Magnetic Microtweezers for High-Throughput Bioseparation in Sub-Nanoliter Droplets.

Multiomics studies at single-cell level require small volume manipulation, high throughput analysis, and multiplexed detection, characteristics that droplet microfluidics can tackle. However, the initial step of molecule bioseparation remains challenging. Here, we describe a unique magnetic device to trap and extract magnetic particles in sub-nanoliter droplets, for compartmentalisation of detection steps. Relying on electrodeposition of NiFe structures and microfluidic manipulation, the extraction of 1 µm diameter magnetic particles was achieved at high throughput (20 droplets per second) with an efficiency close to 100% in 450 pL droplets. The first demonstration of its adaptability to single-cell analysis is demonstrated with the extraction of mRNA. Using a purified nucleic acid solution, this unique magnetic configuration was able to reach a RNA extraction rate of 72%. This is the first demonstration of a physical separation in droplets at high throughput at single-cell scale.

Nanotubes from Lanthanide-Based Misfit-Layered Compounds: Understanding the Growth, Thermodynamic, and Kinetic Stability Limits.

Gaining insights into the kinetics and the thermodynamic limits of nanostructures in high-temperature reactions is crucial for controlling their unique morphology, phase, and structure. Nanotubes from lanthanide-based misfit-layered compounds (MLCs) have been known for more than a decade and were successfully produced mostly via a chemical vapor transport protocol. The MLC nanotubes show diverse structural arrangements and lattice disorders, which could have a salient impact on their properties. Though their structure and charge transfer properties are reasonably well understood, a lack of information on their thermodynamic and kinetic stability limits their scalable synthesis and their applicability in modern technologies. In this study, the growth, thermodynamic stability, and decomposition kinetics of lanthanide-based misfit nanotubes of two model compounds, i.e., (LaS)1.14TaS2 and (SmS)1.19TaS2 are elucidated in detail. The nanotubes were carefully analyzed via atomic resolution electron microscopy imaging and synchrotron-based X-ray and electron diffraction techniques, and the information on their morphology, phase, and structures was deduced. The key insights gained would help to establish the parameters to explore their physio-chemical properties further. Furthermore, this study sheds light on the complex issue of the high-temperature stability of nanotubes and nanostructures in general.

An 8-month adapted motor activity program in a young CMT1A male patient.

Background: It is unclear whether prolonged periods of training can be well tolerated. In Charcot-Marie Tooth disease (CMT). We report the effects of an 8-month, adapted motor activity (AMA) program in a 16-years-old CMT1A male patient. The program included strength, mobility, and balance training (two sessions per week, 1 h per session). Measures: Walking ability and walking velocity (Six-Minute Walking Test-6MWT, Ten Meters Walking Test-10 mW T), balance (Y-Balance Test-YBT, Berg Balance Scale-BBS), functional mobility (Short Physical Performance Battery-Short physical performance battery), fatigue (Checklist Individual strength questionnaire - CIS20R), health and quality of life (Short Form Health Survey 36 questionnaire-SF-36) were evaluated in three moments: before (T0), after 5 (T1) and 8 (T2) months of adapted motor activity. Dorsal and plantar foot flexion strength (Maximal Voluntary Contraction-maximum voluntary contraction) and neuromuscular functions (Electromyography-sEMG, interpolated twitch technique-ITT) were measured at T1 and T2. Results: Relative to T0, an amelioration of walking ability (6MWT, +9,3%) and balance (with improvements on Y-balance composite normalized mean reach of the right and left limb of 15,3% and 8,5%, respectively) was appreciable. Relative to T1, an increase in foot strength in three out of four movements (right plantar flexion, +39,3%, left plantar flexion, +22,7%, left dorsal flexion, 11,5%) was observed. Concerning voluntary muscle activation, a greater recruitment in the left, unlike right, medial gastrocnemius was observed. Conclusion: Results suggest the safety of an 8-month AMA program in a young patient affected by CMT1A.

Cancer-associated fibroblasts actively compress cancer cells and modulate mechanotransduction.

