Metastatic lung adenocarcinoma with BRCA2 mutation and longstanding disease control on olaparib, developing triple negative breast adenocarcinoma with additional BRCA2 reversion mutation: a case report.

BACKGROUND: The BRCA2 gene is a well-known tumor suppressor gene implicated in breast and ovarian cancers. BRCA1/2 mutations can be sensitive to poly ADP-ribose polymerase (PARP) inhibitors such as olaparib. However, some of these patients develop resistance to this treatment and an essential factor contributing to acquired insensitivity is the occurrence of reversion mutations in the BRCA1/2 genes. CASE PRESENTATION: We report the case of a 65-year-old Brazilian female patient who had previously been diagnosed with metastatic lung carcinoma carrying a BRCA2 mutation that had extended to the central nervous system. Following disease progression, olaparib was administered, resulting in a stabilizing effect on her condition for ~ 30 months. During a routine follow-up, a new triple-negative breast tumor was found. Genetic testing revealed the presence of two distinct BRCA2 gene mutations in the breast tumor. The original mutation (p.Val220Ilefs4) led to a frameshift, culminating in the production of a truncated and non-functional BRCA2 protein; the second mutation, K437fs22, rectified the reading frame of exon 11. Consequently, Rad51 could properly bind to BRCA2-an essential protein crucial for DNA repair. This restoration resulted in a functional BRCA2 protein, effectively elucidating the clinical resistance observed in the new breast tumor in this case. CONCLUSIONS: This case report highlights the clinical significance of comprehensive next-generation sequencing analyses for lung adenocarcinomas, both at diagnosis and upon progression. Such analyses enable informed decisions regarding targeted therapies and facilitate a deeper comprehension of resistance mechanisms.

Probing human proteins for short encrypted antimicrobial peptides reveals Hs10, a peptide with selective activity for gram-negative bacteria.

BACKGROUND: Some cationic and amphiphilic α-helical segments of proteins adsorb to prokaryotic membranes when synthesized as individual polypeptide sequences, resulting in broad and potent antimicrobial activity. However, amphiphilicity, a determinant physicochemical property for peptide-membrane interactions, can also be observed in some ß-sheets. METHODS: The software Kamal was used to scan the human reference proteome for short (7-11 amino acid residues) cationic and amphiphilic protein segments with the characteristic periodicity of ß-sheets. Some of the uncovered peptides were chemically synthesized, and antimicrobial assays were conducted. Biophysical techniques were used to probe the molecular interaction of one peptide with phospholipid vesicles, lipopolysaccharides (LPS) and the bacterium Escherichia coli. RESULTS: Thousands of compatible segments were found in human proteins, five were synthesized, and three presented antimicrobial activity in the micromolar range. Hs10, a nonapeptide fragment of the Complement C3 protein, could inhibit only the growth of tested Gram-negative microorganisms, presenting also little cytotoxicity to human fibroblasts. Hs10 interacted with LPS while transitioning from an unstructured segment to a ß-sheet and increased the hydrodynamic radius of LPS particles. This peptide also promoted morphological alterations in E. coli cells. CONCLUSIONS: Data presented herein introduce yet another molecular template to probe proteins in search for encrypted membrane-active segments and demonstrates that, using this approach, short peptides with low cytotoxicity and high selectivity to prokaryotic cells might be obtained. GENERAL SIGNIFICANCE: This work widens the biotechnological potential of the human proteome as a source of antimicrobial peptides with application in human health.

Novel disintegrin-like peptides derived from an amphibian skin cDNA sequence of Hypsiboas punctatus.

Disintegrins comprise a family of small proteins that bind to and alter the physiological function of integrins, especially integrins that mediate platelet aggregation in blood. Here, we report a lysine-glycine-aspartic acid (KGD) disintegrin-like motif present in a 15-amino acid residue peptide identified in a cDNA library of the amphibian Hypsiboas punctatus skin. The original peptide sequence was used as a template from which five new analogs were designed, chemically synthesized by solid phase, and tested for disintegrin activity and tridimensional structural studies using NMR spectroscopy. The original amphibian peptide had no effect on integrin-mediated responses. Nevertheless, derived peptide analogs inhibited integrin-mediated platelet function, including platelet spreading on fibrinogen.

