WFIKKN2 is a bifunctional axon guidance cue that signals through divergent DCC family receptors.

Axon pathfinding is controlled by attractive and repulsive molecular cues that activate receptors on the axonal growth cone, but the full repertoire of axon guidance molecules remains unknown. The vertebrate DCC receptor family contains the two closely related members DCC and Neogenin with prominent roles in axon guidance and three additional, divergent members - Punc, Nope, and Protogenin - for which functions in neural circuit formation have remained elusive. We identified a secreted Punc/Nope/Protogenin ligand, WFIKKN2, which guides mouse peripheral sensory axons through Nope-mediated repulsion. In contrast, WFIKKN2 attracts motor axons, but not via Nope. These findings identify WFIKKN2 as a bifunctional axon guidance cue that acts through divergent DCC family members, revealing a remarkable diversity of ligand interactions for this receptor family in nervous system wiring. One-Sentence Summary: WFIKKN2 is a ligand for the DCC family receptors Punc, Nope, and Prtg that repels sensory axons and attracts motor axons.

Biosensors as diagnostic tools in clinical applications.

Nowadays, the development of new technological solutions in the medical field, in particular biosensors, is a priority and a ground for great scientific and financial investment. From glucose sensors to highly sensible and more precise molecular tools, this biotechnological field has gone through an exponential growth, but still the applications are very limited to the future potential foreseen in the medical area. In the last decade, the advances in the genomic field have permitted the identification of specific biomarkers related to certain diseases, becoming one of the main approaches used in clinical diagnosis. Biomarkers have different clinical values, in the sense that they may provide preventive, predictive, prognostic and therapeutic response related information, not being exclusively used for diagnostic purposes, but also be applied in health management and disease treatment. Therefore, biomarkers allied with biosensors have the potential to revolutionize the way healthcare is managed. The vast choice of bioreceptors such as nucleic acids, antibodies, antigens, enzymes and even whole cells, consents the diagnosis of diseases ranging from viral and bacterial infections to cancer and metabolism disorders. The major appeal of these sensing platforms is that it provides a fast, cost-effective, reliable, highly sensitive and easy way to obtain an earlier clinical diagnosis, which can significantly affect the survival rate or patient's prognosis. This review will explore some of the most recent devices available and its clinical applications.

The neutrophil protein CD177 is a novel PDPN receptor that regulates human cancer-associated fibroblast physiology.

The cancer-associated fibroblast (CAF) marker podoplanin (PDPN) is generally correlated with poor clinical outcomes in cancer patients and thus represents a promising therapeutic target. Despite its biomedical relevance, basic aspects of PDPN biology such as its cellular functions and cell surface ligands remain poorly uncharacterized, thus challenging drug development. Here, we utilize a high throughput platform to elucidate the PDPN cell surface interactome, and uncover the neutrophil protein CD177 as a new binding partner. Quantitative proteomics analysis of the CAF phosphoproteome reveals a role for PDPN in cell signaling, growth and actomyosin contractility, among other processes. Moreover, cellular assays demonstrate that CD177 is a functional antagonist, recapitulating the phenotype observed in PDPN-deficient CAFs. In sum, starting from the unbiased elucidation of the PDPN co-receptome, our work provides insights into PDPN functions and reveals the PDPN/CD177 axis as a possible modulator of fibroblast physiology in the tumor microenvironment.

Whole-genome sequencing reveals insights into the adaptation of French Charolais cattle to Cuban tropical conditions.

