimentation test) and colour of the grain. The abundance of crop residues determined a higher incidence and severity of Zymoseptoria tritici leaf symptoms under the CA system, nevertheless, the late appearance of infection was the main reason for not affecting yield and quality traits. The presence of faba beans (WF) in the rotation significantly reduced leaf symptoms in Z. tritici.

Long noncoding RNAs are emerging players in the epigenetic machinery with key roles in development and diseases. Here we uncover a complex network comprising a promoter-associated noncoding RNA (paRNA), microRNA and epigenetic regulators that controls transcription of the tumour suppressor E-cadherin in epithelial cancers. E-cadherin silencing relies on the formation of a complex between the paRNA and microRNA-guided Argonaute 1 that, together, recruit SUV39H1 and induce repressive chromatin modifications in the gene promoter. A single nucleotide polymorphism (rs16260) linked to increased cancer risk alters the secondary structure of the paRNA, with the risk allele facilitating the assembly of the microRNA-guided Argonaute 1 complex and gene silencing. Collectively, these data demonstrate the role of a paRNA in E-cadherin regulation and the impact of a noncoding genetic variant on its function. Deregulation of paRNA-based epigenetic networks may contribute to cancer and other diseases making them promising targets for drug discovery., Promoter-proximal transcripts have been proposed to act as cis-acting elements regulating transcription. Here, the authors provide evidence that a promoter-proximal RNA, in combination with other epigenetic regulators, controls transcription of E-cadherin in epithelial cancers.

Retinal oxidative damage, associated with an ATP-binding cassette, sub-family A, member 4, also known as ABCA4 gene mutation, has been implicated as a major underlying mechanism for Stargardt disease/fundus flavimaculatus (STG/FF). Recent findings indicate that saffron carotenoid constituents crocins and crocetin may counteract retinal oxidative damage, inflammation and protect retinal cells from apoptosis. This pilot study aimed to evaluate central retinal function following saffron supplementation in STG/FF patients carrying ABCA4 mutations. Methods: in a randomized, double-blind, placebo-controlled study (clinicaltrials.gov: NCT01278277), 31 patients with ABCA4-related STG/FF and a visual acuity >0.25 were randomly assigned to assume oral saffron (20 mg) or placebo over a six month period and then reverted to P or S for a further six month period. Full ophthalmic examinations, as well as central 18° focal electroretinogram (fERG) recordings, were performed at baseline and after six months of either saffron or placebo. The fERG fundamental harmonic component was isolated by Fourier analysis. Main outcome measures were fERG amplitude (in µV) and phase (in degrees). The secondary outcome measure was visual acuity. Results: supplement was well tolerated by all patients throughout follow-up. After saffron, fERG amplitude was unchanged; after placebo, amplitude tended to decrease from baseline (mean change: −0.18 log µV, p < 0.05). Reverting the treatments, amplitude did not change significantly. fERG phase and visual acuity were unchanged throughout follow-up. Conclusions: short-term saffron supplementation was well tolerated and had no detrimental effects on the electroretinographic responses of the central retina and visual acuity. The current findings warrant further long-term clinical trials to assess the efficacy of saffron supplementation in slowing down the progression of central retinal dysfunction in ABCA4-related STG/FF.

We investigated the effects of continuous artificial light exposure on the mouse substantia nigra (SN). A three month exposure of C57Bl/6J mice to white fluorescent light induced a 30% reduction in dopamine (DA) neurons in SN compared to controls, accompanied by a decrease of DA and its metabolites in the striatum. After six months of exposure, neurodegeneration progressed slightly, but the level of DA returned to the basal level, while the metabolites increased with respect to the control. Three month exposure to near infrared LED light (∼710nm) did not alter DA neurons in SN, nor did it decrease DA and its metabolites in the striatum. Furthermore mesencephalic cell viability, as tested by [3H]DA uptake, did not change. Finally, we observed that 710nm LED light, locally conveyed in the rat SN, could modulate the firing activity of extracellular-recorded DA neurons. These data suggest that light can be detrimental or beneficial to DA neurons in SN, depending on the source and wavelength.