Molecular Dynamics of Synthetic Flagelliform Silk Fiber Assembly.

In order to better understand the relationship between Flagelliform (Flag) spider silk molecular structural organization and the mechanisms of fiber assembly, it was designed and produced the Nephilengys cruentata Flag spidroin analogue rNcFlag2222. The recombinant proteins are composed by the elastic repetitive glycine-rich motifs (GPGGX/GGX) and the spacer region, rich in hydrophilic charged amino acids, present at the native silk spidroin. Using different approaches for nanomolecular protein analysis, the structural data of rNcFlag2222 recombinant proteins were compared in its fibrillar and in its fully solvated states. Based on the results was possible to identify the molecular structural dynamics of NcFlag2222 prior to and after fiber formation. Overal rNcFlag2222 shows a mixture of semiflexible and rigid conformations, characterized mostly by the presence of PPII, ß-turn and ß-sheet. These results agree with previous studies and bring insights about the molecular mechanisms that might driven Flag silk fibers assembly and elastomeric behavior.

Contributions of the international plant science community to the fight against infectious diseases in humans-part 2: Affordable drugs in edible plants for endemic and re-emerging diseases.

The fight against infectious diseases often focuses on epidemics and pandemics, which demand urgent resources and command attention from the health authorities and media. However, the vast majority of deaths caused by infectious diseases occur in endemic zones, particularly in developing countries, placing a disproportionate burden on underfunded health systems and often requiring international interventions. The provision of vaccines and other biologics is hampered not only by the high cost and limited scalability of traditional manufacturing platforms based on microbial and animal cells, but also by challenges caused by distribution and storage, particularly in regions without a complete cold chain. In this review article, we consider the potential of molecular farming to address the challenges of endemic and re-emerging diseases, focusing on edible plants for the development of oral drugs. Key recent developments in this field include successful clinical trials based on orally delivered dried leaves of Artemisia annua against malarial parasite strains resistant to artemisinin combination therapy, the ability to produce clinical-grade protein drugs in leaves to treat infectious diseases and the long-term storage of protein drugs in dried leaves at ambient temperatures. Recent FDA approval of the first orally delivered protein drug encapsulated in plant cells to treat peanut allergy has opened the door for the development of affordable oral drugs that can be manufactured and distributed in remote areas without cold storage infrastructure and that eliminate the need for expensive purification steps and sterile delivery by injection.

Contributions of the international plant science community to the fight against human infectious diseases - part 1: epidemic and pandemic diseases.

Infectious diseases, also known as transmissible or communicable diseases, are caused by pathogens or parasites that spread in communities by direct contact with infected individuals or contaminated materials, through droplets and aerosols, or via vectors such as insects. Such diseases cause ˜17% of all human deaths and their management and control places an immense burden on healthcare systems worldwide. Traditional approaches for the prevention and control of infectious diseases include vaccination programmes, hygiene measures and drugs that suppress the pathogen, treat the disease symptoms or attenuate aggressive reactions of the host immune system. The provision of vaccines and biologic drugs such as antibodies is hampered by the high cost and limited scalability of traditional manufacturing platforms based on microbial and animal cells, particularly in developing countries where infectious diseases are prevalent and poorly controlled. Molecular farming, which uses plants for protein expression, is a promising strategy to address the drawbacks of current manufacturing platforms. In this review article, we consider the potential of molecular farming to address healthcare demands for the most prevalent and important epidemic and pandemic diseases, focussing on recent outbreaks of high-mortality coronavirus infections and diseases that disproportionately affect the developing world.

Brazilian Group of Gastrointestinal Tumours' consensus guidelines for the management of oesophageal cancer.