BACKGROUND: In the early 20th century, Cuban farmers imported Charolais cattle (CHFR) directly from France. These animals are now known as Chacuba (CHCU) and have become adapted to the rough environmental tropical conditions in Cuba. These conditions include long periods of drought and food shortage with extreme temperatures that European taurine cattle have difficulty coping with. RESULTS: In this study, we used whole-genome sequence data from 12 CHCU individuals together with 60 whole-genome sequences from six additional taurine, indicus and crossed breeds to estimate the genetic diversity, structure and accurate ancestral origin of the CHCU animals. Although CHCU animals are assumed to form a closed population, the results of our admixture analysis indicate a limited introgression of Bos indicus. We used the extended haplotype homozygosity (EHH) approach to identify regions in the genome that may have had an important role in the adaptation of CHCU to tropical conditions. Putative selection events occurred in genomic regions with a high proportion of Bos indicus, but they were not sufficient to explain adaptation of CHCU to tropical conditions by Bos indicus introgression only. EHH suggested signals of potential adaptation in genomic windows that include genes of taurine origin involved in thermogenesis (ATP9A, GABBR1, PGR, PTPN1 and UCP1) and hair development (CCHCR1 and CDSN). Within these genes, we identified single nucleotide polymorphisms (SNPs) that may have a functional impact and contribute to some of the observed phenotypic differences between CHCU and CHFR animals. CONCLUSIONS: Whole-genome data confirm that CHCU cattle are closely related to Charolais from France (CHFR) and Canada, but also reveal a limited introgression of Bos indicus genes in CHCU. We observed possible signals of recent adaptation to tropical conditions between CHCU and CHFR founder populations, which were largely independent of the Bos indicus introgression. Finally, we report candidate genes and variants that may have a functional impact and explain some of the phenotypic differences observed between CHCU and CHFR cattle.

Intraventricular hemorrhage as clinical presentation of herpes simplex virus encephalitis. A case report and review of the literature.

OBJECTIVE: Herpes simplex virus encephalitis (HSVE) represents the most common cause of sporadic encephalitis in humans. The development of intracerebral hematomas is rare and late during the course of HSVE. To report a case of a patient with HSVE who initially presented a diffuse intracranial hemorrhage with predominant intraventricular bleeding. CASE REPORT: A 66-year-old man was admitted to the Emergency Department with acute headache. Antecedents: alcohol consumption and ethylic hepatopathy. The brain computed tomography showed acute tetraventricular hemorrhage and hydrocephalus. The blood analysis showed pancytopenia and alteration of all hepatic parameters. After external drainage of cerebrospinal fluid the patient presented a worsening of headache, disorientation, mild left hemiparesis, neck stiffness and temperature of 37.6 °C. The cerebrospinal fluid was hemorrhagic, with 3 lymphocytes/mm3, 60 mg/dL of proteins and PCR positive for Herpes simplex virus type 1. The patient improved with intravenous acyclovir, however he experienced several medical complications which caused his dead. DISCUSSION: The patient presented an atypical cerebral bleeding related to HSVE because the development of hematoma was early and the topography of hemorrhage was basically intraventricular. Probably, both atypical characteristics were related to thrombocytopenia and severe coagulation disorder. This case expands the spectrum of cerebrovascular disorders associated with HSVE.

The Immunoglobulin Superfamily Receptome Defines Cancer-Relevant Networks Associated with Clinical Outcome.

Cell surface receptors and their interactions play a central role in physiological and pathological signaling. Despite its clinical relevance, the immunoglobulin superfamily (IgSF) remains uncharacterized and underrepresented in databases. Here, we present a systematic extracellular protein map, the IgSF interactome. Using a high-throughput technology to interrogate most single transmembrane receptors for binding to 445 IgSF proteins, we identify over 500 interactions, 82% previously undocumented, and confirm more than 60 receptor-ligand pairs using orthogonal assays. Our study reveals a map of cell-type-specific interactions and the landscape of dysregulated receptor-ligand crosstalk in cancer, including selective loss of function for tumor-associated mutations. Furthermore, investigation of the IgSF interactome in a large cohort of cancer patients identifies interacting protein signatures associated with clinical outcome. The IgSF interactome represents an important resource to fuel biological discoveries and a framework for understanding the functional organization of the surfaceome during homeostasis and disease, ultimately informing therapeutic development.

SeqBreed: a python tool to evaluate genomic prediction in complex scenarios.

BACKGROUND: Genomic prediction (GP) is a method whereby DNA polymorphism information is used to predict breeding values for complex traits. Although GP can significantly enhance predictive accuracy, it can be expensive and difficult to implement. To help design optimum breeding programs and experiments, including genome-wide association studies and genomic selection experiments, we have developed SeqBreed, a generic and flexible forward simulator programmed in python3. RESULTS: SeqBreed accommodates sex and mitochondrion chromosomes as well as autopolyploidy. It can simulate any number of complex phenotypes that are determined by any number of causal loci. SeqBreed implements several GP methods, including genomic best linear unbiased prediction (GBLUP), single-step GBLUP, pedigree-based BLUP, and mass selection. We illustrate its functionality with Drosophila genome reference panel (DGRP) sequence data and with tetraploid potato genotype data. CONCLUSIONS: SeqBreed is a flexible and easy to use tool that can be used to optimize GP or genome-wide association studies. It incorporates some of the most popular GP methods and includes several visualization tools. Code is open and can be freely modified. Software, documentation, and examples are available at https://github.com/miguelperezenciso/SeqBreed.

