Evaluating intra-fractional tumor motion in lung stereotactic radiotherapy with deep inspiration breath-hold.

PURPOSE: To evaluate the intra-fractional tumor motion in lung stereotactic body radiotherapy (SBRT) with deep inspiration breath-hold (DIBH), and to investigate the adequacy of the current planning target volume (PTV) margins. METHODS: Twenty-eight lung SBRT patients with DIBH were selected in this study. Among the lesions, twenty-three were at right or left lower lobe, two at right middle lobe, and three at right or left upper lobe. Post-treatment gated cone-beam computed tomography (CBCT) was acquired to quantify the intra-fractional tumor shift at each treatment. These obtained shifts were then used to calculate the required PTV margin, which was compared with the current applied margin of 5 mm margin in anterior-posterior (AP) and right-left (RL) directions and 8 mm in superior-inferior (SI) direction. The beam delivery time was prolonged with DIBH. The actual beam delivery time with DIBH (Tbeam_DIBH) was compared with the beam delivery time without DIBH (Tbeam_wo_DIBH) for the corresponding SBRT plan. RESULTS: A total of 113 treatments were analyzed. At six treatments (5.3%), the shifts exceeded the tolerance defined by the current PTV margin. The average shifts were 0.0 ± 1.9 mm, 0.1±1.5 mm, and -0.5 ± 3.7 mm in AP, RL, and SI directions, respectively. The required PTV margins were determined to be 4.5, 3.9, and 7.4 mm in AP, RL, and SI directions, respectively. The average Tbeam_wo_DIBH and Tbeam_DIBH were 2.4 ± 0.4 min and 3.6 ± 1.5 min, respectively. The average treatment slot for lung SBRT with DIBH was 25.3 ± 7.9 min. CONCLUSION: Intra-fractional tumor motion is the predominant source of treatment uncertainties in CBCT-guided lung SBRT with DIBH. The required PTV margin should be determined based on data specific to each institute, considering different techniques and populations. Our data indicate that our current applied PTV margin is adequate, and it is possible to reduce further in the RL direction. The time increase of Tbeam_DIBH, relative to the treatment slot, is not clinically significant.

Isolated Valve Amyloid Deposition in Aortic Stenosis: Potential Clinical and Pathophysiological Relevance.

Amyloid deposition within stenotic aortic valves (AVs) also appears frequent in the absence of cardiac amyloidosis, but its clinical and pathophysiological relevance has not been investigated. We will elucidate the rate of isolated AV amyloid deposition and its potential clinical and pathophysiological significance in aortic stenosis (AS). In 130 patients without systemic and/or cardiac amyloidosis, we collected the explanted AVs during cardiac surgery: 57 patients with calcific AS and 73 patients with AV insufficiency (41 with AV sclerosis and 32 without, who were used as controls). Amyloid deposition was found in 21 AS valves (37%), 4 sclerotic AVs (10%), and none of the controls. Patients with and without isolated AV amyloid deposition had similar clinical and echocardiographic characteristics and survival rates. Isolated AV amyloid deposition was associated with higher degrees of AV fibrosis (p = 0.0082) and calcification (p < 0.0001). Immunohistochemistry analysis suggested serum amyloid A1 (SAA1), in addition to transthyretin (TTR), as the protein possibly involved in AV amyloid deposition. Circulating SAA1 levels were within the normal range in all groups, and no difference was observed in AS patients with and without AV amyloid deposition. In vitro, AV interstitial cells (VICs) were stimulated with interleukin (IL)-1ß which induced increased SAA1-mRNA both in the control VICs (+6.4 ± 0.5, p = 0.02) and the AS VICs (+7.6 ± 0.5, p = 0.008). In conclusion, isolated AV amyloid deposition is frequent in the context of AS, but it does not appear to have potential clinical relevance. Conversely, amyloid deposition within AV leaflets, probably promoted by local inflammation, could play a role in AS pathophysiology.

Myoglobin expression by alternative transcript in different mesenchymal stem cells compartments.