Oesophageal cancer is among the ten most common types of cancer worldwide. More than 80% of the cases and deaths related to the disease occur in developing countries. Local socio-economic, epidemiologic and healthcare particularities led us to create a Brazilian guideline for the management of oesophageal and oesophagogastric junction (OGJ) carcinomas. The Brazilian Group of Gastrointestinal Tumours invited 50 physicians with different backgrounds, including radiology, pathology, endoscopy, nuclear medicine, genetics, oncological surgery, radiotherapy and clinical oncology, to collaborate. This document was prepared based on an extensive review of topics related to heredity, diagnosis, staging, pathology, endoscopy, surgery, radiation, systemic therapy (including checkpoint inhibitors) and follow-up, which was followed by presentation, discussion and voting by the panel members. It provides updated evidence-based recommendations to guide clinical management of oesophageal and OGJ carcinomas in several scenarios and clinical settings.

Seasonal differences in seminal plasma proteins from two bovine breeds adapted to a subtropical climate.

This study was designed to evaluate the seasonal expression of seminal plasma proteins from two bovine breeds adapted to a subtropical climate and their associations with post-thawing sperm and environmental characteristics. Semen samples were obtained three times in summer and three times in winter from four Crioulo Lageano and four Angus bulls. Seminal plasma was obtained by centrifugation, and the other portion of the semen was cryopreserved. Seminal plasma proteins were identified by 2D-nanoUPLC-MSE. Post-thawing assessments of sperm kinetics, morphology and membrane integrity were performed. Environmental data such as air temperature, air humidity and black globe temperature (BGT) were recorded, and the temperature-humidity index (THI) was calculated in summer and winter. Results showed that the climate varied significantly between seasons. Although no statistical differences were observed in semen quality between breeds, the protein profiles varied within and between seasons. We suggest that the most critical proteins in summer affecting sperm characteristics were TIMP-2, DNase, Clusterin, CFAH and GPx6. TIMP-2 and DNase showed a higher abundance in Crioulo Lageano in comparison with Angus, while Clusterin, CFAH and GPx6 presented a lower abundance. To the best of our knowledge, this is the first report of a recently evolved type of glutathione peroxidase, GPx6, in seminal plasma of bovines. In winter, five proteins were considered to be more critical: BSP1, BSP3, CCL2, Sulfhydryl oxidase and TIMP-2. BSP1 and TIMP-2 showed a lower abundance while BSP3, CCL2 and Sulfhydryl oxidase presented a higher abundance in this season in Crioulo Lageano in comparison with Angus.

RESUMO: Este estudo foi desenvolvido para avaliar a expressão sazonal de proteínas plasmáticas seminais de duas raças bovinas adaptadas ao clima subtropical e suas associações com espermatozóides pós-descongelamento e características ambientais. Amostras de sêmen foram obtidas três vezes no verão e três no inverno de quatro touros Crioulo Lageano e quatro Angus. O plasma seminal foi obtido por centrifugação e outra porção do sêmen foi criopreservada. As proteínas plasmáticas seminais foram identificadas por 2D-nanoUPLC-MSE. Foram realizadas avaliações pós-descongelamento da cinética espermática, morfologia e integridade da membrana. Dados ambientais como temperatura do ar, umidade do ar e temperatura do globo negro (BGT) foram registrados, e o índice temperatura-umidade (THI) foi calculado no verão e no inverno. Os resultados mostraram que o clima variou significativamente entre as estações. Embora não tenham sido observadas diferenças estatísticas na qualidade do sêmen entre as raças, os perfis proteicos variaram dentro e entre as estações. Sugerimos que as proteínas mais críticas no verão que afetam as características espermáticas foram TIMP-2, DNase, Clusterin, CFAH e GPx6. TIMP-2 e DNase apresentaram maior abundância em Crioulo Lageano em comparação com Angus, enquanto Clusterin, CFAH e GPx6 apresentaram menor abundância. Até onde sabemos, este é o primeiro relato de um tipo recentemente desenvolvido de glutationa peroxidase, GPx6, no plasma seminal de bovinos. No inverno, cinco proteínas foram consideradas mais críticas: BSP1, BSP3, CCL2, sulfidril oxidase e TIMP-2. BSP1 e TIMP-2 apresentaram menor abundância, enquanto BSP3, CCL2 e Sulfidril oxidase apresentaram maior abundância nesta temporada em Crioulo Lageano em comparação com Angus.