Among the fungi associated with the kiwi wood diseases, the vascular pathogen Phaeoacremonium minimum can infect plants already at nursery stage, without any external symptoms. At the moment, there are not effective control strategies. The effect of soil treatments applicable in organic agriculture was evaluated in two-years experiments on potted kiwi plants artificially inoculated with P. minimum. The soil treatments were based on commercial formulations of iron chelate, silicon, neem paste, Trichoderma afroharzianum strain T22, and cover cropping with selected perennial Poaceae. Cover cropping and iron chelate treatments enhanced the iron availability for the plants and significantly reduced wood necrosis caused by the pathogen. Both treatments also produced an increase of hairy root proliferation, so the plants were able to better cope with stress conditions. Laboratory assays showed the role of iron on the pathogen growth and its pathogenesis enzyme activities.

Di Marco, S., and Osti, F. 2008. Foliar symptom expression of wood decay in Actinidia deliciosa in relation to environmental factors. Plant Dis. 92:1150-1157. Kiwifruit vines (Actinidia deliciosa var. deliciosa) have recently been affected by a new form of decay caused by several fungi that produce different types of wood deterioration in the trunk and cordons. Surveys were conducted over a period of 5 years to investigate epidemiological aspects of the disease in a typical Italian growing area (Emilia-Romagna), where kiwifruit is widely cultivated and where the disease was noted for the first time. The disease was widespread over the kiwifruit growing area surveyed, and its incidence increased over the course of the survey. No relationship was found between vineyard soil characteristics or management methods and the annual incidence of symptomatic vines. Foliar symptoms did not consistently express every season even on obviously infected vines. The time of appearance and the development of the disease were correlated with plant phenology and temperature. In particular, from June to August, temperature seemed to affect the annual incidence of the disease in terms of both symptomatic shoots and symptomatic vines. The aspects in common between the decay of kiwifruit and esca of grapevine could be hypothesized and are discussed.

High density multielectrode arrays (MEAs) based on CMOS technology (CMOS-MEAs) can simultaneously record extracellular spiking activity in neuronal cultures from 4096 closely spaced microelectrodes. This allows for a finer investigation of neuronal network activity compared to conventional MEAs with a few tens of electrodes. However, the sensing properties of these devices differ. To highlight this aspect, here we investigate and discuss the differences observed when quantifying spontaneous synchronized bursting events (SBEs) in datasets acquired with conventional MEAs and high-density MEAs from comparable hippocampal cultures. We found that datasets acquired with high-density MEAs exhibit collective dynamics similar to conventional arrays, but are characterized by a higher percentage of random spikes, i.e. spikes that are not part of a burst, most probably resulting from the larger recording capability. Additionally, the percentage of electrodes that record a burst is remarkably small on high-density MEAs compared to what can be observed on conventional MEAs and SBEs appear to be propagating in time across the electrode array, by involving shorter sequences of spikes per electrode. Overall, these results highlight a lower level of network synchronization involved in SBEs compared to what has been debated for several decades based on conventional MEA recordings from cell cultures.

In order to reduce sea turtle by‐catches on surface drifting long‐lines during professional swordfish (Xiphias gladius) fishing, the relevance of olfactory stimuli in eliciting predation by loggerhead sea turtles (Caretta caretta) was investigated. Choice experiments were run in captivity. Squid‐shaped plastic lures having or lacking scomber odour were presented to 27 specimens (22 immatures and 5 adults). The turtles’ behavioural responses highlighted the importance of chemical clues in eliciting approaching and biting behaviours. This study was carried out within the framework of the EU‐Life Project ‘Urgent conservation measures for C. caretta in the Pelagie islands’ (LIFE 99 NAT/IT/006271).