BACKGROUND: The metabolic phenotype of stem cells is increasingly recognized as a hallmark of their pluripotency with mitochondrial and oxygen-related metabolism playing a not completely defined role in this context. In a previous study, we reported the ectopic expression of myoglobin (MB) in bone marrow-derived hematopoietic stem/progenitor cells. Here, we have extended the analysis to mesenchymal stem cells (MSCs) isolated from different tissues. METHODS: MSCs were isolated from human placental membrane, mammary adipose tissue and dental pulp and subjected to RT-PCR, Western blotting and mass spectrometry to investigate the expression of MB. A combination of metabolic flux analysis and cyto-imaging was used to profile the metabolic phenotype and the mitochondria dynamics in the different MSCs. RESULTS: As for the hematopoietic stem/progenitor cells, the expression of Mb was largely driven by an alternative transcript with the protein occurring both in the monomer and in the dimer forms as confirmed by mass spectrometry analysis. Comparing the metabolic fluxes between neonatal placental membrane-derived and adult mammary adipose tissue-derived MSCs, we showed a significantly more active bioenergetics profile in the former that correlated with a larger co-localization of myoglobin with the mitochondrial compartment. Differences in the structure of the mitochondrial network as well as in the expression of factors controlling the organelle dynamics were also observed between neonatal and adult mesenchymal stem cells. Finally, the expression of myoglobin was found to be strongly reduced following osteogenic differentiation of dental pulp-derived MSCs, while it was upregulated following reprogramming of human fibroblasts to induce pluripotent stem cells. CONCLUSIONS: Ectopic expression of myoglobin in tissues other than muscle raises the question of understanding its function therein. Properties in addition to the canonical oxygen storage/delivery have been uncovered. Finding of Mb expressed via an alternative gene transcript in the context of different stem cells with metabolic phenotypes, its loss during differentiation and recovery in iPSCs suggest a hitherto unappreciated role of Mb in controlling the balance between aerobic metabolism and pluripotency. Understanding how Mb contributes through modulation of the mitochondrial physiology to the stem cell biology paves the way to novel perspectives in regenerative medicine as well as in cancer stem cell therapy.

A Hyperthermoactive-Cas9 Editing Tool Reveals the Role of a Unique Arsenite Methyltransferase in the Arsenic Resistance System of Thermus thermophilus HB27.

Arsenic detoxification systems can be found in a wide range of organisms, from bacteria to humans. In a previous study, we discovered an arsenic-responsive transcriptional regulator in the thermophilic bacterium Thermus thermophilus HB27 (TtSmtB). Here, we characterize the arsenic resistance system of T. thermophilus in more detail. We employed TtSmtB-based pulldown assays with protein extracts from cultures treated with arsenate and arsenite to obtain an S-adenosyl-l-methionine (SAM)-dependent arsenite methyltransferase (TtArsM). In vivo and in vitro analyses were performed to shed light on this new component of the arsenic resistance network and its peculiar catalytic mechanism. Heterologous expression of TtarsM in Escherichia coli resulted in arsenite detoxification at mesophilic temperatures. Although TtArsM does not contain a canonical arsenite binding site, the purified protein does catalyze SAM-dependent arsenite methylation with formation of monomethylarsenites (MMAs) and dimethylarsenites (DMAs). In addition, in vitro analyses confirmed the unique interaction between TtArsM and TtSmtB. Next, a highly efficient ThermoCas9-based genome-editing tool was developed to delete the TtArsM-encoding gene on the T. thermophilus genome and to confirm its involvement in the arsenite detoxification system. Finally, the TtarsX efflux pump gene in the T. thermophilus ΔTtarsM genome was substituted by a gene encoding a stabilized yellow fluorescent protein (sYFP) to create a sensitive genome-based bioreporter system for the detection of arsenic ions. IMPORTANCE We here describe the discovery of an unknown protein by using a proteomics approach with a transcriptional regulator as bait. Remarkably, we successfully obtained a novel type of enzyme through the interaction with a transcriptional regulator controlling the expression of this enzyme. Employing this strategy, we isolated TtArsM, the first thermophilic prokaryotic arsenite methyltransferase, as a new enzyme of the arsenic resistance mechanism in T. thermophilus HB27. The atypical arsenite binding site of TtArsM categorizes the enzyme as the first member of a new arsenite methyltransferase type, exclusively present in the Thermus genus. The enzyme methylates arsenite-producing MMAs and DMAs. Furthermore, we developed an hyperthermophilic Cas9-based genome-editing tool, active up to 65°C. The tool allowed us to perform highly efficient, marker-free modifications (either gene deletion or insertion) in the T. thermophilus genome. With these modifications, we confirmed the critical role of TtArsM in the arsenite detoxification system and developed a sensitive whole-cell bioreporter for arsenic ions. We anticipate that the developed tool can be easily adapted for editing the genomes of other thermophilic bacteria, significantly boosting fundamental and metabolic engineering in hyperthermophilic microorganisms.