Efficacy and Safety of Nivolumab Plus Ipilimumab versus Sunitinib in First-line Treatment of Patients with Advanced Sarcomatoid Renal Cell Carcinoma.

PURPOSE: Patients with advanced renal cell carcinoma with sarcomatoid features (sRCC) have poor prognoses and suboptimal outcomes with targeted therapy. This post hoc analysis of the phase III CheckMate 214 trial analyzed the efficacy of nivolumab plus ipilimumab (NIVO+IPI) versus sunitinib in patients with sRCC. PATIENTS AND METHODS: Patients with sRCC were identified via independent central pathology review of archival tumor tissue or histologic classification per local pathology report. Patients were randomized 1:1 to receive nivolumab (3 mg/kg) plus ipilimumab (1 mg/kg) every 3 weeks (four doses) then nivolumab 3 mg/kg every 2 weeks, or sunitinib 50 mg orally every day (4 weeks; 6-week cycles). Outcomes in patients with sRCC were not prespecified. Endpoints in patients with sRCC and International Metastatic Renal Cell Carcinoma Database Consortium intermediate/poor-risk disease included overall survival (OS), progression-free survival (PFS) per independent radiology review, and objective response rate (ORR) per RECIST v1.1. Safety outcomes used descriptive statistics. RESULTS: Of 1,096 randomized patients in CheckMate 214, 139 patients with sRCC and intermediate/poor-risk disease and six with favorable-risk disease were identified. With 42 months' minimum follow-up in patients with sRCC and intermediate/poor-risk disease, median OS [95% confidence interval (CI)] favored NIVO+IPI [not reached (NR) (25.2-not estimable [NE]); n = 74] versus sunitinib [14.2 months (9.3-22.9); n = 65; HR, 0.45 (95% CI, 0.3-0.7; P = 0.0004)]; PFS benefits with NIVO+IPI were similarly observed [median 26.5 vs. 5.1 months; HR, 0.54 (95% CI, 0.33-0.86; P = 0.0093)]. Confirmed ORR was 60.8% with NIVO+IPI versus 23.1% with sunitinib, with complete response rates of 18.9% versus 3.1%, respectively. No new safety signals emerged. CONCLUSIONS: NIVO+IPI showed unprecedented long-term survival, response, and complete response benefits versus sunitinib in previously untreated patients with sRCC and intermediate/poor-risk disease, supporting the use of first-line NIVO+IPI for this population.See related commentary by Hwang et al., p. 5.

Proteomic analysis of human dental pulp in different clinical diagnosis.

OBJECTIVES: The present study aimed to identify proteins obtained from pulp tissue and correlate with each clinical diagnosis (healthy pulp, inflamed pulp, and necrotic pulp). MATERIALS AND METHODS: A total of forty-five molars were used. Three biological replicas were evaluated. Lysis and sonication were used for protein extraction. Protein quantification was assessed by using the Bradford technique, and shotgun proteome analysis was performed by nanoUPLC-MSE using a Synapt G2 mass spectrometer. Mass spectra data were processed using the Waters PLGS software, and protein identification was done using the human Uniprot database appended to the PLGS search engine. RESULTS: A total of 123 different proteins were identified in all evaluated pulp conditions. Among these, 66 proteins were observed for healthy pulp, 66 for inflamed pulp, and 91 for necrotic pulp. Most protein identification was related to immune response, multi-organism process, platelet activation, and stress in inflamed pulp samples compared to healthy pulp. Proteins related to cellular component organization or biogenesis, developmental process, growth, immune response, multi-organism process, response to stimulus, signaling, stress, and transport were identified in cases of apical periodontitis compared to inflamed pulp. CONCLUSIONS: The progression of the disease to inflamed pulp promoted a high abundance of proteins related to the immune system and stress. Comparing the necrotic pulp with inflamed pulp conditions, a high abundance of proteins was noticed related to metabolism, transport, and response between organisms. CLINICAL RELEVANCE: This finding may assist in future studies of new markers, understanding of tissue engineering, and development of future products.