A Platform for Extracellular Interactome Discovery Identifies Novel Functional Binding Partners for the Immune Receptors B7-H3/CD276 and PVR/CD155.

Receptors expressed on the plasma membrane and their interacting partners critically regulate cellular communication during homeostasis and disease, and as such represent main therapeutic targets. Despite its importance for drug development, receptor-ligand proteomics has remained a daunting field, in part because of the challenges associated to the study of membrane-expressed proteins. Here, to enable sensitive detection of receptor-ligand interactions in high throughput, we implement a new platform, the Conditioned Media AlphaScreen, for interrogation of a library consisting of most single transmembrane human proteins. Using this method to study key immune receptors, we identify and further validate the interleukin receptor IL20RA as the first binding partner for the checkpoint inhibitor B7-H3. Further, KIR2DL5, a natural killer cell protein that had remained orphan, is uncovered as a functional binding partner for the poliovirus receptor (PVR). This interaction is characterized using orthogonal assays, which demonstrate that PVR specifically engages KIR2DL5 on natural killer cells leading to inhibition of cytotoxicity. Altogether, these results reveal unappreciated links between protein families that may importantly influence receptor-driven functions during disease. Applicable to any target of interest, this technology represents a versatile and powerful approach for elucidation of receptor-ligand interactomes, which is essential to understand basic aspects of the biology of the plasma membrane proteins and ultimately inform the development of novel therapeutic strategies.

Corrigendum: The extracellular interactome of the human adenovirus family reveals diverse strategies for immunomodulation.

This corrects the article DOI: 10.1038/ncomms11473.

TREM2 Binds to Apolipoproteins, Including APOE and CLU/APOJ, and Thereby Facilitates Uptake of Amyloid-Beta by Microglia.

Genetic variants of TREM2, a protein expressed selectively by microglia in the brain, are associated with Alzheimer's disease (AD). Starting from an unbiased protein microarray screen, we identified a set of lipoprotein particles (including LDL) and apolipoproteins (including CLU/APOJ and APOE) as ligands of TREM2. Binding of these ligands by TREM2 was abolished or reduced by disease-associated mutations. Overexpression of wild-type TREM2 was sufficient to enhance uptake of LDL, CLU, and APOE in heterologous cells, whereas TREM2 disease variants were impaired in this activity. Trem2 knockout microglia showed reduced internalization of LDL and CLU. ß-amyloid (Aß) binds to lipoproteins and this complex is efficiently taken up by microglia in a TREM2-dependent fashion. Uptake of Aß-lipoprotein complexes was reduced in macrophages from human subjects carrying a TREM2 AD variant. These data link three genetic risk factors for AD and reveal a possible mechanism by which mutant TREM2 increases risk of AD. VIDEO ABSTRACT.

Magneto-nanosensor platform for probing low-affinity protein-protein interactions and identification of a low-affinity PD-L1/PD-L2 interaction.

Substantial efforts have been made to understand the interactions between immune checkpoint receptors and their ligands targeted in immunotherapies against cancer. To carefully characterize the complete network of interactions involved and the binding affinities between their extracellular domains, an improved kinetic assay is needed to overcome limitations with surface plasmon resonance (SPR). Here, we present a magneto-nanosensor platform integrated with a microfluidic chip that allows measurement of dissociation constants in the micromolar-range. High-density conjugation of magnetic nanoparticles with prey proteins allows multivalent receptor interactions with sensor-immobilized bait proteins, more closely mimicking natural-receptor clustering on cells. The platform has advantages over traditional SPR in terms of insensitivity of signal responses to pH and salinity, less consumption of proteins and better sensitivities. Using this platform, we characterized the binding affinities of the PD-1-PD-L1/PD-L2 co-inhibitory receptor system, and discovered an unexpected interaction between the two known PD-1 ligands, PD-L1 and PD-L2.

The extracellular interactome of the human adenovirus family reveals diverse strategies for immunomodulation.

