The molecular chaperone Hsp90 is a component of the cap-binding complex and interacts with the translational repressor Cup during Drosophila oogenesis

In metazoa, the spatio-temporal translation of diverse mRNAs is essential to guarantee proper oocyte maturation and early embryogenesis. The eukaryotic translation initiation factor 4E (eIF4E), which binds the 5′ cap structure of eukaryotic mRNAs, associates with either stimulatory or inhibitory factors to modulate protein synthesis. In order to identify novel factors that might act at the translational level during Drosophila oogenesis, we have undertaken a functional proteomic approach and isolated the product of the Hsp83 gene, the evolutionarily conserved chaperone Hsp90, as a specific component of the cap-binding complex. Here we report that Hsp90 interacts in vitro with the translational repressor Cup. In addition, we show that Hsp83 and cup interact genetically, since lowering Hsp90 activity enhances the oogenesis alterations linked to diverse cup mutant alleles. Hsp90 and Cup co-localize in the cytoplasm of the developing germ-line cells within the germarium, thus suggesting a common function from the earliest stages of oogenesis. Taken together, our data start elucidating the role of Hsp90 during Drosophila female germ-line development and strengthen the idea that Cup has multiple essential functions during egg chamber development

A Hybrid Analytic Hierarchy Process and Likert Scale Approach for the Quality Assessment of Medical Education Programs

The quality assessment of training courses is of utmost importance in the medical education field to improve the quality of the training. This work proposes a hybrid multicriteria decision-making approach based on two methodologies, a Likert scale (LS) and the analytic hierarchy process (AHP), for the quality assessment of medical education programs. On one hand, the qualitative LS method was adopted to estimate the degree of consensus on specific topics; on the other hand, the quantitative AHP technique was employed to prioritize parameters involved in complex decision-making problems. The approach was validated in a real scenario for evaluating healthcare training activities carried out at the Centre of Biotechnology of the National Hospital A.O.R.N. “A. Cardarelli” of Naples (Italy). The rational combination of the two methodologies proved to be a promising decision-making tool for decision makers to identify those aspects of a medical education program characterized by a lower user satisfaction degree (revealed by the LS) and a higher priority degree (revealed by the AHP), potentially suggesting strategies to increase the quality of the service provided and to reduce the waste of resources. The results show how this hybrid approach can provide decision makers with helpful information to select the most important characteristics of the delivered education program and to possibly improve the weakest ones, thus enhancing the whole quality of the training courses

Prebiotic properties of Bacillus coagulans MA-13: production of galactoside hydrolyzing enzymes and characterization of the transglycosylation properties of a GH42 β-galactosidase

Background: The spore-forming lactic acid bacterium Bacillus coagulans MA-13 has been isolated from canned beans manufacturing and successfully employed for the sustainable production of lactic acid from lignocellulosic biomass. Among lactic acid bacteria, B. coagulans strains are generally recognized as safe (GRAS) for human consumption. Low-cost microbial production of industrially valuable products such as lactic acid and various enzymes devoted to the hydrolysis of oligosaccharides and lactose, is of great importance to the food industry. Specifically, α- and β-galactosidases are attractive for their ability to hydrolyze not-digestible galactosides present in the food matrix as well as in the human gastrointestinal tract. Results: In this work we have explored the potential of B. coagulans MA-13 as a source of metabolites and enzymes to improve the digestibility and the nutritional value of food. A combination of mass spectrometry analysis with conventional biochemical approaches has been employed to unveil the intra- and extra- cellular glycosyl hydrolase (GH) repertoire of B. coagulans MA-13 under diverse growth conditions. The highest enzymatic activity was detected on β-1,4 and α-1,6-glycosidic linkages and the enzymes responsible for these activities were unambiguously identified as β-galactosidase (GH42) and α-galactosidase (GH36), respectively. Whilst the former has been found only in the cytosol, the latter is localized also extracellularly. The export of this enzyme may occur through a not yet identified secretion mechanism, since a typical signal peptide is missing in the α-galactosidase sequence. A full biochemical characterization of the recombinant β-galactosidase has been carried out and the ability of this enzyme to perform homo- and hetero-condensation reactions to produce galacto-oligosaccharides, has been demonstrated. Conclusions: Probiotics which are safe for human use and are capable of producing high levels of both α-galactosidase and β-galactosidase are of great importance to the food industry. In this work we have proven the ability of B. coagulans MA-13 to over-produce these two enzymes thus paving the way for its potential use in treatment of gastrointestinal diseases. [Figure not available: see fulltext.

