Giuseppe Gargiulo

Neonicotinoid clothianidin adversely affects insect immunity and promotes replication of a viral pathogen in honey bees

Significance Honey bees are exposed to a wealth of synergistically interacting stress factors, which may induce colony losses often associated with high infection levels of pathogens. Neonicotinoid insecticides have been reported to enhance the impact of pathogens, but the underlying immune alteration is still obscure. In this study we describe the molecular mechanism through which clothianidin adversely affects the insect immune response and promotes replication of a viral pathogen in honey bees bearing covert infections. Our results shed light on a further level of regulation of the immune response in insects and have implications for bee conservation. Large-scale losses of honey bee colonies represent a poorly understood problem of global importance. Both biotic and abiotic factors are involved in this phenomenon that is often associated with high loads of parasites and pathogens. A stronger impact of pathogens in honey bees exposed to neonicotinoid insecticides has been reported, but the causal link between insecticide exposure and the possible immune alteration of honey bees remains elusive. Here, we demonstrate that the neonicotinoid insecticide clothianidin negatively modulates NF-κB immune signaling in insects and adversely affects honey bee antiviral defenses controlled by this transcription factor. We have identified in insects a negative modulator of NF-κB activation, which is a leucine-rich repeat protein. Exposure to clothianidin, by enhancing the transcription of the gene encoding this inhibitor, reduces immune defenses and promotes the replication of the deformed wing virus in honey bees bearing covert infections. This honey bee immunosuppression is similarly induced by a different neonicotinoid, imidacloprid, but not by the organophosphate chlorpyriphos, which does not affect NF-κB signaling. The occurrence at sublethal doses of this insecticide-induced viral proliferation suggests that the studied neonicotinoids might have a negative effect at the field level. Our experiments uncover a further level of regulation of the immune response in insects and set the stage for studies on neural modulation of immunity in animals. Furthermore, this study has implications for the conservation of bees, as it will contribute to the definition of more appropriate guidelines for testing chronic or sublethal effects of pesticides used in agriculture.

Apoptosis of nurse cells at the late stages of oogenesis of Drosophila melanogaster

Abstract In Drosophila a remarkable feature of oogenesis is the regression of the nurse cells after dumping their cytoplasmic contents into the oocyte. We have studied the nature of this process at the late stages of egg chamber development. In egg chambers DAPI staining shows highly condensed chromatin from stage 12 and TUNEL labelling shows DNA fragmentation up to stage 14. Gel electrophoresis of the end-labelled DNA, extracted from isolated egg chambers at the same stages of development, shows a ladder typical of apoptotic nuclei. This provides evidence that, during Drosophila oogenesis, the nurse cells undergo apoptosis. Apoptotic nuclei have also been detected in dumping-defective egg chambers, indicating that the cytoplasmic depletion of nurse cells is concurrent with but apparently not the cause of the process.

Mapping the intrinsic curvature and flexibility along the DNA chain

The energy of DNA deformation plays a crucial and active role in its packaging and its function in the cell. Considerable effort has gone into developing methodologies capable of evaluating the local sequence-directed curvature and flexibility of a DNA chain. These studies thus far have focused on DNA constructs expressly tailored either with anomalous flexibility or curvature tracts. Here we demonstrate that these two structural properties can be mapped also along the chain of a “natural” DNA with any sequence on the basis of its scanning force microscope (SFM) images. To know the orientation of the sequence of the investigated DNA molecules in their SFM images, we prepared a palindromic dimer of the long DNA molecule under study. The palindromic symmetry also acted as an internal gauge of the statistical significance of the analysis carried out on the SFM images of the dimer molecules. It was found that although the curvature modulus is not efficient in separating static and dynamic contributions to the curvature of the population of molecules, the curvature taken with its direction (its sign in two dimensions) permits the direct separation of the intrinsic curvature from the flexibility contributions. The sequence-dependent flexibility seems to vary monotonically with the chains intrinsic curvature; the chain rigidity was found to modulate as its local thermodynamic stability and does not correlate with the dinucleotide chain rigidities evaluation made from x-ray data by other authors.

Building up the Drosophila eggshell: First of all the eggshell genes must be transcribed

The Drosophila eggshell provides a model system for studying the assembly of extracellular matrix. Eggshell formation is a complex process that requires time‐coordinated synthesis, cleavage, and transport of various proteins and finally cross‐linking mediated by particular functional domains. It has been suggested that the eggshell can act as a storage site for spatial cues involved in embryonic pattern formation. Its structural components are synthesized in the somatic follicle cells in a precise temporally and spatially regulated manner. This review will summarize our knowledge of eggshell gene expression. We will discuss the amplification of the chorion gene clusters and the data acquired on the expression patterns and the regulatory elements controlling transcription of eggshell genes. We will then focus on the findings that correlate follicular epithelium patterning and eggshell gene expression, and discuss the interesting perspectives of an involvement in eggshell assembly of embryonic patterning cues. Developmental Dynamics 237:2061–2072, 2008.