Therapeutic targeting of Lyn kinase to treat chorea-acanthocytosis.

Chorea-Acanthocytosis (ChAc) is a devastating, little understood, and currently untreatable neurodegenerative disease caused by VPS13A mutations. Based on our recent demonstration that accumulation of activated Lyn tyrosine kinase is a key pathophysiological event in human ChAc cells, we took advantage of Vps13a-/- mice, which phenocopied human ChAc. Using proteomic approach, we found accumulation of active Lyn, γ-synuclein and phospho-tau proteins in Vps13a-/- basal ganglia secondary to impaired autophagy leading to neuroinflammation. Mice double knockout Vps13a-/- Lyn-/- showed normalization of red cell morphology and improvement of autophagy in basal ganglia. We then in vivo tested pharmacologic inhibitors of Lyn: dasatinib and nilotinib. Dasatinib failed to cross the mouse brain blood barrier (BBB), but the more specific Lyn kinase inhibitor nilotinib, crosses the BBB. Nilotinib ameliorates both Vps13a-/- hematological and neurological phenotypes, improving autophagy and preventing neuroinflammation. Our data support the proposal to repurpose nilotinib as new therapeutic option for ChAc patients.

An integrated transcriptomic and proteomic approach to identify the main Torymus sinensis venom components.

During oviposition, ectoparasitoid wasps not only inject their eggs but also a complex mixture of proteins and peptides (venom) in order to regulate the host physiology to benefit their progeny. Although several endoparasitoid venom proteins have been identified, little is known about the components of ectoparasitoid venom. To characterize the protein composition of Torymus sinensis Kamijo (Hymenoptera: Torymidae) venom, we used an integrated transcriptomic and proteomic approach and identified 143 venom proteins. Moreover, focusing on venom gland transcriptome, we selected additional 52 transcripts encoding putative venom proteins. As in other parasitoid venoms, hydrolases, including proteases, phosphatases, esterases, and nucleases, constitute the most abundant families in T. sinensis venom, followed by protease inhibitors. These proteins are potentially involved in the complex parasitic syndrome, with different effects on the immune system, physiological processes and development of the host, and contribute to provide nutrients to the parasitoid progeny. Although additional in vivo studies are needed, initial findings offer important information about venom factors and their putative host effects, which are essential to ensure the success of parasitism.

Tyrosine Phosphorylation Modulates Peroxiredoxin-2 Activity in Normal and Diseased Red Cells.

Peroxiredoxin-2 (Prx2) is the third most abundant cytoplasmic protein in red blood cells. Prx2 belongs to a well-known family of antioxidants, the peroxiredoxins (Prxs), that are widely expressed in mammalian cells. Prx2 is a typical, homodimeric, 2-Cys Prx that uses two cysteine residues to accomplish the task of detoxifying a vast range of organic peroxides, H2O2, and peroxynitrite. Although progress has been made on functional characterization of Prx2, much still remains to be investigated on Prx2 post-translational changes. Here, we first show that Prx2 is Tyrosine (Tyr) phosphorylated by Syk in red cells exposed to oxidation induced by diamide. We identified Tyr-193 in both recombinant Prx2 and native Prx2 from red cells as a specific target of Syk. Bioinformatic analysis suggests that phosphorylation of Tyr-193 allows Prx2 conformational change that is more favorable for its peroxidase activity. Indeed, Syk-induced Tyr phosphorylation of Prx2 enhances in vitro Prx2 activity, but also contributes to Prx2 translocation to the membrane of red cells exposed to diamide. The biologic importance of Tyr-193 phospho-Prx2 is further supported by data on red cells from a mouse model of humanized sickle cell disease (SCD). SCD is globally distributed, hereditary red cell disorder, characterized by severe red cell oxidation due to the pathologic sickle hemoglobin. SCD red cells show Tyr-phosphorylated Prx2 bound to the membrane and increased Prx2 activity when compared to healthy erythrocytes. Collectively, our data highlight the novel link between redox related signaling and Prx2 function in normal and diseased red cells.

