Dual angiopoietin-2 and VEGFA inhibition elicits antitumor immunity that is enhanced by PD-1 checkpoint blockade.

Pathological angiogenesis is a hallmark of cancer and a therapeutic target. Vascular endothelial growth factor A (VEGFA) and angiopoietin-2 (ANGPT2; also known as ANG2) are proangiogenic cytokines that sustain tumor angiogenesis and limit antitumor immunity. We show that combined ANGPT2 and VEGFA blockade by a bispecific antibody (A2V) provided superior therapeutic benefits, as compared to the single agents, in both genetically engineered and transplant tumor models, including metastatic breast cancer (MMTV-PyMT), pancreatic neuroendocrine tumor (RIP1-Tag2), and melanoma.

Advances in pancreatic islet monolayer culture on glass surfaces enable super-resolution microscopy and insights into beta cell ciliogenesis and proliferation.

A robust and reproducible method for culturing monolayers of adherent and well-spread primary islet cells on glass coverslips is required for detailed imaging studies by super-resolution and live-cell microscopy. Guided by an observation that dispersed islet cells spread and adhere well on glass surfaces in neuronal co-culture and form a monolayer of connected cells, we demonstrate that in the absence of neurons, well-defined surface coatings combined with components of neuronal culture media collectively support robust attachment and growth of primary human or rat islet cells as monolayers on glass surfaces. The islet cell monolayer cultures on glass stably maintain distinct mono-hormonal insulin+, glucagon+, somatostatin+ and PP+ cells and glucose-responsive synchronized calcium signaling as well as expression of the transcription factors Pdx-1 and NKX-6.

Selective Targeting of SH2 Domain-Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies.

The binding of Src-homology 2 (SH2) domains to phosphotyrosine (pY) sites is critical for the autoinhibition and substrate recognition of the eight Src family kinases (SFKs). The high sequence conservation of the 120 human SH2 domains poses a significant challenge to selectively perturb the interactions of even the SFK SH2 family against the rest of the SH2 domains. We have developed synthetic binding proteins, termed monobodies, for six of the SFK SH2 domains with nanomolar affinity.

Cell-free reconstitution reveals centriole cartwheel assembly mechanisms.

How cellular organelles assemble is a fundamental question in biology. The centriole organelle organizes around a nine-fold symmetrical cartwheel structure typically ∼100 nm high comprising a stack of rings that each accommodates nine homodimers of SAS-6 proteins. Whether nine-fold symmetrical ring-like assemblies of SAS-6 proteins harbour more peripheral cartwheel elements is unclear.

Evaluating Nanoparticle Binding to Blood Compartment Immune Cells in High-Throughput with Flow Cytometry.

Nanoparticles are increasingly being utilized for in vivo applications, where they are implemented as carriers for drugs, contrast agents for noninvasive medical imaging, or delivery vehicles for macromolecular agents such as DNA or proteins. However, they possess many physical and chemical properties that cause them to become rapidly recognized by the immune system as a foreign body, leading to their clearance and elimination, even before they may accumulate to critical concentrations at anatomic and cellular sites of action. The techniques described in this chapter aim to identify potential interactions of test, fluorescently tagged nano-formulations with circulating immune cells, with the goal of predicting potentially problematic formulations that may be rapidly cleared following in vivo administration.

Transcellular Pathways in Lymphatic Endothelial Cells Regulate Changes in Solute Transport by Fluid Stress.

Rationale: The transport of interstitial fluid and solutes into lymphatic vessels is important for maintaining interstitial homeostasis and delivering antigens and soluble factors to the lymph node for immune surveillance. Transendothelial transport across lymphatic endothelial cells (LECs) is commonly considered to occur paracellularly, or between cell-cell junctions, and driven by local pressure and concentration gradients. However, emerging evidence suggests that LECs also play active roles in regulating interstitial solute balance and can scavenge and store antigens, raising the possibility that vesicular or transcellular pathways may be important in lymphatic solute transport.

Differential signaling networks of Bcr-Abl p210 and p190 kinases in leukemia cells defined by functional proteomics.