Viruses encode secreted and cell-surface expressed proteins essential to modulate host immune defenses and establish productive infections. However, to date there has been no systematic study of the extracellular interactome of any human virus. Here we utilize the E3 proteins, diverse and rapidly evolving transmembrane-containing proteins encoded by human adenoviruses, as a model system to survey the extracellular immunomodulatory landscape. From a large-scale protein interaction screen against a microarray of more than 1,500 human proteins, we find and validate 51 previously unidentified virus-host interactions. Our results uncover conserved strategies as well as substantial diversity and multifunctionality in host targeting within and between viral species. Prominent modulation of the leukocyte immunoglobulin-like and signalling lymphocyte activation molecule families and a number of inhibitory receptors were identified as hubs for viral perturbation, suggesting unrecognized immunoregulatory strategies. We describe a virus-host extracellular interaction map of unprecedented scale that provides new insights into viral immunomodulation.

Operational redundancy in axon guidance through the multifunctional receptor Robo3 and its ligand NELL2.

Axon pathfinding is orchestrated by numerous guidance cues, including Slits and their Robo receptors, but it remains unclear how information from multiple cues is integrated or filtered. Robo3, a Robo family member, allows commissural axons to reach and cross the spinal cord midline by antagonizing Robo1/2-mediated repulsion from midline-expressed Slits and potentiating deleted in colorectal cancer (DCC)-mediated midline attraction to Netrin-1, but without binding either Slits or Netrins. We identified a secreted Robo3 ligand, neural epidermal growth factor-like-like 2 (NELL2), which repels mouse commissural axons through Robo3 and helps steer them to the midline. These findings identify NELL2 as an axon guidance cue and establish Robo3 as a multifunctional regulator of pathfinding that simultaneously mediates NELL2 repulsion, inhibits Slit repulsion, and facilitates Netrin attraction to achieve a common guidance purpose.

Baculovirus display for discovery of low-affinity extracellular receptor-ligand interactions using protein microarrays.

When used in conjunction with multivalent protein probes, protein microarrays offer a robust technology for discovery of low-affinity extracellular protein-protein interactions. Probes for receptor-matching screens generally consist of purified extracellular domains fused to affinity tags. Given that approximately two-thirds of extracellular proteins are transmembrane domain-containing proteins, it would be desirable to develop a system to express and display probe receptors in a native-like membrane environment. Toward this end, we evaluated baculovirus display as a platform for generating multivalent probes for protein microarray screens. Virion particles were generated displaying single-transmembrane domain receptors BTLA, CD200, and EFNB2, representing a range of affinities for their interacting partners. Virions directly labeled with Cy5 fluorophore were screened against a microarray containing more than 600 extracellular proteins, and the results were compared with data derived from soluble Fc protein or probe-coated protein A microbeads. An optimized protocol employing a blocking step with a nonrelated probe-expressing control baculovirus allowed identification of the expected interactions with a signal-to-noise ratio similar to or higher than those obtained with the other formats. Our results demonstrate that baculovirus display is suitable for detection of high- and low-affinity extracellular protein-protein interactions on protein microarrays. This platform eliminates the need for protein purification and provides a native-like lipid environment for membrane-associated receptors.

Defining the ligand specificity of the deleted in colorectal cancer (DCC) receptor.

The growth and guidance of many axons in the developing nervous system require Netrin-mediated activation of Deleted in Colorectal Cancer (DCC) and other still unknown signaling cues. Commissural axon guidance defects are more severe in DCC mutant mice than Netrin-1 mutant mice, suggesting additional DCC activating signals besides Netrin-1 are involved in proper axon growth. Here we report that interaction screens on extracellular protein microarrays representing over 1,000 proteins uniquely identified Cerebellin 4 (CBLN4), a member of the C1q-tumor necrosis factor (TNF) family, and Netrin-1 as extracellular DCC-binding partners. Immunofluorescence and radio-ligand binding studies demonstrate that Netrin-1 competes with CBLN4 binding at an overlapping site within the membrane-proximal fibronectin domains (FN) 4-6 of DCC and binds with ∼5-fold higher affinity. CBLN4 also binds to the DCC homolog, Neogenin-1 (NEO1), but with a lower affinity compared to DCC. CBLN4-null mice did not show a defect in commissural axons of the developing spinal cord but did display a transient increase in the number of wandering axons in the brachial plexus, consistent with a role in axon guidance. Overall, the data solidifies CBLN4 as a bona fide DCC ligand and strengthens its implication in axon guidance.