During tumor progression, cancer-associated fibroblasts (CAFs) accumulate in tumors and produce an excessive extracellular matrix (ECM), forming a capsule that enwraps cancer cells. This capsule acts as a barrier that restricts tumor growth leading to the buildup of intratumoral pressure. Combining genetic and physical manipulations in vivo with microfabrication and force measurements in vitro, we found that the CAFs capsule is not a passive barrier but instead actively compresses cancer cells using actomyosin contractility. Abrogation of CAFs contractility in vivo leads to the dissipation of compressive forces and impairment of capsule formation. By mapping CAF force patterns in 3D, we show that compression is a CAF-intrinsic property independent of cancer cell growth. Supracellular coordination of CAFs is achieved through fibronectin cables that serve as scaffolds allowing force transmission. Cancer cells mechanosense CAF compression, resulting in an altered localization of the transcriptional regulator YAP and a decrease in proliferation. Our study unveils that the contractile capsule actively compresses cancer cells, modulates their mechanical signaling, and reorganizes tumor morphology.

Multiomic Droplet-Based Assay for Ultralow Input Samples.

The extraction and separation of cellular compounds are crucial steps in numerous biological protocols, particularly in multiomics studies, where several cellular modalities are examined simultaneously. While magnetic particle extraction is commonly used, it may not be applicable for ultralow input samples. Microfluidics has made possible the analysis of rare or low-materiality samples such as circulating tumor cells or single cells through miniaturization of numerous protocols. In this study, a microfluidics workflow for separating different cellular modalities from ultralow input samples is presented. This approach is based on magnetic tweezers technology, allowing the extraction and resuspension of magnetic particles between consecutive nanoliter droplets to perform multistep assays on small volumes. The ability to separate and recover mRNA and gDNA in samples containing less than 10 cells is demonstrated, achieving separation efficiency comparable to the one obtained with conventional pipetting but with a significantly lower amount of starting material, typically 1-2 orders of magnitude less.

Improved Electrochemical Performance of NTs-WS2@C Nanocomposites for Lithium-Ion and Sodium-Ion Batteries.

Even though WS2 nanotubes (NTs-WS2) have great potential as anode materials for lithium-ion batteries (LIBs) and sodium-ion batteries (SIBs) thanks to their unusual layered structure, their conductivity and cycling stability are far from satisfactory. To tackle these issues, carbon-coated WS2 (NTs-WS2@C) nanocomposites were prepared through a facile synthesis method that involved precipitating a carbon precursor (20% sucrose) on WS2 nanotubes, followed by annealing treatment under an argon environment. Thanks to the presence of highly conductive and mechanically robust carbon on the outer surface, NTs-WS2@C nanocomposites show improved electrochemical performance compared with bare NTs-WS2. After 60 cycles at 80 mA g-1 current density, the cells display high capacities of 305 mAh g-1 in LIBs and 152 mAh g-1 in SIBs, respectively. As the current density increases to 600 mA g-1, it provides specific capacities of 209 and 115 mAh g-1, correspondingly. The enhanced electrochemical performance in LIBs and SIBs is primarily attributed to the synergistic effects of the tubular architecture of WS2, carbon network and stable nanocomposite structure, which can effectively constrain volume variation during the metal ions intercalation/deintercalation processes.

Signature of Ultrafast Formation and Annihilation of Polaronic States in a Layered Ferromagnet.

The strong interaction between charge and lattice vibration gives rise to a polaron, which has a profound effect on optical and transport properties of matters. In magnetic materials, polarons are involved in spin dependent transport, which can be potentially tailored for spintronic and opto-spintronic device applications. Here, we identify the signature of ultrafast formation of polaronic states in CrBr3. The polaronic states are long-lived, having a lifetime on the time scale of nanoseconds to microseconds, which coincides with the emission lifetime of ∼4.3 µs. Transition of the polaronic states is strongly screened by the phonon, generating a redshift of the transition energy ∼0.2 eV. Moreover, energy-dependent localization of polaronic states is discovered followed by transport/annihilation properties. These results shed light on the nature of the polarons and their formation and transport dynamics in layered magnetic materials, which paves the way for the rational design of two-dimensional magnetic devices.

