Large T cell clones expressing immune checkpoints increase during multiple myeloma evolution and predict treatment resistance.

Tumor recognition by T cells is essential for antitumor immunity. A comprehensive characterization of T cell diversity may be key to understanding the success of immunomodulatory drugs and failure of PD-1 blockade in tumors such as multiple myeloma (MM). Here, we use single-cell RNA and T cell receptor sequencing to characterize bone marrow T cells from healthy adults (n = 4) and patients with precursor (n = 8) and full-blown MM (n = 10). Large T cell clones from patients with MM expressed multiple immune checkpoints, suggesting a potentially dysfunctional phenotype. Dual targeting of PD-1 + LAG3 or PD-1 + TIGIT partially restored their function in mice with MM. We identify phenotypic hallmarks of large intratumoral T cell clones, and demonstrate that the CD27- and CD27+ T cell ratio, measured by flow cytometry, may serve as a surrogate of clonal T cell expansions and an independent prognostic factor in 543 patients with MM treated with lenalidomide-based treatment combinations.

In vivo screening characterizes chromatin factor functions during normal and malignant hematopoiesis.

Cellular differentiation requires extensive alterations in chromatin structure and function, which is elicited by the coordinated action of chromatin and transcription factors. By contrast with transcription factors, the roles of chromatin factors in differentiation have not been systematically characterized. Here, we combine bulk ex vivo and single-cell in vivo CRISPR screens to characterize the role of chromatin factor families in hematopoiesis. We uncover marked lineage specificities for 142 chromatin factors, revealing functional diversity among related chromatin factors (i.e. barrier-to-autointegration factor subcomplexes) as well as shared roles for unrelated repressive complexes that restrain excessive myeloid differentiation. Using epigenetic profiling, we identify functional interactions between lineage-determining transcription factors and several chromatin factors that explain their lineage dependencies. Studying chromatin factor functions in leukemia, we show that leukemia cells engage homeostatic chromatin factor functions to block differentiation, generating specific chromatin factor-transcription factor interactions that might be therapeutically targeted. Together, our work elucidates the lineage-determining properties of chromatin factors across normal and malignant hematopoiesis.

Circulating Tumor and Immune Cells for Minimally Invasive Risk Stratification of Smoldering Multiple Myeloma.

PURPOSE: Early intervention in smoldering multiple myeloma (SMM) requires optimal risk stratification to avoid under- and overtreatment. We hypothesized that replacing bone marrow (BM) plasma cells (PC) for circulating tumor cells (CTC), and adding immune biomarkers in peripheral blood (PB) for the identification of patients at risk of progression due to lost immune surveillance, could improve the International Myeloma Working Group 20/2/20 model. EXPERIMENTAL DESIGN: We report the outcomes of 150 patients with SMM enrolled in the iMMunocell study, in which serial assessment of tumor and immune cells in PB was performed every 6 months for a period of 3 years since enrollment. RESULTS: Patients with >0.015% versus ≤0.015% CTCs at baseline had a median time-to-progression of 17 months versus not reached (HR, 4.9; P < 0.001). Presence of >20% BM PCs had no prognostic value in a multivariate analysis that included serum free light-chain ratio >20, >2 g/dL M-protein, and >0.015% CTCs. The 20/2/20 and 20/2/0.015 models yielded similar risk stratification (C-index of 0.76 and 0.78). The combination of the 20/2/0.015 model with an immune risk score based on the percentages of SLAN+ and SLAN- nonclassical monocytes, CD69+HLADR+ cytotoxic NK cells, and CD4+CXCR3+ stem central memory T cells, allowed patient' stratification into low, intermediate-low, intermediate-high, and high-risk disease with 0%, 20%, 39%, and 73% rates of progression at 2 years. CONCLUSIONS: This study showed that CTCs outperform BM PCs for assessing tumor burden. Additional analysis in larger series are needed to define a consensus cutoff of CTCs for minimally invasive stratification of SMM.

3D Printable Dynamic Hydrogel: As Simple as it Gets!