A bioinformatic study of antimicrobial peptides identified in the Black Soldier Fly (BSF) Hermetia illucens (Diptera: Stratiomyidae).

Antimicrobial peptides (AMPs) play a key role in the innate immunity, the first line of defense against bacteria, fungi, and viruses. AMPs are small molecules, ranging from 10 to 100 amino acid residues produced by all living organisms. Because of their wide biodiversity, insects are among the richest and most innovative sources for AMPs. In particular, the insect Hermetia illucens (Diptera: Stratiomyidae) shows an extraordinary ability to live in hostile environments, as it feeds on decaying substrates, which are rich in microbial colonies, and is one of the most promising sources for AMPs. The larvae and the combined adult male and female H. illucens transcriptomes were examined, and all the sequences, putatively encoding AMPs, were analysed with different machine learning-algorithms, such as the Support Vector Machine, the Discriminant Analysis, the Artificial Neural Network, and the Random Forest available on the CAMP database, in order to predict their antimicrobial activity. Moreover, the iACP tool, the AVPpred, and the Antifp servers were used to predict the anticancer, the antiviral, and the antifungal activities, respectively. The related physicochemical properties were evaluated with the Antimicrobial Peptide Database Calculator and Predictor. These analyses allowed to identify 57 putatively active peptides suitable for subsequent experimental validation studies.

Loss of Function of the Gene Encoding the Histone Methyltransferase KMT2D Leads to Deregulation of Mitochondrial Respiration.

KMT2D encodes a methyltransferase responsible for histone 3 lysine 4 (H3K4) mono-/di-methylation, an epigenetic mark correlated with active transcription. Here, we tested the hypothesis that KMT2D pathogenic loss-of-function variants, which causes the Kabuki syndrome type 1, could affect the mitochondrial metabolic profile. By using Seahorse technology, we showed a significant reduction of the mitochondrial oxygen consumption rate as well as a reduction of the glycolytic flux in both Kmt2d knockout MEFs and skin fibroblasts of Kabuki patients harboring heterozygous KMT2D pathogenic variants. Mass-spectrometry analysis of intermediate metabolites confirmed alterations in the glycolytic and TCA cycle pathways. The observed metabolic phenotype was accompanied by a significant increase in the production of reactive oxygen species. Measurements of the specific activities of the mitochondrial respiratory chain complexes revealed significant inhibition of CI (NADH dehydrogenase) and CIV (cytochrome c oxidase); this result was further supported by a decrease in the protein content of both complexes. Finally, we unveiled an impaired oxidation of glucose and larger reliance on long-chain fatty acids oxidation. Altogether, our findings clearly indicate a rewiring of the mitochondrial metabolic phenotype in the KMT2D-null or loss-of-function context that might contribute to the development of Kabuki disease, and represents metabolic reprogramming as a potential new therapeutic approach.

The interaction between the F55 virus-encoded transcription regulator and the RadA host recombinase reveals a common strategy in Archaea and Bacteria to sense the UV-induced damage to the host DNA.

Sulfolobus spindle-shaped virus 1 is the only UV-inducible member of the virus family Fuselloviridae. Originally isolated from Saccharolobus shibatae B12, it can also infect Saccharolobus solfataricus. Like the CI repressor of the bacteriophage λ, the SSV1-encoded F55 transcription repressor acts as a key regulator for the maintenance of the SSV1 carrier state. In particular, F55 binds to tandem repeat sequences located within the promoters of the early and UV-inducible transcripts. Upon exposure to UV light, a temporally coordinated pattern of gene expression is triggered. In the case of the better characterized bacteriophage λ, the switch from lysogenic to lytic development is regulated by a crosstalk between the virus encoded CI repressor and the host RecA, which regulates also the SOS response. For SSV1, instead, the regulatory mechanisms governing the switch from the carrier to the induced state have not been completely unravelled. In this study we have applied an integrated biochemical approach based on a variant of the EMSA assay coupled to mass spectrometry analyses to identify the proteins associated with F55 when bound to its specific DNA promoter sequences. Among the putative F55 interactors, we identified RadA and showed that the archaeal molecular components F55 and RadA are functional homologs of bacteriophage λ (factor CI) and Escherichia coli (RecA) system.

TRIM8 interacts with KIF11 and KIFC1 and controls bipolar spindle formation and chromosomal stability.