Pollinator convergence and the nature of species' boundaries in sympatric Sardinian Ophrys (Orchidaceae)

Background and Aims In the sexually deceptive Ophrys genus, species isolation is generally considered ethological and occurs via different, specific pollinators, but there are cases in which Ophrys species can share a common pollinator and differ in pollen placement on the body of the insect. In that condition, species are expected to be reproductively isolated through a pre-mating mechanical barrier. Here, the relative contribution of pre- vs. post-mating barriers to gene flow among two Ophrys species that share a common pollinator and can occur in sympatry is studied. Methods A natural hybrid zone on Sardinia between O. iricolor and O. incubacea, sharing Andrena morio as pollinator, was investigated by analysing floral traits involved in pollinator attraction as odour extracts both for non-active and active compounds and for labellum morphology. The genetic architecture of the hybrid zone was also estimated with amplified fragment length polymorphism (AFLP) markers, and pollination fitness and seed set of both parental species and their hybrids in the sympatric zone were estimated by controlled crosses. Key Results Although hybrids were intermediate between parental species in labellum morphology and non-active odour compounds, both parental species and hybrids produced a similar odour bouquet for active compounds. However, hybrids produced significantly lower fruit and seed set than parental species, and the genetic architecture of the hybrid zone suggests that they were mostly first-generation hybrids. Conclusions The two parental species hybridize in sympatry as a consequence of pollinator overlap and weak mechanical isolation, but post-zygotic barriers reduce hybrid frequency and fitness, and prevent extensive introgression. These results highlight a significant contribution of late post-mating barriers, such as chromosomal divergence, for maintaining reproductive isolation, in an orchid group for which pre-mating barriers are often considered predominan

Apoptosome-deficient cells lose cytochrome c through proteasomal degradation but survive by autophagy-dependent glycolysis

Cytochrome c release from mitochondria promotes apoptosome formation and caspase activation. The question as to whether mitochondrial permeabilization kills cells via a caspase-independent pathway when caspase activation is prevented is still open. Here we report that proneural cells of embryonic origin, when induced to die but rescued by apoptosome inactivation are deprived of cytosolic cytochrome c through proteasomal degradation. We also show that, in this context, those cells keep generating ATP by glycolysis for a long period of time and that they keep their mitochondria in a depolarized state that can be reverted. Moreover, under these conditions, such apoptosome-deficient cells activate a Beclin 1-dependent autophagy pathway to sustain glycolytic-dependent ATP production. Our findings contribute to elucidating what the point-of-no-return in apoptosis is. They also help in clarifying the issue of survival of apoptosome-deficient proneural cells under stress conditions. Unraveling this issue could be highly relevant for pharmacological intervention and for therapies based on neural stem cell transfer in the treatment of neurological disorders

New label-free methods for protein relative quantification applied to the investigation of an animal model of Huntington Disease

Spectral Counts approaches (SpCs) are largely employed for the comparison of protein expression profiles in label-free (LF) differential proteomics applications. Similarly, to other comparative methods, also SpCs based approaches require a normalization procedure before Fold Changes (FC) calculation. Here, we propose new Complexity Based Normalization (CBN) methods that introduced a variable adjustment factor (f), related to the complexity of the sample, both in terms of total number of identified proteins (CBN(P)) and as total number of spectral counts (CBN(S)). Both these new methods were compared with the Normalized Spectral Abundance Factor (NSAF) and the Spectral Counts log Ratio (Rsc), by using standard protein mixtures. Finally, to test the robustness and the effectiveness of the CBNs methods, they were employed for the comparative analysis of cortical protein extract from zQ175 mouse brains, model of Huntington Disease (HD), and control animals (raw data available via ProteomeXchange with identifier PXD017471). LF data were also validated by western blot and MRM based experiments. On standard mixtures, both CBN methods showed an excellent behavior in terms of reproducibility and coefficients of variation (CVs) in comparison to the other SpCs approaches. Overall, the CBN(P) method was demonstrated to be the most reliable and sensitive in detecting small differences in protein amounts when applied to biological samples

New label-free methods for protein relative quantification applied to the investigation of an animal model of Huntington Disease