3D printing technology offers a vast range of applications for tissue engineering applications. Over the past decade a vast range of new equipment has been developed; while, 3D printable biomaterials, especially hydrogels, are investigated to fit the printability requirements. The current candidates for bioprinting often require post-printing cross-linking to maintain their shape. On the other hand, dynamic hydrogels are considered as the most promising candidate for this application with their extrudability and self-healing properties. However, it proves to be very difficult to match the required rheological in a simple material. Here, this study presents for the first time the simplest formulation of a dynamic hydrogel based on thiol-functionalized hyaluronic acid formulated with gold ions that fulfill all the requirements to be printed without the use of external stimuli, as judged by the rheological studies. The printability is also demonstrated with a 3D printer allowing for the printing of the dynamic hydrogel as it is, achieving 3D construct with a relatively good precision and up to 24 layers, corresponding to 10 mm high. This material is the simplest 3D printable hydrogel and its mixture with cells and biological compounds is expected to open a new era in 3D bioprinting.

Preneoplastic somatic mutations including MYD88L265P in lymphoplasmacytic lymphoma.

Normal cell counterparts of solid and myeloid tumors accumulate mutations years before disease onset; whether this occurs in B lymphocytes before lymphoma remains uncertain. We sequenced multiple stages of the B lineage in elderly individuals and patients with lymphoplasmacytic lymphoma, a singular disease for studying lymphomagenesis because of the high prevalence of mutated MYD88. We observed similar accumulation of random mutations in B lineages from both cohorts and unexpectedly found MYD88L265P in normal precursor and mature B lymphocytes from patients with lymphoma. We uncovered genetic and transcriptional pathways driving malignant transformation and leveraged these to model lymphoplasmacytic lymphoma in mice, based on mutated MYD88 in B cell precursors and BCL2 overexpression. Thus, MYD88L265P is a preneoplastic event, which challenges the current understanding of lymphomagenesis and may have implications for early detection of B cell lymphomas.

Nanoscale Wetting of Single Viruses.

The epidemic spread of many viral infections is mediated by the environmental conditions and influenced by the ambient humidity. Single virus particles have been mainly visualized by atomic force microscopy (AFM) in liquid conditions, where the effect of the relative humidity on virus topography and surface cannot be systematically assessed. In this work, we employed multi-frequency AFM, simultaneously with standard topography imaging, to study the nanoscale wetting of individual Tobacco Mosaic virions (TMV) from ambient relative humidity to water condensation (RH > 100%). We recorded amplitude and phase vs. distance curves (APD curves) on top of single virions at various RH and converted them into force vs. distance curves. The high sensitivity of multifrequency AFM to visualize condensed water and sub-micrometer droplets, filling gaps between individual TMV particles at RH > 100%, is demonstrated. Dynamic force spectroscopy allows detecting a thin water layer of thickness ~1 nm, adsorbed on the outer surface of single TMV particles at RH < 60%.

A Probabilistic Model for Crystal Growth Applied toProtein Deposition at the Microscale.

A probabilistic discrete model for 2D protein crystal growth is presented. This model takesinto account the available space and can describe growing processes of a different nature due to theversatility of its parameters, which gives the model great flexibility. The accuracy of the simulation istested against a real recrystallization experiment, carried out with the bacterial protein SbpA fromLysinibacillus sphaericus CCM2177, showing high agreement between the proposed model and theactual images of the crystal growth. Finally, it is also discussed how the regularity of the interface(i.e., the curve that separates the crystal from the substrate) affects the evolution of the simulation.

Phytotoxicity of wear debris from traditional and innovative brake pads.

Traffic-related emissions include gas and particles that can alter air quality and affect human and environmental health. Limited studies have demonstrated that particulate debris thrown off from brakes are toxic to higher plants. The acute phytotoxicity of brake pad wear debris (BPWD) investigated using cress seeds grown in soil contaminated with increasing concentrations of debris. Two types of pads were used: a commercially available phenol based pad and an innovative cement-based pad developed within of the LIFE+ COBRA project. The results suggested that even through the BPWD generated by the two pads were similar in and morphology, debris from traditional pads were more phytotoxic than that from cementitious pads, causing significant alterations in terms of root elongation and loss of plasma membrane integrity.