The faithful inheritance of chromosomes is essential for the propagation of organisms. In eukaryotes, central to this process is the mitotic spindle. Recently, we have identified TRIM8 as a gene aberrantly expressed in gliomas whose expression reduces the clonogenic potential in the patients' glioma cells. TRIM8 encodes an E3 ubiquitin ligase involved in various pathological processes, including hypertrophy, antiviral defense, encephalopathy, and cancer development. To gain insights into the TRIM8 functions, we characterized the TRIM8 interactome in primary mouse embryonic neural stem cells using proteomics. We found that TRIM8 interacts with KIFC1, and KIF11/Eg5, two master regulators of mitotic spindle assembly and cytoskeleton reorganization. By exploring the TRIM8 role in the mitotic spindle machinery, we showed that TRIM8 localizes at the mitotic spindle during mitosis and plays a role in centrosome separation at the beginning of mitosis with a subsequent delay of the mitotic progression and impact on chromosomal stability.

Venomics of the ectoparasitoid wasp Bracon nigricans.

BACKGROUND: Venom is one of the most important sources of regulation factors used by parasitic Hymenoptera to redirect host physiology in favour of the developing offspring. This has stimulated a number of studies, both at functional and "omics" level, which, however, are still quite limited for ectophagous parasitoids that permanently paralyze and suppress their victims (i.e., idiobiont parasitoids). RESULTS: Here we present a combined transcriptomic and proteomic study of the venom of the generalist idiobiont wasp Bracon nigricans, an ectophagous larval parasitoid of different lepidopteran species, for which we recently described the host regulation strategy and the functional role of the venom in the induction of physiological changes in parasitized hosts. The experimental approach used led to the identification of the main components of B. nigricans venom involved in host regulation. Enzymes degrading lipids, proteins and carbohydrates are likely involved in the mobilization of storage nutrients from the fat body and may concurrently be responsible for the release of neurotoxic fatty acids inducing paralysis, and for the modulation of host immune responses. CONCLUSION: The present work contributes to fill the gap of knowledge on venom composition in ectoparasitoid wasps, and, along with our previous physiological study on this species, provides the foundation on which to develop a functional model of host regulation, based both on physiological and molecular data. This paves the way towards a better understanding of parasitism evolution in the basal lineages of Hymenoptera and to the possible exploitation of venom as source of bioinsecticidal molecules.

Evolution of an insect immune barrier through horizontal gene transfer mediated by a parasitic wasp.

Genome sequencing data have recently demonstrated that eukaryote evolution has been remarkably influenced by the acquisition of a large number of genes by horizontal gene transfer (HGT) across different kingdoms. However, in depth-studies on the physiological traits conferred by these accidental DNA acquisitions are largely lacking. Here we elucidate the functional role of Sl gasmin, a gene of a symbiotic virus of a parasitic wasp that has been transferred to an ancestor of the moth species Spodoptera littoralis and domesticated. This gene is highly expressed in circulating immune cells (haemocytes) of larval stages, where its transcription is rapidly boosted by injection of microorganisms into the body cavity. RNAi silencing of Sl gasmin generates a phenotype characterized by a precocious suppression of phagocytic activity by haemocytes, which is rescued when these immune cells are incubated in plasma samples of control larvae, containing high levels of the encoded protein. Proteomic analysis demonstrates that the protein Sl gasmin is released by haemocytes into the haemolymph, where it opsonizes the invading bacteria to promote their phagocytosis, both in vitro and in vivo. Our results show that important physiological traits do not necessarily originate from evolution of pre-existing genes, but can be acquired by HGT events, through unique pathways of symbiotic evolution. These findings indicate that insects can paradoxically acquire selective advantages with the help of their natural enemies.

The extreme hyper-reactivity of Cys94 in lysozyme avoids its amorphous aggregation.

Many proteins provided with disulfide bridges in the native state undergo amorphous irreversible aggregation when these bonds are not formed. Here we show that egg lysozyme displays a clever strategy to prevent this deleterious aggregation during the nascent phase when disulfides are still absent. In fact, when the reduced protein assembles into a molten globule state, its cysteines acquire strong hyper-reactivity towards natural disulfides. The most reactive residue, Cys94, reacts with oxidized glutathione (GSSG) 3000 times faster than an unperturbed protein cysteine. A low pKa of its sulfhydryl group (6.6/7.1) and a productive complex with GSSG (KD = 0.3 mM), causes a fast glutathionylation of this residue (t1/2 = 3 s) and a complete inhibition of the protein aggregation. Other six cysteines display 70 times higher reactivity toward GSSG. The discovery of extreme hyper-reactivity in cysteines only devoted to structural roles opens new research fields for Alzheimer's and Parkinson diseases.