Development of a Graphene-Based Biosensor for Detecting Recombinant Cyanovirin-N.

We present a graphene-based biosensor selective to recombinant cyanovirin-N (rCV-N), an antiviral protein that has proven to be an effective microbicide to inhibit HIV replication. We modified the graphene monolayer devices with 1-pyrenebutanoic acid succinimidyl ester, which interacts with both graphene and the primary and secondary amines of antibodies. By monitoring the change in the electrical resistance of the device, we were able to detect rCV-N in solutions in the range of 0.01 to 10 ng/mL, and found that the detection limit was 0.45 pg/mL, which is much smaller than that obtained with currently available techniques. This is important for applications of this microbicide against HIV, since it may be produced at a large scale from soya bean seeds processed using the available industrial processing technologies. The sensor showed high sensitivity, selectivity, and reproducibility.

Brazilian Group of Gastrointestinal Tumours' consensus guidelines for the management of gastric cancer.

Gastric cancer is among the ten most common types of cancer worldwide. Most cases and deaths related to the disease occur in developing countries. Local socio-economic, epidemiologic and healthcare particularities led us to create a Brazilian guideline for the management of gastric carcinomas. The Brazilian Group of Gastrointestinal Tumors (GTG) invited 50 physicians with different backgrounds, including radiology, pathology, endoscopy, nuclear medicine, genetics, oncological surgery, radiotherapy and clinical oncology, to collaborate. This document was prepared based on an extensive review of topics related to heredity, diagnosis, staging, pathology, endoscopy, surgery, radiation, systemic therapy and follow-up, which was followed by presentation, discussion, and voting by the panel members. It provides updated evidence-based recommendations to guide clinical management of gastric carcinomas in several scenarios and clinical settings.

Genetic switches designed for eukaryotic cells and controlled by serine integrases.

Recently, new serine integrases have been identified, increasing the possibility of scaling up genomic modulation tools. Here, we describe the use of unidirectional genetic switches to evaluate the functionality of six serine integrases in different eukaryotic systems: the HEK 293T cell lineage, bovine fibroblasts and plant protoplasts. Moreover, integrase activity was also tested in human cell types of therapeutic interest: peripheral blood mononuclear cells (PBMCs), neural stem cells (NSCs) and undifferentiated embryonic stem (ES) cells. The switches were composed of plasmids designed to flip two different genetic parts driven by serine integrases. Cell-based assays were evaluated by measurement of EGFP fluorescence and by molecular analysis of attL/attR sites formation after integrase functionality. Our results demonstrate that all the integrases were capable of inverting the targeted DNA sequences, exhibiting distinct performances based on the cell type or the switchable genetic sequence. These results should support the development of tunable genetic circuits to regulate eukaryotic gene expression.

Proteomics unravels new candidate genes for Meloidogyne resistance in wild Arachis.