Electrospinning of pyrazole-isothiazole derivatives: nanofibers from small molecules.

We investigate the electrospinning of small molecules, specifically designed peptide derivatives of the pyrazole-isothiazole scaffold. Such non-natural peptides enhance the spectrum of fundamental materials used for electrospinning. Unlike standard electrospun materials, our peptides are not polymeric, but able to aggregate in solution and especially during processing. They contain donor/acceptor groups that can form hydrogen bonds, and groups that are able to generate π-stacking interactions, which are known as important requirements for assembly processes. The pyrazole-isothiazole derivatives were synthesized by means of a 1,3-dipolar cycloaddition reaction, which is completely regioselective, affording only one isomer. We demonstrate that our compounds can be electrospun from fluoroalcohol solution into solid, quasi-endless micro- and nanofibers. The electrospinnability varies substantially, depending on the amino acids linked to the scaffold. Some compounds provide only short fibers, while Fmoc-glycyl-(N-benzyl)-pyrazole-isothiazole-tert-butyl carboxylate-1,1-dioxide forms continuous, homogenous, and bead-free fibers (droplet-like beads are a common problem in electrospinning). We analyzed the compounds and the fibers with various spectroscopic techniques (MS, IR and Raman). Electrospinning does not change chemical composition and configuration, suggesting the monomeric form of the compounds even in the fibers. Interestingly, we found that the stereochemistry of the scaffold can affect the ability of the peptide to be electrospun.

Catalysis of a 1,3-dipolar reaction by distorted DNA incorporating a heterobimetallic platinum(ii) and copper(ii) complex.

A novel catalytic system based on covalently modified DNA is described. This catalyst promotes 1,3-dipolar reactions between azomethine ylides and maleimides. The catalytic system is based on the distortion of the double helix of DNA by means of the formation of Pt(ii) adducts with guanine units. This distortion, similar to that generated in the interaction of DNA with platinum chemotherapeutic drugs, generates active sites that can accommodate N-metallated azomethine ylides. The proposed reaction mechanism, based on QM(DFT)/MM calculations, is compatible with thermally allowed concerted (but asynchronous) [π4s + π2s] mechanisms leading to the exclusive formation of racemic endo-cycloadducts.

Key factors of scanning a plant virus with AFM in air and aqueous solution.

For tobacco mosaic virus (TMV) as a model virus, this article shows typical issues of scanning soft biological matter by atomic force microscopy (AFM). TMV adsorbed on chemically different flat surfaces, gold, mica, and APDMES-functionalized silicon, is studied in air and aqueous environment. In air, the TMV particles arrangement shows some variety, depending on the substrate. The height of TMV is reduced to 13.7, 15.8, and 15.6 nm, for gold, APDMES, and mica, respectively while the width is about ∼30 nm due to the influence of the tip radius. In aqueous solution, the surface charges of the virus and the solid support play an important role in the virus adsorption process. While deposition on negatively charged mica is favored only at low pH values, it is shown that positively charged APDMES functionalized silicon can be a suitable substrate to work with at neutral pHs. The effects of cantilever oscillation's free amplitude (A0 ) and the amplitude set-point (A) are also assessed here. While high A0 prompt reversible deformation of TMV in measurements performed in air, irreversible damage of the virus in liquid conditions (water) is observed using stiff cantilevers (0.35 N m-1 ) and high A0 (81 nm), leading to a 6 nm reduction in the height of TMV after the first scan. Finally, low values of the amplitude set-point (A/A0 = 0.3), which means applying higher forces to the sample, also brings the damage of TMV virus assemblies, reducing its monolayer roughness to 0.3 nm. Microsc. Res. Tech. 80:18-29, 2017. © 2016 Wiley Periodicals, Inc.

Multifrequency Force Microscopy of Helical Protein Assembly on a Virus.