The two major isoforms of the oncogenic Bcr-Abl tyrosine kinase, p210 and p190, are expressed upon the Philadelphia chromosome translocation. p210 is the hallmark of chronic myelogenous leukemia, whereas p190 occurs in the majority of B-cell acute lymphoblastic leukemia. Differences in protein interactions and activated signaling pathways that may be associated with the different diseases driven by p210 and p190 are unknown.

Zika virus causes supernumerary foci with centriolar proteins and impaired spindle positioning.

Zika virus (ZIKV) causes congenital microcephaly. Although ZIKV can impair cell cycle progression and provoke apoptosis, which probably contributes to disease aetiology through depletion of neural progenitor cells, additional cellular mechanisms may be important. Here, we investigated whether ZIKV infection alters centrosome number and spindle positioning, because such defects are thought to be at the root of inherited primary autosomal recessive microcephaly (MCPH).

SMiLE-seq identifies binding motifs of single and dimeric transcription factors.

Resolving the DNA-binding specificities of transcription factors (TFs) is of critical value for understanding gene regulation. Here, we present a novel, semiautomated protein-DNA interaction characterization technology, selective microfluidics-based ligand enrichment followed by sequencing (SMiLE-seq). SMiLE-seq is neither limited by DNA bait length nor biased toward strong affinity binders; it probes the DNA-binding properties of TFs over a wide affinity range in a fast and cost-effective fashion.

Peroxiredoxin 1 Protects Telomeres from Oxidative Damage and Preserves Telomeric DNA for Extension by Telomerase.

Oxidative damage of telomeres can promote cancer, cardiac failure, and muscular dystrophy. Specific mechanisms protecting telomeres from oxidative damage have not been described. We analyzed telomeric chromatin composition during the cell cycle and show that the antioxidant enzyme peroxiredoxin 1 (PRDX1) is enriched at telomeres during S phase.

Origins of Brain Tumor Macrophages.

The ontogeny of brain-tumor-associated macrophages is poorly understood. New findings indicate that both resident microglia and blood-derived monocytes generate the pool of macrophages that infiltrate brain tumors of either primary or metastatic origin.

The matricellular protein CYR61 promotes breast cancer lung metastasis by facilitating tumor cell extravasation and suppressing anoikis.

Matricellular proteins play multiple roles in primary tumor growth, local invasion and tumor angiogenesis. However, their contribution to metastasis and the putative mechanisms involved are less well characterized. In ER-negative human breast cancer, elevated expression levels of the matricellular protein Cysteine-rich angiogenic inducer 61 (CYR61) are associated with more aggressive progression.

SNP2TFBS - a database of regulatory SNPs affecting predicted transcription factor binding site affinity.

SNP2TFBS is a computational resource intended to support researchers investigating the molecular mechanisms underlying regulatory variation in the human genome. The database essentially consists of a collection of text files providing specific annotations for human single nucleotide polymorphisms (SNPs), namely whether they are predicted to abolish, create or change the affinity of one or several transcription factor (TF) binding sites. A SNP's effect on TF binding is estimated based on a position weight matrix (PWM) model for the binding specificity of the corresponding factor.

Tandem affinity purification of AtTERT reveals putative interaction partners of plant telomerase in vivo.

The life cycle of telomerase involves dynamic and complex interactions between proteins within multiple macromolecular networks. Elucidation of these associations is a key to understanding the regulation of telomerase under diverse physiological and pathological conditions from telomerase biogenesis, through telomere recruitment and elongation, to its non-canonical activities outside of telomeres. We used tandem affinity purification coupled to mass spectrometry to build an interactome of the telomerase catalytic subunit AtTERT, using Arabidopsis thaliana suspension cultures.

Designer matrices for intestinal stem cell and organoid culture.

Epithelial organoids recapitulate multiple aspects of real organs, making them promising models of organ development, function and disease. However, the full potential of organoids in research and therapy has remained unrealized, owing to the poorly defined animal-derived matrices in which they are grown. Here we used modular synthetic hydrogel networks to define the key extracellular matrix (ECM) parameters that govern intestinal stem cell (ISC) expansion and organoid formation, and show that separate stages of the process require different mechanical environments and ECM components.