Arachis stenosperma is a wild peanut relative exclusive to South America that harbors high levels of resistance against several pathogens, including the peanut root-knot nematode (RKN) Meloidogyne arenaria. In this study, a proteomic survey of A. stenosperma-M. arenaria interaction using 2-DE and LC-MS/MS identified approximately 1400 proteins, out of which 222 were differentially abundant (DAPs) when RKN inoculated root samples were compared to the control. Most of these DAPs were assigned to functional categories related to plant responses to pathogens including stress, glycolysis, redox and tricarboxylic acid cycle. The comparison between the transcriptome (RNA-Seq) and proteome expression changes, showed that almost 55% of these DAPs encode genes with a similar expression trend to their protein counterparts. Most of these genes were induced during RKN infection and some were related to plant defense, such as MLP-like protein 34 (MLP34), cinnamoyl-CoA reductase 1 (CCR1), enolase (ENO), alcohol dehydrogenase (ADH) and eukaryotic translation initiation factor 5A (eIF5A). The overexpression of AsMLP34 in Agrobacterium rhizogenes transgenic roots in a susceptible peanut cultivar showed a reduction in the number of M. arenaria galls and egg masses, indicating that AsMLP34 is a promising candidate gene to be exploited in breeding programs for RKN control in peanut. SIGNIFICANCE: The use of an integrated approach to compare plant-nematode transcriptional and translational data enabled the identification of a new gene, AsMLP34, for Meloidogyne resistance.

Intragenic antimicrobial peptides (IAPs) from human proteins with potent antimicrobial and anti-inflammatory activity.

Following the treads of our previous works on the unveiling of bioactive peptides encrypted in plant proteins from diverse species, the present manuscript reports the occurrence of four proof-of-concept intragenic antimicrobial peptides in human proteins, named Hs IAPs. These IAPs were prospected using the software Kamal, synthesized by solid phase chemistry, and had their interactions with model phospholipid vesicles investigated by differential scanning calorimetry and circular dichroism. Their antimicrobial activity against bacteria, yeasts and filamentous fungi was determined, along with their cytotoxicity towards erythrocytes. Our data demonstrates that Hs IAPs are capable to bind model membranes while attaining α-helical structure, and to inhibit the growth of microorganisms at concentrations as low as 1µM. Hs02, a novel sixteen residue long internal peptide (KWAVRIIRKFIKGFIS-NH2) derived from the unconventional myosin 1h protein, was further investigated in its capacity to inhibit lipopolysaccharide-induced release of TNF-α in murine macrophages. Hs02 presented potent anti-inflammatory activity, inhibiting the release of TNF-α in LPS-primed cells at the lowest assayed concentration, 0.1 µM. A three-dimensional solution structure of Hs02 bound to DPC micelles was determined by Nuclear Magnetic Resonance. Our work exemplifies how the human genome can be mined for molecules with biotechnological potential in human health and demonstrates that IAPs are actual alternatives to antimicrobial peptides as pharmaceutical agents or in their many other putative applications.

Identification and characterization of phospholipases A2 from the skin secretion of Pithecopus azureus anuran.

The present work reports the isolation, characterization and the complete sequence of a phospholipase A2 (PLA2) present in the skin secretion of Pithecopus azureus. Among several peptides and small proteins previously described by our group from some species belonging to this amphibian genus (formerly named Phyllomedusa), a 15 kDa N-glycosylated protein showing PLA2 activity was purified, assayed, sequenced and named Pa-PLA2. The Pithecopus azureus skin phospholipase A2 polypeptide chain is composed by 125 amino acid residues linked by seven disulfide bonds and two N-glycosylated sites (N67 and N108). The Pa-PLA2 enzymatic activity was qualitatively evaluated and compared to classical viperid PLA2 showing that both, native and deglycosylated Pa-PLA2 forms, are catalytically functional. The tridimensional molecular model of Pa-PLA2 indicates that the observed glycan moieties are suggestively placed far from the active site of that enzyme and therefore having little or no significant role on the direct interaction of the Pa-PLA2 catalytic pocket and its substrates.

Pan Proteome of Xanthomonas campestris pv. campestris Isolates Contrasting in Virulence.