Noncanonical inflammasome activation by intracellular LPS independent of TLR4.

Gram-negative bacteria including Escherichia coli, Citrobacter rodentium, Salmonella typhimurium, and Shigella flexneri are sensed in an ill-defined manner by an intracellular inflammasome complex that activates caspase-11. We show that macrophages loaded with synthetic lipid A, E. coli lipopolysaccharide (LPS), or S. typhimurium LPS activate caspase-11 independently of the LPS receptor Toll-like receptor 4 (TLR4). Consistent with lipid A triggering the noncanonical inflammasome, LPS containing a divergent lipid A structure antagonized caspase-11 activation in response to E. coli LPS or Gram-negative bacteria. Moreover, LPS-mutant E. coli failed to activate caspase-11. Tlr4(-/-) mice primed with TLR3 agonist polyinosinic:polycytidylic acid [poly(I:C)] to induce pro-caspase-11 expression were as susceptible as wild-type mice were to sepsis induced by E. coli LPS. These data unveil a TLR4-independent mechanism for innate immune recognition of LPS.

Protein microarrays for identification of novel extracellular protein-protein interactions.

Functional protein microarrays offer the capability for high-throughput protein interaction analysis and have long promised to be a powerful tool for understanding protein interactions at the proteome scale. Although popular techniques for protein-protein interaction mapping like yeast-two-hybrid and affinity-purification mass spectrometry have performed well for identifying intracellular protein-protein interactions, the study of interactions between extracellular proteins has remained challenging for these methods. Instead, the use of protein microarrays appears to be a robust and efficient method for the identification of interactions among the members of this class of protein. This unit describes methods for extracellular protein microarray production, screening, and analysis. A protocol is described for enhanced detection of low-affinity interactions by generating multivalent complexes using Fc-fusion bait proteins and protein A microbeads, along with a statistical method for hit scoring and identification of nonspecific interactions.

A requirement for wild-type Ras isoforms in mutant KRas-driven signalling and transformation.

The mutant forms of KRas, NRas and HRas drive the initiation and progression of a number of human cancers, but less is known about the role of WT (wild-type) Ras alleles and isoforms in cancer. We used zinc-finger nucleases targeting HRas and NRas to modify both alleles of these genes in the mutant KRas-driven Hec1A endometrial cancer cell line, which normally expresses WT copies of these genes. The disruption of either WT isoform of Ras compromised growth-factor-dependent signalling through the ERK (extracellular-signal-regulated kinase) pathway. In addition, the disruption of HRas hindered the activation of Akt and subsequent downstream signalling. This was associated with decreased proliferation, increased apoptosis and decreased anchorage-independent growth in the HRas-disrupted cells. However, xenograft tumour growth was not significantly affected by the disruption of either NRas or HRas. As expected, deleting the mutant allele of KRas abolished tumour growth, whereas deletion of the remaining WT copy of KRas increased the tumorigenic properties of these cells; deleting a single copy of either HRas or NRas did not mimic this effect. The present study demonstrates that the WT copies of HRas, NRas and KRas play unique roles in the context of mutant KRas-driven tumours.

Novel IAC-LC-ESI-MS(2) analytical set-up for ochratoxin A determination in pork.

A reliable exposure assessment of mycotoxin contamination relies on their unambiguous identification and accurate quantification. With such purpose, a new analytical methodology was developed for evaluation of ochratoxin A (OTA) contamination in pork. Briefly OTA extraction from minced muscle samples involved the use of acidified methanol, ultrasound treatment and centrifugation, followed by an immunoaffinity column (IAC) clean-up step before mass spectrometric detection (precursor-to-fragment transitions: m/z 404→m/z 358 and m/z 404→m/z 386) in positive ESI mode using SRM scanning. The method combines green chemistry principles (e.g. absence of highly toxic solvents commonly used in this matrix) with a validation according to the guidelines laid down by the 2002/657/EC European Decision parameters: recoveries varied between 98.5% and 100.6%, limits of detection (LOD) and quantification (LOQ) were estimated at 0.06 and 0.19 µg/kg, whereas decision limit (CCα) and detection capability (CCß) were 0.01 and 0.50 µg/kg, respectively. The proposed analytical set-up was successfully applied to twenty pork samples commercially acquired.