High-resolution microscopy techniques have been extensively used to investigate the structure of soft, biological matter at the nanoscale, from very thin membranes to small objects, like viruses. Electron microscopy techniques allow for obtaining extraordinary resolution by averaging signals from multiple identical structures. In contrast, atomic force microscopy (AFM) collects data from single entities. Here, it is possible to finely modulate the interaction with the samples, in order to be sensitive to their top surface, avoiding mechanical deformations. However, most biological surfaces are highly curved, such as fibers or tubes, and ultimate details of their surface are in the vicinity of steep height variations. This limits lateral resolution, even when sharp probes are used. We overcome this problem by using multifrequency force microscopy on a textbook example, the Tobacco Mosaic Virus (TMV). We achieved unprecedented resolution in local maps of amplitude and phase shift of the second excited mode, recorded together with sample topography. Our data, which combine multifrequency imaging and Fourier analysis, confirm the structure deduced from averaging techniques (XRD, cryoEM) for surface features of single virus particles, down to the helical pitch of the coat protein subunits, 2.3 nm. Remarkably, multifrequency AFM images do not require any image postprocessing.

On the molecular interaction between albumin and ibuprofen: An AFM and QCM-D study.

The adsorption of proteins on surfaces often results in a change of their structural behavior and consequently, a loss of bioactivity. One experimental method to study interactions on a molecular level is single molecular force spectroscopy that permits to measure forces down to the pico-newton range. In this work, the binding force between human serum albumin (HSA), covalently immobilized on glutaraldehyde modified gold substrates, and ibuprofen sodium salt was studied by means of single molecular force spectroscopy. First of all, a protocol was established to functionalize atomic force microscopy (AFM) tips with ibuprofen. The immobilization protocol was additionally tested by quartz crystal microbalance with dissipation (QCM-D) and contact angle measurements. AFM was used to characterize the adsorption of HSA on gold substrates, which lead to a packed monolayer of thickness slightly lower than the reported value in solution. Finally, single molecule spectroscopy results were used to characterize the binding force between albumin and ibuprofen and calculate the distance of the transition state (0.6 nm) and the dissociation rate constant (0.055 s(-1)). The results might indicate that part of the adsorbed protein still preserves its functionality upon adsorption.

Making novel bio-interfaces through bacterial protein recrystallization on biocompatible polylactide derivative films.

Fabrication of novel bio-supramolecular structures was achieved by recrystallizing the bacterial surface protein SbpA on amorphous and semicrystalline polylactide derivatives. Differential scanning calorimetry showed that the glass transition temperature (T(g)) for (poly-L-lactide)-PLLA, poly(L,D-lactide)-PDLLA, poly(lactide-co-glycolide)-PLGA and poly(lactide-co-caprolactone)-PLCL was 63 °C, 53 °C, 49 °C and 15 °C, respectively. Tensile stress-strain tests indicated that PLLA, PLGA, and PDLLA had a glassy behaviour when tested below T(g). The obtained Young modulus were 1477 MPa, 1330 MPa, 1306 MPa, and 9.55 MPa for PLLA, PLGA, PDLLA, and PLCL, respectively. Atomic force microscopy results confirmed that SbpA recrystallized on every polymer substrate exhibiting the native S-layer P4 lattice (a = b = 13 nm, γ = 90°). However, the polymer substrate influenced the domain size of the S-protein crystal, with the smallest size for PLLA (0.011 µm(2)), followed by PDLLA (0.034 µm(2)), and PLGA (0.039 µm(2)), and the largest size for PLCL (0.09 µm(2)). quartz crystal microbalance with dissipation monitoring (QCM-D) measurements indicated that the adsorbed protein mass per unit area (~1800 ng cm(-2)) was independent of the mechanical, thermal, and crystalline properties of the polymer support. The slowest protein adsorption rate was observed for amorphous PLCL (the polymer with the weakest mechanical properties and lowest T(g)). QCM-D also monitored protein self-assembly in solution and confirmed that S-layer formation takes place in three main steps: adsorption, self-assembly, and crystal reorganization. Finally, this work shows that biodegradable polylactide derivatives films are a suitable support to form robust biomimetic S-protein layers.