The multifunctional polydnavirus TnBVANK1 protein: impact on host apoptotic pathway.

Toxoneuron nigriceps (Hymenoptera, Braconidae) is an endophagous parasitoid of the larval stages of the tobacco budworm, Heliothis virescens (Lepidoptera, Noctuidae). The bracovirus associated with this wasp (TnBV) is currently being studied. Several genes expressed in parasitised host larvae have been isolated and their possible roles partly elucidated. TnBVank1 encodes an ankyrin motif protein similar to insect and mammalian IκB, an inhibitor of the transcription nuclear factor κB (NF-κB). Here we show that, when TnBVank1 was stably expressed in polyclonal Drosophila S2 cells, apoptosis is induced. Furthermore, we observed the same effects in haemocytes of H. virescens larvae, after TnBVank1 in vivo transient transfection, and in haemocytes of parasitised larvae. Coimmunoprecipitation experiments showed that TnBVANK1 binds to ALG-2 interacting protein X (Alix/AIP1), an interactor of apoptosis-linked gene protein 2 (ALG-2). Using double-immunofluorescence labeling, we observed the potential colocalization of TnBVANK1 and Alix proteins in the cytoplasm of polyclonal S2 cells. When Alix was silenced by RNA interference, TnBVANK1 was no longer able to cause apoptosis in both S2 cells and H. virescens haemocytes. Collectively, these results indicate that TnBVANK1 induces apoptosis by interacting with Alix, suggesting a role of TnBVANK1 in the suppression of host immune response observed after parasitisation by T. nigriceps.

A Novel Pathogenic BRCA1 Splicing Variant Produces Partial Intron Retention in the Mature Messenger RNA.

About 10% of all breast cancers arise from hereditary mutations that increase the risk of breast and ovarian cancers; and about 25% of these are associated with the BRCA1 or BRCA2 genes. The identification of BRCA1/BRCA2 mutations can enable physicians to better tailor the clinical management of patients; and to initiate preventive measures in healthy carriers. The pathophysiological significance of newly identified variants poses challenges for genetic counseling. We characterized a new BRCA1 variant discovered in a breast cancer patient during BRCA1/2 screening by next-generation sequencing. Bioinformatic predictions; indicating that the variant is probably pathogenetic; were verified using retro-transcription of the patient's RNA followed by PCR amplifications performed on the resulting cDNA. The variant causes the loss of a canonic donor splice site at position +2 in BRCA1 intron 21; and consequently the partial retention of 156 bp of intron 21 in the patient's transcript; which demonstrates that this novel BRCA1 mutation plays a pathogenetic role in breast cancer. These findings enabled us to initiate appropriate counseling and to tailor the clinical management of this family. Lastly; these data reinforce the importance of studying the effects of sequence variants at the RNA level to verify their potential role in disease onset.

Identification of major Toxoneuron nigriceps venom proteins using an integrated transcriptomic/proteomic approach.

Endoparasitoids in the order Hymenoptera are natural enemies of several herbivorous insect pest species. During oviposition they inject a mixture of factors, which include venom, into the host, ensuring the successful parasitism and the development of their progeny. Although these parasitoid factors are known to be responsible for host manipulation, such as immune system suppression, little is known about both identity and function of the majority of their venom components. To identify the major proteins of Toxoneuron nigriceps (Hymenoptera: Braconidae) venom, we used an integrated transcriptomic and proteomic approach. The tandem-mass spectrometric (LC-MS/MS) data combined with T. nigriceps venom gland transcriptome used as a reference database resulted in the identification of a total of thirty one different proteins. While some of the identified proteins have been described in venom from several parasitoids, others were identified for the first time. Among the identified proteins, hydrolases constituted the most abundant family followed by transferases, oxidoreductases, ligases, lyases and isomerases. The hydrolases identified in the T. nigriceps venom glands included proteases, peptidases and glycosidases, reported as common components of venom from several parasitoid species. Taken together, the identified proteins included factors that could potentially inhibit the host immune system, manipulate host physiological processes and host development, as well as provide nutrients to the parasitoid progeny, degrading host tissues by specific hydrolytic enzymes. The venom decoding provides us with information about the identity of candidate venom factors which could contribute to the success of parasitism, together with other maternal and embryonic factors.