Spectral Counts approaches (SpCs) are largely employed for the comparison of protein expression profiles in label-free (LF) differential proteomics applications. Similarly, to other comparative methods, also SpCs based approaches require a normalization procedure before Fold Changes (FC) calculation. Here, we propose new Complexity Based Normalization (CBN) methods that introduced a variable adjustment factor (f), related to the complexity of the sample, both in terms of total number of identified proteins (CBN(P)) and as total number of spectral counts (CBN(S)). Both these new methods were compared with the Normalized Spectral Abundance Factor (NSAF) and the Spectral Counts log Ratio (Rsc), by using standard protein mixtures. Finally, to test the robustness and the effectiveness of the CBNs methods, they were employed for the comparative analysis of cortical protein extract from zQ175 mouse brains, model of Huntington Disease (HD), and control animals (raw data available via ProteomeXchange with identifier PXD017471). LF data were also validated by western blot and MRM based experiments. On standard mixtures, both CBN methods showed an excellent behavior in terms of reproducibility and coefficients of variation (CVs) in comparison to the other SpCs approaches. Overall, the CBN(P) method was demonstrated to be the most reliable and sensitive in detecting small differences in protein amounts when applied to biological samples

Identification and Functional Characterization of Toxoneuron nigriceps Ovarian Proteins Involved in the Early Suppression of Host Immune Response

The endophagous parasitoid Toxoneuron nigriceps (Viereck) (Hymenoptera, Braconidae) of the larval stages of the tobacco budworm Heliothis virescens (Fabricius) (Lepidoptera, Noctuidae) injects the egg, the venom, the calyx fluid, which includes a Polydnavirus (T. nigriceps BracoVirus: TnBV) and the Ovarian Proteins (OPs) into the host body during oviposition. The host metabolism and immune system are disrupted prematurely shortly after parasitization by the combined action of the TnBV, venom, and OPs. OPs are involved in the early suppression of host immune response, before TnBV infects and expresses its genes in the host tissues. In this work, we evaluated the effect of HPLC fractions deriving from in toto OPs. Two fractions caused a reduction in hemocyte viability and were subsequently tested to detect changes in hemocyte morphology and functionality. The two fractions provoked severe oxidative stress and actin cytoskeleton disruption, which might explain the high rate of hemocyte mortality, loss of hemocyte functioning, and hence the host\u2019s reduced hemocyte encapsulation ability. Moreover, through a transcriptome and proteomic approach we identify the proteins of the two fractions: eight proteins were identified that might be involved in the observed host hemocyte changes. Our findings will contribute to a better understanding of the secreted ovarian components and their role in parasitoid wasp strategy for evading host immune responses

What antarctic plants can tell us about climate changes: Temperature as a driver for metabolic reprogramming

Global warming is strongly affecting the maritime Antarctica climate and the consequent melting of perennial snow and ice covers resulted in increased colonization by plants. Colobanthus quitensis is a vascular plant highly adapted to the harsh environmental conditions of Antarctic Peninsula and understanding how the plant is responding to global warming is a new challenging target for modern cell physiology. To this aim, we performed differential proteomic analysis on C. quitensis plants grown in natural conditions compared to plants grown for one year inside open top chambers (OTCs) which determine an increase of about 4 °C at midday, mimicking the effect of global warming. A thorough analysis of the up and downregulated proteins highlighted an extensive metabolism reprogramming leading to enhanced photoprotection and oxidative stress control as well as reduced content of cell wall components. Overall, OTCs growth seems to be advantageous for C. quitensis plants which could benefit from a better CO2 diffusion into the mesophyll and a reduced ROS‐mediated photodamage

Hb Vanvitelli: A new unstable α-globin chain variant causes undiagnosed chronic haemolytic anaemia when co-inherited with deletion − α3.7

Hb variants are structurally abnormal haemoglobins which can originate a wide range of phenotypes from clinically silent conditions to very severe disorders. In many cases, diagnosis is very difficult due to the instability of Hb mutants or the occurrence of misleading symptoms, such as cyanosis or hypoxia. Here we report the case of a young female with undiagnosed chronic haemolytic anaemia and low oxygen saturation in the absence of respiratory distress. High performance liquid chromatography showed the occurrence of an abnormal peak in the HbA2 region, which disappeared few days after blood sampling. Genetic analysis of both α genes revealed the −α3.7 deletion in heterozygous state and a novel mutation c.130 T > C leading to the substitution of Phenylalanine at codon 43 with Leucine in the α1 gene. This substitution originated a new Hb variant, named Hb Vanvitelli, with a molecular mass of 15,092.2 ± 0.4 Da. Biochemical and laboratory tests described a hyper unstable Hb variant with altered oxygen affinity that was clinically significant only when co-inherited with genetic defects affecting the α2 locus. This case highlights the genetic complexity and diagnostic pitfalls of Hb variants, defined “experiments of nature” which can generate severe clinical conditions