Developing neuronal systems intrinsically generate coordinated spontaneous activity that propagates by involving a large number of synchronously firing neurons. In vivo, waves of spikes transiently characterize the activity of developing brain circuits and are fundamental for activity-dependent circuit formation. In vitro, coordinated spontaneous spiking activity, or network bursts (NBs), interleaved within periods of asynchronous spikes emerge during the development of 2D and 3D neuronal cultures. Several studies have investigated this type of activity and its dynamics, but how a neuronal system generates these coordinated events remains unclear. Here, we investigate at a cellular level the generation of network bursts in spontaneously active neuronal cultures by exploiting high-resolution multielectrode array recordings and computational network modelling. Our analysis reveals that NBs are generated in specialized regions of the network (functional neuronal communities) that feature neuronal links with high cross-correlation peak values, sub-millisecond lags and that share very similar structural connectivity motifs providing recurrent interactions. We show that the particular properties of these local structures enable locally amplifying spontaneous asynchronous spikes and that this mechanism can lead to the initiation of NBs. Through the analysis of simulated and experimental data, we also show that AMPA currents drive the coordinated activity, while NMDA and GABA currents are only involved in shaping the dynamics of NBs. Overall, our results suggest that the presence of functional neuronal communities with recurrent local connections allows a neuronal system to generate spontaneous coordinated spiking activity events. As suggested by the rules used for implementing our computational model, such functional communities might naturally emerge during network development by following simple constraints on distance-based connectivity., Author summary Coordinated spontaneous spiking activity is fundamental for the normal formation of brain circuits during development. However, how ensembles of neurons generate these events remains unclear. To address this question, in the present study, we investigated the network properties that might be required to a neuronal system for the generation of these spontaneous waves of spikes. We performed our study on spontaneously active neuronal cell cultures using high-resolution electrical recordings and a computational network model developed to reproduce our experimental data both quantitatively and qualitatively. Through the analysis of both experimental and simulated data, we found that network bursts are initiated in regions of the network, or “functional communities”, characterized by particular local connectivity properties. We also found that these regions can amplify the background asynchronous spiking activity preceding a network burst and, in this way, can give rise to coordinated spiking events. As a whole, our results suggest the presence of functional communities of neurons in a developing neuronal system that might naturally emerge by following simple constraints on distance-based connectivity. These regions are most likely required for the generation of the spontaneous coordinated activity that can drive activity-dependent circuit formation.

The MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites (CFSs) during early mitosis to trigger DNA-repair synthesis that ensures faithful chromosome segregation. Here, we show that these DNA transactions are promoted by RECQ5 DNA helicase in a manner dependent on its Ser727 phosphorylation by CDK1. Upon replication stress, RECQ5 associates with CFSs in early mitosis through its physical interaction with MUS81 and promotes MUS81-dependent mitotic DNA synthesis. RECQ5 depletion or mutational inactivation of its ATP-binding site, RAD51-interacting domain, or phosphorylation site causes excessive binding of RAD51 to CFS loci and impairs CFS expression. This leads to defective chromosome segregation and accumulation of CFS-associated DNA damage in G1 cells. Biochemically, RECQ5 alleviates the inhibitory effect of RAD51 on 3'-flap DNA cleavage by MUS81-EME1 through its RAD51 filament disruption activity. These data suggest that RECQ5 removes RAD51 filaments stabilizing stalled replication forks at CFSs and hence facilitates CFS cleavage by MUS81-EME1.

Several studies link disease progression, recurrence, and treatment failures to the cancer stem-like cell (CSC) subpopulation within the heterogeneous tumor cell population. Myc is a transcription factor having a central function in stem cell biology and in human cancers. Hence, Myc represents an attractive target to develop CSC-specific therapies. Recent findings suggest that Myc transcription can be silenced using an RNA interference (RNAi)–based strategy that targets noncoding promoter-associated RNA (paRNA) overlapping the transcription start site. In this study, we investigated the effects of silencing Myc transcription on prostate CSC in cell culture and xenograft models of human prostate cancer. Treatment with an effective promoter-targeting siRNA reduced the fraction of CSCs, leading to reduced self-renewal, tumor-initiating, and metastatic capability. Combined analysis of stem-like cells and senescence markers indicated that Myc silencing triggered a phenotypic shift and senescence in the CSC subpopulation. Notably, systemic delivery of the promoter-targeting siRNA in the xenograft model produced a striking suppression in the development of prostate tumors. Our results support a pivotal role for Myc in CSC maintenance and show that Myc targeting via RNAi-based transcriptional silencing can trigger CSC senescence and loss of their tumor-initiating capability. More generally, our findings demonstrate the efficacy of RNAi-based transcriptional strategies and the potential to target regulatory noncoding paRNAs for therapeutic applications. Cancer Res; 73(22); 6816–27. ©2013 AACR.