Magnetic Nanocomposites of Coated Ferrites/MOF as Pesticide Adsorbents.

Magnetic metal-organic frameworks (MMOFs) are gaining increased attention as emerging adsorbents/water remediation agents. Herein, a facile development of novel MMOFs comprised of coated ferrite nanoparticles (MNPs) and UiO-66 metal-organic framework is reported. In specific, coated Co- and Zn-doped ferrite magnetic nanoparticles were synthesized as building block while the metal-organic framework was grown in the presence of MNPs via a semi-self-assembly approach. The utilization of coated MNPs facilitated the conjugation and stands as a novel strategy for fabricating MMOFs with increased stability and an explicit structure. MMOFs were isolated with 13-25 nm crystallites sizes, 244-332 m2/g specific surface area (SSA) and 22-42 emu/g saturation magnetization values. Establishing the UiO-66 framework via the reported semi-self-assembly resulted in roughly 70% reduction in both magnetic properties and SSA, compared with the initial MNPs building blocks and UiO-66 framework, respectively. Nonetheless, the remaining 30% of the magnetization and SSA was adequate for successful and sufficient adsorption of two different pesticides, 2,4-Dichlorophenoxyacetic acid (2,4-D) and 2,4,5-Trichlorophenoxyacetic acid (2,4,5-T), while the recovery with a commercial magnet and reuse were also found to be effective. Adsorption and kinetic studies for all three MMOFs and both pesticides were performed, and data were fitted to Langmuir-Freundlich isotherm models.

Chiral Phonons and Giant Magneto-Optical Effect in CrBr3 2D Magnet.

Phonons with chirality determine the optical helicity of inelastic light scattering processes due to their nonzero angular momentum. Here it is shown that 2D magnetic CrBr3 hosts chiral phonons at the Brillouin-zone center. These chiral phonons are linear combinations of the doubly-degenerate Eg phonons, and the phonon eigenmodes exhibit clockwise and counterclockwise rotational vibrations corresponding to angular momenta of l = ± 1. Such Eg chiral phonons completely switch the polarization of incident circularly polarized light. On the other hand, the non-degenerate non-chiral Ag phonons display a giant magneto-optical effect under an external out-of-plane magnetic field, rotating the plane of polarization of the scattered linearly polarized light. The corresponding degree of polarization of the scattered light changes from 91% to -68% as the magnetic field strength increases from 0 to 5 T. In contrast, the chiral Eg modes display no field dependence. The results lay a foundation for the study of phonon chirality and magneto-optical phenomena in 2D magnetic materials, as well as their related applications, such as the phonon Hall effect, topological photonics, and Raman lasing.

Sprinkler irrigation in the production of safe rice by soils heavily polluted by arsenic and cadmium.

Among the factors affecting the bioaccumulation of As and Cd in rice, a key role is played by the irrigation methods. The sprinkler irrigation (SP), optimized for rice in Sardinia, Italy, applied to several rice genotypes over many years has produced no differences in yields in comparison to what observed using the traditional continuous flooding irrigation method (CF). Because all the previous SP trials have been performed just on one, unpolluted soil, the principal aim of this study is to ascertain the effectiveness of SP to simultaneously minimize the bioaccumulation of As and Cd in rice grain even in soils severely polluted by As and/or Cd. Hence, a Carnise rice genotype was cultivated in an open field in: i) an unpolluted soil; ii) a soil polluted with 55 mg kg-1 of As; iii) a soil polluted with 40 mg kg-1 of Cd; iv) a soil polluted with 50 mg kg-1 of As and 50 mg kg-1 of Cd. In the worst condition of pollution, the amounts of total As and Cd measured in the kernels using a fully validated ICP-MS method is 90 ± 10 µg kg-1 and 50 ± 20 µg kg-1, respectively, i.e. less than 50% and the 25% of the maximum concentration set for these elements in rice by the European Community (200 µg kg-1 for the inorganic As and the total amount of Cd, respectively). SP might represent a simple and valuable tool able to produce safe rice also from soils where the traditional irrigation might produce inedible rice only.