Fully sequenced genomes of Xanthomonas campestris pv. campestris (Xcc) strains are reported. However, intra-pathovar differences are still intriguing and far from clear. In this work, the contrasting virulence between two isolates of Xcc - Xcc51 (more virulent) and XccY21 (less virulent) is evaluated by determining their pan proteome profiles. The bacteria are grown in NYG and XVM1 (optimal for induction of hrp regulon) broths and collected at the max-exponential growth phase. Shotgun proteomics reveals a total of 329 proteins when Xcc isolates are grown in XVM1. A comparison of both profiles reveals 47 proteins with significant abundance fluctuations, out of which, 39 show an increased abundance in Xcc51 and are mainly involved in virulence/adaptation mechanisms, genetic information processing, and membrane receptor/iron transport systems, such as BfeA, BtuB, Cap, Clp, Dcp, FyuA, GroEs, HpaG, Tig, and OmpP6. Several differential proteins are further analyzed by qRT-PCR, which reveals a similar expression pattern to the protein abundance. The data shed light on the complex Xcc pathogenicity mechanisms and point out a set of proteins related to the higher virulence of Xcc51. This information is essential for the development of more efficient strategies aiming at the control of black rot disease.

Meloidogyne incognita PASSE-MURAILLE (MiPM) Gene Encodes a Cell-Penetrating Protein That Interacts With the CSN5 Subunit of the COP9 Signalosome.

The pathogenicity of phytonematodes relies on secreted virulence factors to rewire host cellular pathways for the benefits of the nematode. In the root-knot nematode (RKN) Meloidogyne incognita, thousands of predicted secreted proteins have been identified and are expected to interact with host proteins at different developmental stages of the parasite. Identifying the host targets will provide compelling evidence about the biological significance and molecular function of the predicted proteins. Here, we have focused on the hub protein CSN5, the fifth subunit of the pleiotropic and eukaryotic conserved COP9 signalosome (CSN), which is a regulatory component of the ubiquitin/proteasome system. We used affinity purification-mass spectrometry (AP-MS) to generate the interaction network of CSN5 in M. incognita-infected roots. We identified the complete CSN complex and other known CSN5 interaction partners in addition to unknown plant and M. incognita proteins. Among these, we described M. incognita PASSE-MURAILLE (MiPM), a small pioneer protein predicted to contain a secretory peptide that is up-regulated mostly in the J2 parasitic stage. We confirmed the CSN5-MiPM interaction, which occurs in the nucleus, by bimolecular fluorescence complementation (BiFC). Using MiPM as bait, a GST pull-down assay coupled with MS revealed some common protein partners between CSN5 and MiPM. We further showed by in silico and microscopic analyses that the recombinant purified MiPM protein enters the cells of Arabidopsis root tips in a non-infectious context. In further detail, the supercharged N-terminal tail of MiPM (NTT-MiPM) triggers an unknown host endocytosis pathway to penetrate the cell. The functional meaning of the CSN5-MiPM interaction in the M. incognita parasitism is discussed. Moreover, we propose that the cell-penetrating properties of some M. incognita secreted proteins might be a non-negligible mechanism for cell uptake, especially during the steps preceding the sedentary parasitic phase.