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

The ON and OFF pathways that emerge at the first synapse in the retina are generally thought to be streamed in parallel to higher visual areas, but recent work shows cross talk at the level of retinal ganglion cells. The ON pathway drives inhibitory inputs onto some OFF ganglion cells, such that these neurons show “push-pull” convergence of OFF-excitation and ON-disinhibition. In this study we measure the spatial receptive field of excitatory and inhibitory inputs to OFF-sustained (OFF-S) retinal ganglion cells of mouse, establish how contrast adaptation modulates excitatory and inhibitory synaptic inputs, and show the pharmacology of the inhibitory inputs. We find that the spatial tuning properties of excitatory and inhibitory inputs are sufficient to determine the spatial profile of the spike output and that high spatial acuity may be particularly reliant on disinhibitory circuits. Contrast adaptation reduced excitation to OFF-S ganglion cells, as expected, and also unmasked an asymmetry in inhibitory inputs: disinhibition at light-off was immune to contrast adaptation, but inhibition at light-on was substantially reduced. In pharmacological experiments we confirm that inhibitory inputs are partly mediated by glycine, but our measurements also suggest a substantial role for GABA. Our observations therefore reveal functional diversity in the inhibitory inputs to OFF ganglion cells and suggest that in addition to enhancing operational range these inputs help shape the spatial receptive fields of ganglion cells.

Esca is a serious disease, caused mainly by two vascular fungi, Phaeomoniella chlamydospora and Phaeoacremonium aleophilum , in a complex infective process that has not yet been fully elucidated. The cellulose-degrading system of these two fungi was examined and was found to be unable to degrade crystalline cellulose. When iron was added to the culture medium, however, Pa. chlamydospora and Pm. aleophilum were able to degrade crystalline cellulose: the iron formed hydroxyl radicals that made up for the lack of specific enzyme activity of the fungi. The iron did not affect the activity of the other enzymes present, such as the endoglucanases. It is shown that iron-dependent processes of a non-enzymatic nature occur, and that these amplify the pathogenic capacity of Pa. chlamydospora and Pm. aleophilum , supplementing the pathogenic capacity of the enzymes. The manner in which these non-enzymatic processes can help to further our understanding of the nature of interaction between grapevine and the esca pathogens is discussed.

Early sensory experience shapes the functional and anatomical connectivity of neuronal networks. Light deprivation alters synaptic transmission and modifies light response properties in the visual system, from retinal circuits to higher visual centers. These effects are more pronounced during a critical period in juvenile life and are mostly reversed by restoring normal light conditions. Here we show that complete light deprivation, from birth to periods beyond the critical period, permanently modifies the receptive field properties of retinal ganglion cells. Visual deprivation reduced both the strength of light responses in ganglion cells and their receptive field size. Light deprivation produced an imbalance in the ratio of inhibitory to excitatory inputs, with a shift toward larger inhibitory conductances. Ganglion cell receptive fields in visually deprived animals showed a spatial mismatch of inhibitory and excitatory inputs and inhibitory inputs were highly scattered over the receptive field. These results indicate that visual experience early in life is critical for the refinement of retinal circuits and for appropriate signaling of the spatiotemporal properties of visual stimuli, thus influencing the response properties of neurons in higher visual centers and their processing of visual information.

Recent data challenged the assumption that light has little effect on retina development. Here, we report evidence that dark rearing permanently changes the synaptic input to GCs. A reduced spontaneous postsynaptic currents (SPSCs) frequency was found in retinal GCs from rats born and raised in the dark for three months. Glutamate antagonists (CNQX and AP-5) reversibly reduced SPSCs frequency in control and dark-reared (DR) retinae. The GABA antagonist picrotoxin (PTX) reduced SPSCs frequency in control retinas, but increased SPSCs frequency in DR, mainly by presynaptic action on excitatory currents. In DR animals exposed to normal cyclic light for 3 months, SPSCs frequency remained lower then in control rats and increased following PTX, suggesting that long-term dark rearing induces permanent modifications of the retinal circuitry. Our results strongly support the idea that light stimulation plays a role in establishing normal synaptic input to GCs.