Apoptosis mapping in space and time of 3D tumor ecosystems reveals transmissibility of cytotoxic cancer death.

The emerging tumor-on-chip (ToC) approaches allow to address biomedical questions out of reach with classical cell culture techniques: in biomimetic 3D hydrogels they partially reconstitute ex vivo the complexity of the tumor microenvironment and the cellular dynamics involving multiple cell types (cancer cells, immune cells, fibroblasts, etc.). However, a clear bottleneck is the extraction and interpretation of the rich biological information contained, sometime hidden, in the cell co-culture videos. In this work, we develop and apply novel video analysis algorithms to automatically measure the cytotoxic effects on human cancer cells (lung and breast) induced either by doxorubicin chemotherapy drug or by autologous tumor-infiltrating cytotoxic T lymphocytes (CTL). A live fluorescent dye (red) is used to selectively pre-stain the cancer cells before co-cultures and a live fluorescent reporter for caspase activity (green) is used to monitor apoptotic cell death. The here described open-source computational method, named STAMP (spatiotemporal apoptosis mapper), extracts the temporal kinetics and the spatial maps of cancer death, by localizing and tracking cancer cells in the red channel, and by counting the red to green transition signals, over 2-3 days. The robustness and versatility of the method is demonstrated by its application to different cell models and co-culture combinations. Noteworthy, this approach reveals the strong contribution of primary cancer-associated fibroblasts (CAFs) to breast cancer chemo-resistance, proving to be a powerful strategy to investigate intercellular cross-talks and drug resistance mechanisms. Moreover, we defined a new parameter, the 'potential of death induction', which is computed in time and in space to quantify the impact of dying cells on neighbor cells. We found that, contrary to natural death, cancer death induced by chemotherapy or by CTL is transmissible, in that it promotes the death of nearby cancer cells, suggesting the release of diffusible factors which amplify the initial cytotoxic stimulus.

Single-cell proteomic and transcriptomic analysis of macrophage heterogeneity using SCoPE2.

BACKGROUND: Macrophages are innate immune cells with diverse functional and molecular phenotypes. This diversity is largely unexplored at the level of single-cell proteomes because of the limitations of quantitative single-cell protein analysis. RESULTS: To overcome this limitation, we develop SCoPE2, which substantially increases quantitative accuracy and throughput while lowering cost and hands-on time by introducing automated and miniaturized sample preparation. These advances enable us to analyze the emergence of cellular heterogeneity as homogeneous monocytes differentiate into macrophage-like cells in the absence of polarizing cytokines. SCoPE2 quantifies over 3042 proteins in 1490 single monocytes and macrophages in 10 days of instrument time, and the quantified proteins allow us to discern single cells by cell type. Furthermore, the data uncover a continuous gradient of proteome states for the macrophages, suggesting that macrophage heterogeneity may emerge in the absence of polarizing cytokines. Parallel measurements of transcripts by 10× Genomics suggest that our measurements sample 20-fold more protein copies than RNA copies per gene, and thus, SCoPE2 supports quantification with improved count statistics. This allowed exploring regulatory interactions, such as interactions between the tumor suppressor p53, its transcript, and the transcripts of genes regulated by p53. CONCLUSIONS: Even in a homogeneous environment, macrophage proteomes are heterogeneous. This heterogeneity correlates to the inflammatory axis of classically and alternatively activated macrophages. Our methodology lays the foundation for automated and quantitative single-cell analysis of proteins by mass spectrometry and demonstrates the potential for inferring transcriptional and post-transcriptional regulation from variability across single cells.

YS-TaS2 and YxLa1-xS-TaS2 (0 ≤ x ≤ 1) Nanotubes: A Family of Misfit Layered Compounds.