Rilotumumab plus epirubicin, cisplatin, and capecitabine as first-line therapy in advanced MET-positive gastric or gastro-oesophageal junction cancer (RILOMET-1): a randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: Rilotumumab is a fully human monoclonal antibody that selectively targets the ligand of the MET receptor, hepatocyte growth factor (HGF). We aimed to assess the efficacy, safety, and pharmacokinetics of rilotumumab combined with epirubicin, cisplatin, and capecitabine, and to assess potential biomarkers, in patients with advanced MET-positive gastric or gastro-oesophageal junction adenocarcinoma. METHODS: This multicentre, randomised, double-blind, placebo-controlled, phase 3 study was done at 152 centres in 27 countries. We recruited adults (aged ≥18 years) with unresectable locally advanced or metastatic gastric or gastro-oesophageal junction adenocarcinoma, an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, MET-positive tumours (≥25% of tumour cells with membrane staining of ≥1+ staining intensity), and evaluable disease, who had not received previous systemic therapy. Eligible patients were randomly assigned (1:1) via a computerised voice response system to receive rilotumumab 15 mg/kg intravenously or placebo in combination with open-label chemotherapy (epirubicin 50 mg/m2 intravenously; cisplatin 60 mg/m2 intravenously; capecitabine 625 mg/m2 orally twice daily) in 21-day cycles for up to ten cycles. After completion of chemotherapy, patients continued to receive rilotumumab or placebo monotherapy until disease progression, intolerability, withdrawal of consent, or study termination. Randomisation was stratified by disease extent and ECOG performance status. Both patients and physicians were masked to study treatment assignment. The primary endpoint was overall survival, analysed by intention to treat. We report the final analysis. This study is registered with ClinicalTrials.gov, number NCT01697072. FINDINGS: Between Nov 7, 2012, and Nov 21, 2014, 609 patients were randomly assigned to rilotumumab plus epirubicin, cisplatin, and capecitabine (rilotumumab group; n=304) or placebo plus epirubicin, cisplatin, and capecitabine (placebo group; n=305). Study treatment was stopped early after an independent data monitoring committee found a higher number of deaths in the rilotumumab group than in the placebo group; all patients in the rilotumumab group subsequently discontinued all study treatment. Median follow-up was 7·7 months (IQR 3·6-12·0) for patients in the rilotumumab group and 9·4 months (5·3-13·1) for patients in the placebo group. Median overall survival was 8·8 months (95% CI 7·7-10·2) in the rilotumumab group compared with 10·7 months (9·6-12·4) in the placebo group (stratified hazard ratio 1·34, 95% CI 1·10-1·63; p=0·003). The most common grade 3 or worse adverse events in the rilotumumab and placebo groups were neutropenia (86 [29%] of 298 patients vs 97 [32%] of 299 patients), anaemia (37 [12%] vs 43 [14%]), and fatigue (30 [10%] vs 35 [12%]). The frequency of serious adverse events was similar in the rilotumumab and placebo groups (142 [48%] vs 149 [50%]). More deaths due to adverse events occurred in the rilotumumab group than the placebo group (42 [14%] vs 31 [10%]). In the rilotumumab group, 33 (11%) of 298 patients had fatal adverse events due to disease progression, and nine (3%) had fatal events not due to disease progression. In the placebo group, 23 (8%) of 299 patients had fatal adverse events due to disease progression, and eight (3%) had fatal events not due to disease progression. INTERPRETATION: Ligand-blocking inhibition of the MET pathway with rilotumumab is not effective in improving clinical outcomes in patients with MET-positive gastric or gastro-oesophageal adenocarcinoma. FUNDING: Amgen.

Comparative NanoUPLC-MSE analysis between magainin I-susceptible and -resistant Escherichia coli strains.

In recent years the antimicrobial peptides (AMPs) have been prospected and designed as new alternatives to conventional antibiotics. Indeed, AMPs have presented great potential toward pathogenic bacterial strains by means of complex mechanisms of action. However, reports have increasingly emerged regarding the mechanisms by which bacteria resist AMP administration. In this context, we performed a comparative proteomic study by using the total bacterial lysate of magainin I-susceptible and -resistant E. coli strains. After nanoUPLC-MSE analyses we identified 742 proteins distributed among the experimental groups, and 25 proteins were differentially expressed in the resistant strains. Among them 10 proteins involved in bacterial resistance, homeostasis, nutrition and protein transport were upregulated, while 15 proteins related to bacterial surface modifications, genetic information and ß-lactams binding-protein were downregulated. Moreover, 60 exclusive proteins were identified in the resistant strains, among which biofilm and cell wall formation and multidrug efflux pump proteins could be observed. Thus, differentially from previous studies that could only associate single proteins to AMP bacterial resistance, data here reported show that several metabolic pathways may be related to E. coli resistance to AMPs, revealing the crucial role of multiple "omics" studies in order to elucidate the global molecular mechanisms involved in this resistance.