Treatment strategies for coronary in-stent restenosis: systematic review and hierarchical Bayesian network meta-analysis of 24 randomised trials and 4880 patients

Study question What is the most safe and effective interventional treatment for coronary in-stent restenosis? Methods In a hierarchical Bayesian network meta-analysis, PubMed, Embase, Scopus, Cochrane Library, Web of Science, ScienceDirect, and major scientific websites were screened up to 10 August 2015. Randomised controlled trials of patients with any type of coronary in-stent restenosis (either of bare metal stents or drug eluting stents; and either first or recurrent instances) were included. Trials including multiple treatments at the same time in the same group or comparing variants of the same intervention were excluded. Primary endpoints were target lesion revascularisation and late lumen loss, both at six to 12 months. The main analysis was complemented by network subanalyses, standard pairwise comparisons, and subgroup and sensitivity analyses. Study answer and limitations Twenty four trials (4880 patients), including seven interventional treatments, were identified. Compared with plain balloons, bare metal stents, brachytherapy, rotational atherectomy, and cutting balloons, drug coated balloons and drug eluting stents were associated with a reduced risk of target lesion revascularisation and major adverse cardiac events, and with reduced late lumen loss. Treatment ranking indicated that drug eluting stents had the highest probability (61.4%) of being the most effective for target lesion vascularisation; drug coated balloons were similarly indicated as the most effective treatment for late lumen loss (probability 70.3%). The comparative efficacy of drug coated balloons and drug eluting stents was similar for target lesion revascularisation (summary odds ratio 1.10, 95% credible interval 0.59 to 2.01) and late lumen loss reduction (mean difference in minimum lumen diameter 0.04 mm, 95% credible interval −0.20 to 0.10). Risks of death, myocardial infarction, and stent thrombosis were comparable across all treatments, but these analyses were limited by a low number of events. Trials had heterogeneity regarding investigation periods, baseline characteristics, and endpoint reporting, with a lack of information at long term follow-up. Direct and indirect evidence was also inconsistent for the comparison between drug eluting stents and drug coated balloons. What this study adds Compared with other currently available interventional treatments for coronary in-stent restenosis, drug coated balloons and drug eluting stents are associated with superior clinical and angiographic outcomes, with a similar comparative efficacy. Funding, competing interests, data sharing This study received no external funding. The authors declare no competing interests. No additional data available.

Moderate and Severe Preoperative Chronic Kidney Disease Worsen Clinical Outcomes After Transcatheter Aortic Valve Implantation Meta-Analysis of 4992 Patients

Background—There is a conflicting evidence on safety and efficacy of transcatheter aortic valve implantation in patients with preoperative chronic kidney disease (CKD). Therefore, we conducted a meta-analysis on the impact of CKD on outcomes after transcatheter aortic valve implantation. Methods and Results—Nine studies including 4992 patients were analyzed. Overall preoperative CKD (stages 3–5) significantly increased early (odds ratio [OR], 1.44; 95% confidence interval [CI], 1.08–1.94 and OR, 1.66; 95% CI, 1.04–2.67) and 1-year (OR, 1.66; 95% CI, 1.23–2.25 and OR, 1.32; 95% CI, 1.06–1.63) all-cause and cardiovascular mortality, respectively. Moderate CKD (stage 3) alone also increased early and 1-year all-cause mortality (OR, 1.43; 95% CI, 1.10–1.85 and OR, 1.41; 95% CI, 1.13–1.74). CKD stages 4 to 5 and 3 compared with stages 1 to 2 increased early stroke (OR, 2.67; 95% CI, 1.53–4.65 and OR, 1.66; 95% CI, 1.09–2.52), acute kidney injury (OR, 2.09; 95% CI, 1.17–3.72 and OR, 1.32; 95% CI, 1.09–1.60) and need for dialysis (OR, 5.92; 95% CI, 2.46–14.27 and OR, 1.55; 95% CI, 0.65–3.70), in the absence of significant differences in contrast medium administration (mean difference, −26.07; 95% CI, −53.00 to 0.85 and mean difference, −0.42; 95% CI, −16.10 to 15.26). Bleeding (life-threatening or major) was nonsignificantly increased in CKD 3 to 5 compared with CKD 1 to 2, but significantly increased in most severe patients (CKD 4–5 versus CKD 1–2: OR, 1.66; 95% CI, 1.13–2.44; CKD 4–5 versus CKD 3: OR, 1.68; 95% CI, 1.27–2.24). Conclusions—Both moderate and severe preoperative CKD significantly worsen transcatheter aortic valve implantation prognosis. Future studies on risk evaluation, prevention, and postoperative management are needed.