Influence of surface chemistry and protein concentration on the adsorption rate and S-layer crystal formation.

Bacterial crystalline surface layers (S-layers) are the outermost envelope of prokaryotic organisms representing the simplest biological membranes developed during evolution. In this context, the bacterial protein SbpA has already shown its intrinsic ability to reassemble on different substrates forming protein crystals of square lattice symmetry. In this work, we present the interaction between the bacterial protein SbpA and five self-assembled monolayers carrying methyl (CH(3)), hydroxyl (OH), carboxylic acid (COOH) and mannose (C(6)H(12)O(6)) as functional groups. Protein adsorption and S-layer formation have been characterized by atomic force microscopy (AFM) while protein adsorption kinetics, mass uptake and the protein layer viscoelastic properties were investigated with quartz crystal microbalance with dissipation monitoring (QCM-D). The results indicate that the protein adsorption rate and crystalline domain area depend on surface chemistry and protein concentration. Furthermore, electrostatic interactions tune different protein rate adsorption and S-layer recrystallization pathways. Electrostatic interactions induce faster adsorption rate than hydrophobic or hydrophilic interactions. Finally, the shear modulus and the viscosity of the recrystallized S-layer on CH(3)C(6)S, CH(3)C(11)S and COOHC(11)S substrates were calculated from QCM-D measurements. Protein-protein interactions seem to play a main role in the mechanical stability of the formed protein (crystal) bilayer.

Waterborne polyurethane-acrylic hybrid nanoparticles by miniemulsion polymerization: applications in pressure-sensitive adhesives.

Waterborne polyurethane-acrylic hybrid nanoparticles for application as pressure-sensitive adhesives (PSAs) were prepared by one-step miniemulsion polymerization. The addition of polyurethane to a standard waterborne acrylic formulation results in a large increase in the cohesive strength and hence a much higher shear holding time (greater than seven weeks at room temperature), which is a very desirable characteristic for PSAs. However, with the increase in cohesion, there is a decrease in the relative viscous component, and hence there is a decrease in the tack energy. The presence of a small concentration of methyl methacrylate (MMA) in the acrylic copolymer led to phase separation within the particles and created a hemispherical morphology. The tack energy was particularly low in the hybrid containing MMA because of the effects of lower energy dissipation and greater cross-linking. These results highlight the great sensitivity of the viscoelastic and adhesive properties to the details of the polymer network architecture and hence to the precise composition and synthesis conditions.

Surface dependence of protein nanocrystal formation.

The self-assembly kinetics and nanocrystal formation of the bacterial surface-layer-protein SbpA are studied with a combination of quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM). Silane coupling agents, aminopropyltriethoxysilane (APTS) and octadecyltrichlorosilane (OTS), are used to vary the protein-surface interaction in order to induce new recrystallization pathways. The results show that the final S-layer crystal lattice parameters (a = b = 14 nm, gamma = 90 degrees ), the layer thickness (15 nm), and the adsorbed mass density (1700 ng cm(-2)) are independent of the surface chemistry. Nevertheless, the adsorption rate is five times faster on APTS and OTS than on SiO(2,) strongly affecting protein nucleation and growth. As a consequence, protein crystalline domains of 0.02 microm(2) for APTS and 0.05 microm(2) for OTS are formed, while for silicon dioxide the protein domains have a typical size of about 32 microm(2). In addition, more-rigid crystalline protein layers are formed on hydrophobic substrates. In situ AFM experiments reveal three different kinetic steps: adsorption, self-assembly, and crystalline-domain reorganization. These steps are corroborated by frequency-dissipation curves. Finally, it is shown that protein adsorption is a diffusion-driven process. Experiments at different protein concentrations demonstrate that protein adsorption saturates at 0.05 mg mL(-1) on silane-coated substrates and at 0.07 mg mL(-1) on hydrophilic silicon dioxide.