We present the analysis of a family of nanotubes (NTs) based on the quaternary misfit layered compound (MLC) YxLa1-xS-TaS2. The NTs were successfully synthesized within the whole range of possible compositions via the chemical vapor transport technique. In-depth analysis of the NTs using electron microscopy and spectroscopy proves the in-phase (partial) substitution of La by Y in the (La,Y)S subsystem and reveals structural changes compared to the previously reported LaS-TaS2 MLC-NTs. The observed structure can be linked to the slightly different lattice parameters of LaS and YS. Raman spectroscopy and infrared transmission measurements reveal the tunability of the plasmonic and vibrational properties. Density-functional theory calculations showed that the YxLa1-xS-TaS2 MLCs are stable in all compositions. Moreover, the calculations indicated that substitution of La by Sc atoms is electronically not favorable, which explains our failed attempt to synthesize these MLC and NTs thereof.

Synthesis and characterization of quaternary La(Sr)S-TaS2 misfit-layered nanotubes.

Misfit-layered compounds (MLCs) are formed by the combination of different lattices and exhibit intriguing structural and morphological characteristics. MLC Sr x La1- x S-TaS2 nanotubes with varying Sr composition (10, 20, 40, and 60 Sr atom %, corresponding to x = 0.1, 0.2, 0.4 and 0.6, respectively) were prepared in the present study and systematically investigated using a combination of high-resolution electron microscopy and spectroscopy. These studies enable detailed insight into the structural aspects of these phases to be gained at the atomic scale. The addition of Sr had a significant impact on the formation of the nanotubes with higher Sr content, leading to a decrease in the yield of the nanotubes. This trend can be attributed to the reduced charge transfer between the rare earth/S unit (La x Sr1- x S) and the TaS2 layer in the MLC which destabilizes the MLC lattice. The influence of varying the Sr content in the nanotubes was systematically studied using Raman spectroscopy. Density functional theory calculations were carried out to support the experimental observations.

An overview of the recent advances in inorganic nanotubes.

Advanced nanomaterials play a prominent role in nanoscience and nanotechnology developments, opening new frontiers in these areas. Among these nanomaterials, due to their unique characteristics and enhanced chemical and physical properties, inorganic nanotubes have been considered one of the most interesting nanostructures. In recent years, important progress has been achieved in the production and study of these nanomaterials, including boron nitride, transition metal dichalcogenide nanotubular structures, misfit-based nanotubes and other hybrid/doped nanotubular objects. This review is devoted to the in-depth analysis of recent studies on the synthesis, atomic structures, properties and applications of inorganic nanotubes and related nanostructures. Particular attention is paid to the growth mechanism of these nanomaterials. This is a crucial point for the challenges ahead related to the mass production of high-quality defect-free nanotubes for a variety of applications.

Synthesis and Characterization of Nanotubes from Misfit (LnS)1+y TaS2 (Ln=Pr, Sm, Gd, Yb) Compounds.

The synthesis and characterization of nanotubes from misfit layered compounds (MLCs) of the type (LnS)1+y TaS2 (denoted here as LnS-TaS2 ; Ln=Pr, Sm, Gd, and Yb), not reported before, are described (the bulk compound YbS-LaS2 was not previously documented). Transmission electron microscopy and selected area electron diffraction showed that the interlayer spacing along the c axis decreased with an increase in the atomic number of the lanthanide atom, which suggested tighter interaction between the LnS layer and TaS2 for the late lanthanides. The Raman spectra of the tubules were studied and compared to those of the bulk MLC compounds. Similar to the bulk MLCs, the Raman spectra could be divided into the low-frequency modes (110-150 cm-1 ) of the LnS lattice and the high-frequency modes (250-400 cm-1 ) of the TaS2 lattice. The Raman spectra indicated that the vibrational lattice modes of the strained layers in the tubes were stiffer than those in the bulk compounds. Furthermore, the modes of the late lanthanides were higher in energy than those of the earlier lanthanides, which suggested larger charge transfer between the LnS and TaS2 layers for the late lanthanides. Polarized Raman measurements showed the expected binodal intensity profile (antenna effect). The intensity ratio of the Raman signal showed that the E2g mode of TaS2 was more sensitive to the light-polarization effect than its A1g mode. These nanotubes are expected to reveal interesting low-temperature quasi-1D transport behavior.

Resolution improvement of 3D stereo-lithography through the direct laser trajectory programming: Application to microfluidic deterministic lateral displacement device.