Chemical Genetic Screen Identifies Natural Products that Modulate Centriole Number.

Centrioles are microtubule-based organelles found in most eukaryotic cells and that are critical for the formation of cilia and flagella, as well as of centrosomes in animal cells. The number of centrioles must be strictly regulated in proliferating cells in order to ensure genome integrity upon cell division. Despite their importance, however, the mechanisms governing centriole assembly and number control remain incompletely understood, owing in part to a paucity of available small-molecule compounds for dissection and alteration of the underlying processes.

Analysis of Mammary Gland Phenotypes by Transplantation of the Genetically Marked Mammary Epithelium.

The mammary gland is the only organ to undergo most of its development after birth and therefore particularly attractive for studying developmental processes. In the mouse, powerful tissue recombination techniques are available that can be elegantly combined with the use of different genetically engineered mouse models to study development and differentiation in vivo.In this chapter, we describe how epithelial intrinsic gene function can by discerned by grafting mammary epithelial cells of different genotypes to wild-type recipients.

KAT2A/KAT2B-targeted acetylome reveals a role for PLK4 acetylation in preventing centrosome amplification.

Lysine acetylation is a widespread post-translational modification regulating various biological processes. To characterize cellular functions of the human lysine acetyltransferases KAT2A (GCN5) and KAT2B (PCAF), we determined their acetylome by shotgun proteomics. One of the newly identified KAT2A/2B substrate is polo-like kinase 4 (PLK4), a key regulator of centrosome duplication.

Perivascular Macrophages Limit Permeability.

Objective: Perivascular cells, including pericytes, macrophages, smooth muscle cells, and other specialized cell types, like podocytes, participate in various aspects of vascular function. However, aside from the well-established roles of smooth muscle cells and pericytes, the contributions of other vascular-associated cells are poorly understood. Our goal was to ascertain the function of perivascular macrophages in adult tissues under nonpathological conditions.

Nucleolin Targeting Impairs the Progression of Pancreatic Cancer and Promotes the Normalization of Tumor Vasculature.

Pancreatic cancer is a highly aggressive tumor, mostly resistant to the standard treatments. Nucleolin is overexpressed in cancers and its inhibition impairs tumor growth. Herein, we showed that nucleolin was overexpressed in human specimens of pancreatic ductal adenocarcinoma (PDAC) and that the overall survival significantly increased in patients with low levels of nucleolin.

Influence of Rotational Nucleosome Positioning on Transcription Start Site Selection in Animal Promoters.

The recruitment of RNA-Pol-II to the transcription start site (TSS) is an important step in gene regulation in all organisms. Core promoter elements (CPE) are conserved sequence motifs that guide Pol-II to the TSS by interacting with specific transcription factors (TFs). However, only a minority of animal promoters contains CPEs.

HRD Motif as the Central Hub of the Signaling Network for Activation Loop Autophosphorylation in Abl Kinase.

A number of structural factors modulate the activity of Abelson (Abl) tyrosine kinase, whose deregulation is often related to oncogenic processes. First, only the open conformation of the Abl kinase domain's activation loop (A-loop) favors ATP binding to the catalytic cleft. In this regard, the trans-autophosphorylation of the Y412 residue, which is located along the A-loop, favors the stability of the open conformation, in turn enhancing Abl activity.

High-resolution 3D analysis of mouse small-intestinal stroma.

Here we detail a protocol for whole-mount immunostaining of mouse small-intestinal villi that can be used to generate high-resolution 3D images of all gut cell types, including blood and lymphatic vessel cells, neurons, smooth muscle cells, fibroblasts and immune cells. The procedure describes perfusion, fixation, dissection, immunostaining, mounting, clearing, confocal imaging and quantification, using intestinal vasculature as an example. As intestinal epithelial cells prevent visualization with some antibodies, we also provide an optional protocol to remove these cells before fixation.