PURPOSE. To test whether the saffron extract (Crocus sativus L.) given as a dietary supplement counteracts the effects of continuous light exposure in the albino rat retina. METHODS. Three experimental groups of Sprague–Dawley rats were used. Experimental animals were prefed either saffron or -carotene (1 mg extract/kg/d) before they were exposed to bright continuous light (BCL) for 24 hours. Flash electroretinograms (fERGs) were recorded in control and treated rats the day before and 1 week after light exposure. At the end of the second recording session, the animals were killed and the retinas were quickly removed, fixed, cryosectioned, and labeled so that the thickness of the outer nuclear layer (ONL) could be analyzed. Changes in protein level and cellular localization of fibroblast growth factor (FGF)2 were determined by Western blot analysis and retinal immunohistochemistry, respectively. In a second series of experiments, rats were killed at the end of light exposure, and the amount of apoptotic figures in the ONL was assessed by terminal transferase-mediated deoxyuridine triphosphate (d-UTP)-biotin nick-end labeling (TUNEL). BCL induced DNA fragmentation, characteristic of dying cells, almost exclusively in the photoreceptor layer. The rate of photoreceptor death induced by BCL is expressed as the frequency of TUNEL-positive profiles per millimeter. RESULTS. The photoreceptor layer was largely preserved in saffron-treated animals because it was the fERG response. In addition, the rate of photoreceptor death induced by BCL appeared drastically reduced in treated animals. In -carotene prefeeding experiments, morphologic analysis showed preservation of the ONL similar to that obtained with saffron prefeeding, whereas the fERG response was unrecordable. Western blot analysis showed that exposure to light induced a strong upregulation of FGF2 in control and -carotene–treated rats, but s no change was noted in saffron-treated rats. CONCLUSIONS. These results show that saffron may protect photoreceptors from retinal stress, maintaining both morphology and function and probably acting as a regulator of programmed cell death. (Invest Ophthalmol Vis Sci. 2008;49:1254 –1261)

Prostate cancer is the most frequent epithelial cancer in elderly men in Western countries. Depending on the stage, prostate cancer treatment requires surgery, hormone therapy, radio- and chemo-therapy. c-MYC is a transcription factor activated by mitogenic signaling pathways and playing a central function in stem cell biology. Over-activity of c-Myc plays a central role in tumorigenesis by affecting cell proliferation, metabolic adaptation and survival. Amplification of c-MYC is one of the most common genetic alterations occurring in cancer genomes. c-Myc expression is significantly elevated in invasive prostate adenocarcinomas compared to benign prostatic hyperplasia and normal prostate tissue. Particularly, c-Myc is highly expressed in prostate cancer cells having the CD44+/CD24- phenotype, which is described as a hallmark of cancer progenitor/stem cells (CSCs). Importantly, several studies link therapy resistance and disease progression to the CSC subpopulation within the highly heterogeneous cellular composition of the tumor. CSCs are likely the tumor initiating population that produces metastasis and disease recurrence. These lines of evidence suggest a central role of c-Myc in the maintenance of CSC compartment in human tumors and justify c-Myc as a therapeutic target. Previously, we showed that the c-Myc gene could be silenced with small interfering RNAs (siRNA) targeting its promoter. Interestingly, single transfection with myc targeting siRNA (myc-siRNA) induced long lasting effects on cell proliferation and clonogenicity, indicative of a persistent loss of proliferative and clonogenic potential. In this study, we investigated the effects of transcriptional silencing of c-Myc on the CSC subpopulation in human prostate cancer cell lines in vitro and in vivo. We found that treatment with myc-siRNA significantly reduced the fraction of CSCs defined by expression of stem cell surface markers CD44 and CD24, in vitro sphere forming ability and self-renewal. Furthermore, combined analysis of senescence and cell surface markers showed that senescence occurred prevalently in the CD44+/CD24- cell subpopulation leading to its depletion, and reduced self renewal capability, in vivo tumorigenicity and metastatic potential. Moreover, repeated intraperitoneal administration of myc-siRNA over a 4-week period reduced tumor growth in a xenogeneic prostate cancer model. These results are consistent with the role of c-Myc in the maintenance of the CSC subpopulation in human prostate tumors and suggest that c-Myc downregulation induces an irreversible loss of clonogenic and tumor-initiating capability linked to the induction of cell senescence in CSCs. Our findings show also that an RNAi-based transcriptional therapy directed to genes directly involved in the maintenance of the cancer stem cells could be an efficient approach to block progression and relapse of prostate cancer. Citation Format: Gianluca Civenni, Anastasia Malek, Domenico Albino, Manuela Sarti, Sandra Pinton, Stefano Di Marco, Giuseppina M. Carbone, Carlo V. Catapano. Transcriptional silencing of c-Myc by promoter-directed siRNA efficiently target prostate cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3307. doi:10.1158/1538-7445.AM2013-3307