The vast majority of current microfluidic devices are produced using soft lithography, a technique with strong limitations regarding the fabrication of three-dimensional architectures. Additive manufacturing holds great promises to overcome these limitations, but conventional machines still lack the resolution required by most microfluidic applications. 3D printing machines based on two-photon lasers, in contrast, have the needed resolution but are too limited in speed and size of the global device. Here we demonstrate how the resolution of conventional stereolithographic machines can be improved by a direct programming of the laser path and can contribute to bridge the gap between the two above technologies, allowing the direct printing of features between 10 and 100 µm, corresponding to a large fraction of microfluidic applications. This strategy allows to achieve resolutions limited only by the physical size of the laser beam, decreasing by a factor at least 2× the size of the smallest features printable, and increasing their reproducibility by a factor 5. The approach was applied to produce an open microfluidic device with the reversible seal, integrating periodical patterns using the simple motifs, and validated by the fabrication of a deterministic lateral displacement particles sorting device. The sorting of polystyrene beads (diameter: 20 µm and 45 µm) was achieved with a specificity >95%, comparable with that achieved with arrays prepared by microlithography.

Microfluidic platform combining droplets and magnetic tweezers: application to HER2 expression in cancer diagnosis.

The development of precision medicine, together with the multiplication of targeted therapies and associated molecular biomarkers, call for major progress in genetic analysis methods, allowing increased multiplexing and the implementation of more complex decision trees, without cost increase or loss of robustness. We present a platform combining droplet microfluidics and magnetic tweezers, performing RNA purification, reverse transcription and amplification in a fully automated and programmable way, in droplets of 250nL directly sampled from a microtiter-plate. This platform decreases sample consumption about 100 fold as compared to current robotized platforms and it reduces human manipulations and contamination risk. The platform's performance was first evaluated on cell lines, showing robust operation on RNA quantities corresponding to less than one cell, and then clinically validated with a cohort of 21 breast cancer samples, for the determination of their HER2 expression status, in a blind comparison with an established routine clinical analysis.

Variation of milk components in the Italian Brown cattle.

The aim of this study was to evaluate the variations of protein, casein, saturated (SFA), unsaturated (UFA), monounsaturated (MUFA), polyunsaturated (PUFA) fatty acids contents and cheese yield in the milk of two groups of Italian Brown cows conventionally reared in indoor period of housing or consuming pasture during the summer months in 2008 and 2013. Milk components were obtained from samples collected during the national routine (conventionally reared) and 'extraordinary' (pasture period) milk recording scheme in herds located near Sondrio (Lombardia, Italy). Milk samples were processed with the MilkoScanTM FT6000 for the identification of milk casein, SFA, UFA, MUFA and PUFA composition. The groups were analysed separately per year and the environmental factors affecting milk protein, casein, and fatty acids contents (pasture/indoor, parity, data of sampling, days in milk, days from collection to analysis) were included in the MIXED procedure of SAS 9.3. A total of 778 milk samples were available, including 234 records from indoor and 544 observations from pasture feeding. Pasture intake affected the content of casein (%) and the proportion of fat in milk (g/100 g), enhancing milk casein levels (from 2.90 to 3) and reducing the concentration of milk SFA in milk from grazing cows (from 2.29 to 1.92). Additionally, the cheese yield was calculated as 'kg of cheese per 100 kg of milk' and resulted to be 10.4 and 12 in 2008 from milk of cows reared indoor and with pasture based diet, respectively. The dairy industry should take advantage of the milk production during grazing periods from which high quality products may be obtained.

Photocatalytic Activity of Au/TiO2 Photocatalysts for H2 Evolution: Role of the Au Nanoparticles as a Function of the Irradiation Wavelength.

An investigation of hydrogen production with a series of Au/TiO2 photocatalysts reveals that the Au nanoparticles play different roles depending on the wavelength of the light irradiation. Under visible-light irradiation, the photoactivity is primarily controlled by the intensity of the Au surface plasmon band, whereas under UV irradiation the Au nanoparticles act as co-catalysts with TiO2 .