Proteome analysis of human amniotic mesenchymal stem cells (hA-MSCs) reveals impaired antioxidant ability, cytoskeleton and metabolic functionality in maternal obesity.

Maternal obesity increases the risk of obesity and/or obesity-related diseases in the offspring of animal models. The aim of this study was to identify metabolic dysfunctions that could represent an enhanced risk for human obesity or obesity-related diseases in newborn or in adult life, similar to what occurs in animal models. To this aim, we studied the proteome of 12 obese (Ob-) and 6 non-obese (Co-) human amniotic mesenchymal stem cells (hA-MSCs) obtained from women at delivery by cesarean section (pre-pregnancy body mass index [mean ± SD]: 42.7 ± 7.7 and 21.3 ± 3.3 kg/m(2), respectively). The proteome, investigated by two-dimensional fluorescence difference gel electrophoresis/mass spectrometry, revealed 62 differently expressed proteins in Ob- vs Co-hA-MSCs (P < 0.05), nine of which were confirmed by western blotting. Bioinformatics analysis showed that these 62 proteins are involved in several statistically significant pathways (P < 0.05), including the stress response, cytoskeleton and metabolic pathways. Oxidative stress was shown to be an early triggering factor of tissue fat accumulation and obesity-related disorders in the offspring of obese animal models. Our finding of a reduced stress response in Ob-hA-MSCs suggests that a similar mechanism could occur also in humans. Long-term follow-up studies of newborns of obese mothers are required to verify this hypothesis.

Deglycosylation Step to Improve the Identification of Egg Proteins in Art Samples.

A deglycosylation step using Peptide-N-Glycosidase F (PNGaseF) has been introduced in a standard proteomic protocol to more confidently identify egg based binders. The ingenuity of introducing a PNGaseF digestion was aimed at removing the molecular hindrance, made up by the heavily glycosylated egg proteins, before the protease(s) hydrolysis. This novelty in the protocol resulted in obtaining a significant increase of proteolytic egg peptides thus improving the quality and reliability of egg identification in artwork samples. The protocol has been set up on paint replicas and successfully tested on two historical samples of different origin.

Dosimetric experience with 2 commercially available multilumen balloon-based brachytherapy to deliver accelerated partial-breast irradiation.

The purpose of this work was to report dosimetric experience with 2 kinds of multilumen balloon (MLB), 5-lumen Contura MLB (C-MLB) and 4-lumen MammoSite MLB (MS-MLB), to deliver accelerated partial-breast irradiation, and compare the ability to achieve target coverage and control skin and rib doses between 2 groups of patients treated with C-MLB and MS-MLB brachytherapy. C-MLB has 5 lumens, the 4 equal-spaced peripheral lumens are 5 mm away from the central lumen. MS-MLB has 4 lumens, the 3 equal-spaced peripheral lumens are 3 mm away from the central lumen. In total, 43 patients were treated, 23 with C-MLB, and 20 with MS-MLB. For C-MLB group, 8 patients were treated with a skin spacing < 7 mm and 12 patients with rib spacing < 7 mm. For MS-MLB group, 2 patients were treated with a skin spacing < 7 mm and 5 patients with rib spacing < 7 mm. The dosimetric goals were (1) ≥ 95% of the prescription dose (PD) covering ≥ 95% of the target volume (V(95%) ≥ 95%), (2) maximum skin dose ≤ 125% of the PD, (3) maximum rib dose ≤ 145% of the PD (if possible), and (4) the V(150%) ≤ 50 cm(3) and V(200%) ≤ 10 cm(3). All dosimetric criteria were met concurrently in 82.6% of C-MLB patients, in 80.0% of MS-MLB patients, and in 81.4% of all 43 patients. For each dosimetric parameter, t-test of these 2 groups showed p > 0.05. Although the geometric design of C-MLB is different from that of MS-MLB, both applicators have the ability to shape the dose distribution and to provide good target coverage, while limiting the dose to skin and rib. No significant difference was observed between the 2 patient groups in terms of target dose coverage and dose to organs at risk.