Cell Survival and Polarity of Drosophila Follicle Cells Require the Activity of Ecdysone Receptor B1 Isoform

Proper assembly and maintenance of epithelia are critical for normal development and homeostasis. Here, using the Drosophila ovary as a model, we identify a role for the B1 isoform of the ecdysone receptor (EcR-B1) in this process. We performed a reverse genetic analysis of EcR-B1 function during oogenesis and demonstrate that silencing of this receptor isoform causes loss of integrity and multilayering of the follicular epithelium. We show that multilayered follicle cells lack proper cell polarity with altered distribution of apical and basolateral cell polarity markers including atypical-protein kinase C (aPKC), Discs-large (Dlg), and Scribble (Scrib) and aberrant accumulation of adherens junctions and F-actin cytoskeleton. We find that the EcR-B1 isoform is required for proper follicle cell polarity both during early stages of oogenesis, when follicle cells undergo the mitotic cell cycle, and at midoogenesis when these cells stop dividing and undergo several endocycles. In addition, we show that the EcR-B1 isoform is required during early oogenesis for follicle cell survival and that disruption of its function causes apoptotic cell death induced by caspase.

Bivalirudin versus heparin with or without glycoprotein IIb/IIIa inhibitors in patients with STEMI undergoing primary PCI: An updated meta-analysis of 10,350 patients from five randomized clinical trials

Aims: To evaluate the impact of bivalirudin versus heparin on efficacy and safety outcomes of ST-segment elevation myocardial infarction (STEMI) patients undergoing primary percutaneous coronary intervention (PCI) and to explore the impact of differential use (bailout vs. routine) of glycoprotein IIb/IIIa inhibitors (GPI). Methods and Results: Five randomized controlled trials encompassing 10,350 patients were included. Primary efficacy and safety endpoints were all-cause death and major bleeding, respectively. All-cause death at 30 days did not significantly differ with bivalirudin compared to heparin (odds ratio (OR) 0.97, 95% confidence interval (CI) 0.74–1.28; P=0.84). Major bleeding was significantly reduced by bivalirudin compared to heparin (OR 0.58, 95% CI 0.40–0.85; P=0.005). Bivalirudin use was associated with non-significantly different rates of 30-day definite stent thrombosis (ST) (OR 1.71, 95% CI 0.84–3.49; P=0.14), albeit with higher rates of acute ST (OR 3.55, 95% CI 1.67–7.56; P=0.001) and non-significantly different rates of subacute ST (OR 0.86, 95% CI 0.46–1.61; P=0.64). There were non-significant differences in the 30-day rates of reinfarction (OR 1.47, 95% CI 0.94–2.30; P=0.10) and cardiovascular death (OR 0.76, 95% CI 0.56–1.02; P=0.07). There were no significant interactions between bailout versus routine GPI use in the heparin arm for any of the safety or efficacy outcomes (all Pinteraction>0.10). Conclusions: Bivalirudin compared with heparin was associated with comparable 30-day rates of mortality with reduced major bleeding, at the price of an increased risk of acute ST, with non-significant differences in the overall 30-day rates of ST and reinfarction. Intended use of GPI in the heparin arm did not significantly modify the treatment effects of bivalirudin. Given the important differences between trials, as well as evolution in technique and adjunct pharmacotherapy, further randomized trials are warranted to discriminate whether there are substantial safety and efficacy differences between these agents during primary PCI in STEMI.

EcR-B1 and Usp nuclear hormone receptors regulate expression of the VM32E eggshell gene during Drosophila oogenesis

Ecdysone signaling plays key roles in Drosophila oogenesis, as its activity is required at multiple steps during egg chamber maturation. Recently, its involvement has been reported on eggshell production by controlling chorion gene transcription and amplification. Here, we present evidence that ecdysone signaling also controls the expression of the eggshell gene VM32E, whose product is a component of vitelline membrane and endochorion layers. Specifically blocking the function of the different Ecdysone receptor (EcR) isoforms we demonstrate that EcR-B1 is responsible for ecdysone-mediated VM32E transcriptional regulation. Moreover, we show that the EcR partner Ultraspiracle (Usp) is also necessary for VM32E expression. By analyzing the activity of specific VM32E regulatory regions in usp(2) clones we identify the promoter region mediating ecdysone-dependent VM32E expression. By in vitro binding assay and site-directed mutagenesis we demonstrate that this region contains a Usp binding site necessary for VM32E regulation. Our results further support the crucial role of ecdysone signaling in controlling transcription of eggshell structural genes and suggest that the heterodimeric complex EcR-B1/Usp mediates the ecdysone-dependent VM32E transcriptional activation in the main body follicle cells.

Drosophila vitelline membrane cross-linking requires the fs(1)Nasrat, fs(1)polehole and chorion genes activities

Abstract. During the final step of Drosophila vitelline membrane formation, the structural proteins composing this layer become cross-linked by covalent bonds. In the present report, we analyzed the vitelline membrane cross-linking in mutants having defects either in this layer or in the chorionic layers. In the fs(1)Nasrat and fs(1)polehole mutant alleles conferring defects in vitelline membrane formation, disruption of vitelline membrane cross-linking was observed, indicating the involvement of these two genes in the process. On the contrary, in the fs(1)Nasrat and fs(1)polehole alleles showing defects only at the termini of the embryo the vitelline membrane is properly formed, confirming a multifunctional activity of their gene products. Altered vitelline membrane cross-linking was also detected in a mutant of the chorion protein gene Cp36 and in the chorion amplification mutant fs(1)K1214, suggesting a role of the structural components of chorion layers in the process of vitelline membrane hardening.